RAFAEL RIBEIRO ALMEIDA

Transcrição

1 RAFAEL RIBEIRO ALMEIDA Reconhecimento entre clados e efeito supressor induzido por vacinas de DNA codificando peptídeos conservados e promíscuos do grupo M do HIV-1 Tese apresentada à Faculdade de Medicina da Universidade de São Paulo para obtenção de título de Doutor em Ciências Programa de Alergia e Imunopatologia Orientador: Prof. Dr. Edecio Cunha Neto SÃO PAULO 2014

2 RAFAEL RIBEIRO ALMEIDA Reconhecimento entre clados e efeito supressor induzido por vacinas de DNA codificando peptídeos conservados e promíscuos do grupo M do HIV-1 Tese apresentada à Faculdade de Medicina da Universidade de São Paulo para obtenção de título de Doutor em Ciências Programa de Alergia e Imunopatologia Orientador: Prof. Dr. Edecio Cunha Neto SÃO PAULO 2014

3 Dados Internacionais de Catalogação na Publicação (CIP) Preparada pela Biblioteca da Faculdade de Medicina da Universidade de São Paulo reprodução autorizada pelo autor Almeida, Rafael Ribeiro Reconhecimento entre clados e efeito supressor induzido por vacinas de DNA codificando peptídeos conservados e promíscuos do grupo M do HIV-1 / Rafael Ribeiro Almeida. -- São Paulo, Tese (doutorado)--faculdade de Medicina da Universidade de São Paulo. Programa de Alergia e Imunopatologia. Orientador: Edecio Cunha Neto. Descritores: 1.HIV-1 2.Vacinas de DNA 3.Linfócitos T CD4-positivos 4.Sequência consenso 5.Imunidade cruzada 6.Gene classe II do complexo de histocompatibilidade (MHC) 7.Imunossupressão 8.Camundongos 9.Humanos 10.Infecções por HIV USP/FM/DBD-217/14

4 Aos meus pais, Otacílio e Mércia, pelo apoio e compreensão.

5 Agradecimentos Ao meu orientador Prof. Dr. Edecio Cunha-Neto pelos ensinamentos, compreensão, paciência e pela presença constante em todo o desenvolvimento deste trabalho. Ao Prof. Dr. Jorge Kalil pelas importantes sugestões fornecidas nas reuniões de quinta-feira. Ao Prof. Dr. Douglas F. Nixon pelos ensinamentos e discussões enriquecedoras. Aos amigos do Biotério de Experimentação, Dr. Edilberto Postól e Luiz Mundel, pelo companheirismo e ajuda na condução dos experimentos. Aos colegas do grupo HIV/vacina, Vinicius e Susan, pelo auxílio na condução dos experimentos e pela amizade durante esses anos. Aos amigos e colegas de laboratório (LIM60 e LIM19) que direta ou indiretamente contribuíram para o desenvolvimento deste trabalho, proporcionando momentos de discussão científica e também momentos de grande descontração e diversão. À Fundação de Amparo à Pesquisa do Estado de São Paulo, FAPESP.

6 Esta tese está de acordo com as seguintes normas, em vigor no momento desta publicação: Referências: adaptado de International Committee of Medical Journals Editors (Vancouver). Universidade de São Paulo. Faculdade de Medicina. Divisão de Biblioteca e Documentação. Guia de apresentação de dissertações, teses e monografias. Elaborado por Anneliese Carneiro da Cunha, Maria Julia de A. L. Freddi, Maria F. Crestana, Marinalva de Souza Aragão, Suely Campos Cardoso, Valéria Vilhena. 3a ed. São Paulo: Divisão de Biblioteca e Documentação; Abreviaturas dos títulos dos periódicos de acordo com List of Journals Indexed in Index Medicus.

7 SUMÁRIO Lista de abreviaturas, símbolos e siglas... I Lista de tabelas... V Lista de figuras... VI Resumo... VII Abstract... VIII 1. Introdução AIDS Epidemiologia Origem e diversidade genética do HIV Ciclo replicativo do HIV Patogênese da infecção pelo HIV Resposta imunológica contra o HIV Estratégias vacinais contra o HIV Objetivos Objetivo geral Objetivos específicos Avaliar a capacidade da vacina HIVBr27 em induzir respostas de células T CD4 + com reatividade cruzada entre subtipos do HIV Avaliar a capacidade de ligação dos peptídeos codificados pelas vacinas HIVBr27 e HIVenv7 a moléculas HLA de classe II e MHC de classe II murinas Avaliar a frequência de reconhecimento dos peptídeos codificados pelas vacinas HIVBr27 e HIVenv7 por pacientes infectados pelo HIV Avaliar a imunossupressão induzida pela vacina HIVenv Avaliar a capacidade protetora da vacina HIVBr Metodologia Transformação de bactérias DH5α com plasmídeos vacinais Extração e purificação do DNA plasmidial Peptídeos Teste de ligação peptídeos-hla de classe II Camundongos e imunizações Suspensão de células esplênicas... 28

8 3.7. Proliferação celular contra peptídeos do HIV ELISPOT de IFN-γ Desafio com vírus Vaccinia recombinante Resultados A imunização com HIVBr27 induz resposta imune celular contra peptídeos de diferentes subtipos do HIV A imunização com a vacina HIVBr27 protege parcialmente contra infecção pelo vírus Vaccinia recombinante codificando Gag e Pol do HIV Os peptídeos codificados por HIVBr27 se ligam a múltiplas moléculas HLA de classe II Os peptídeos codificados por HIVBr27 são reconhecidos por células de pacientes infectados com HIV A vacina HIVenv7 não apresenta propriedades imunossupressoras consistentes Os peptídeos codificados por HIVenv7 se ligam a múltiplas moléculas HLA de classe II Os peptídeos codificados por HIVenv7 são reconhecidos por células de pacientes infectados com HIV Discussão Conclusões Anexo Anexo A Aprovação do Comitê de Ética em Pesquisa Anexo B Aval para utilização de amostras de pacientes infectados pelo HIV Referências bibliográficas Publicações... 72

9 I LISTA DE ABREVIATURAS, SÍMBOLOS E SIGLAS % porcentagem aa ADCC AEC AIDS APC ART BFA aminoácidos citotoxicidade celular dependente de anticorpo 3-amino-9-ethylcarbazole Acquired Immune Deficiency Syndrome (Síndrome da Imunodeficiência Adquirida) Allophycocyanin (aloficocianina) Antiretroviral therapy (Terapia antirretroviral) Brefeldina CAPPesq Comissão de Ética para Análise de Projetos de Pesquisa CCR5 C-C chemokine receptor type 5 (receptor de quimiocina C-C do tipo 5) CD CDC CFSE CO 2 Con A CTL Cluster of Differentiation (designação de grupos) Centers for disease control and prevention (Centro para Controle e Prevenção de Doenças) Carboxyfluorescein succinimidyl ester (carboxifluoresceína succinimidil éster) Dióxido de carbono Concanavalina A Cytotoxic T limphocytes (linfócitos T citotóxicos) CXCR4 CXC chemokine Receptor type 4 (receptor de quimiocina CXC do tipo 4) DMSO DNA EDTA ELISPOT Fmoc Dimetil-sulfóxido deoxyribonucleic acid (Ácido desoxirribonucléico) Ethylenediamine tetraacetic acid (ácido tetracético etilenodiamínico) Enzyme-linked immunospot assay N-9-fluorenilmetoxicarbonil

10 II CRFs g GALT gp HIV-1 HIV-2 HLA HRP IFN-γ IFN-α Circulating recombinant forms (Formas recombinants circulantes) Gramas Tecido linfóide associado à mucosa intestinal Glicoproteína Human immunodeficiency vírus type 1 (Vírus da Imunodeficiêcia Humana do tipo 1) Human immunodeficiency vírus type 2 (Vírus da Imunodeficiêcia Humana do tipo 2) Human leukocyte antigen (Antígeno leucocitário humano) Horseradish peroxidase Interferon gama Interferon alfa IL-2 Interleucina 2 IMT InCor KHCO 3 LB LIM LPS LTNP mdcs mg Instituto de Medicina Tropical Instituto do Coração Bicarbonato de potássio Lúria Bertani Laboratório de Investigação Médica Lipopolissacarídeo Long term non progressors (Não progressores por longo tempo) Mieloid dendritic cells (células dendríticas mielóides) Miligramas MHC Major histocompatibility complex (Complexo principal de histocompatibilidade) MIP-1α Macrophage Inflammatory Protein alpha (proteína alfa inflamatória de macrófagos)

11 III MIP-1β ml mm mrna NaOH ng NH 4 Cl NK NKT o C OMS PBS pdcs PE PercP ph R10 RANTES RhCMV RNA rpm RPMI RT SFB Macrophage Inflammatory Protein betha (proteína beta inflamatória de macrófagos) Mililitro Milimolar Messenger ribonucleic acid (Ácido ribonucleico mensageiro) Hidróxido de sódio Nanogramas Cloreto de amonio Natural killer (matadoras naturais) Natural Killer T cells Grau centígrado Organização Mundial da Saúde Phosphate buffered saline (tampão salina-fosfato) Plasmocitoid dendritic cells (células dendríticas plasmocitóides) Phycoerythrin (ficoeritrina) Peridinin chlorophyll protein (proteína clorofil peridina) Potencial hidrogeniônico Meio RPMI suplementado com 10% de SFB Chemokine (C-C motif) ligand 5 (Ligante de quimiocina 5 (motivo C-C) Citomegalovírus símio Ribonucleic acid (Ácido ribonucleic) Rotações por minuto Roswell Park Memorial Institute Reverse transcriptase Soro Fetal Bovino

12 IV SIV SPF Simian Immunodeficieny Virus (vírus da imunodeficiência símia) Specific pathogen free (livre de patógenos específicos) TEPITOPE TLR UFS vol μg μl Algoritmo de predição de epitopos restritos a moléculas HLA-DR Toll like receptor Unidades formadoras de spots Volume Micrograma Microlitro

13 V LISTA DE TABELAS Tabela 1. Peptídeos variantes do HIV-1 e previsão de ligação a moléculas HLA-DR...32 Tabela 2. Os peptídeos codificados por HIVBr27 se ligam a múltiplas moléculas HLA-DR...36 Tabela 3. Os peptídeos codificados por HIVBr27 se ligam a múltiplas moléculas HLA-DP...37 Tabela 4. Os peptídeos codificados por HIVBr27 se ligam a múltiplas moléculas HLA-DQ...38 Tabela 5. Os peptídeos codificados por HIVBr27 se ligam a moléculas IA b e IA d...39 Tabela 6. Dados clínicos dos pacientes HIV Tabela 7. Os peptídeos codificados por HIVenv7 se ligam a múltiplas moléculas HLA-DR...46 Tabela 8. Os peptídeos codificados por HIVenv7 se ligam a múltiplas moléculas HLA-DP...47 Tabela 9. Os peptídeos codificados por HIVenv7 se ligam a múltiplas moléculas HLA-DQ...48 Tabela 10. Os peptídeos codificados por HIVenv7 se ligam a múltiplas moléculas IA b e IA d...49

14 VI LISTA DE FIGURAS Figura 1. Distribuição global de subtipos e formas recombinantes do grupo M do HIV Figura 2. Ciclo replicativo do HIV...9 Figura 3. Infecção pelo HIV-1 e resposta imunológica...14 Figura 4. A imunização com HIVBr27 induz resposta imune celular contra diferentes subtipos do HIV Figura 5. A imunização com HIVBr27 protege parcialmente contra desafio com vírus Vaccinia recombinante codificando Gag e Pol do HIV Figura 6. Os peptídeos codificados por HIVBr27 são reconhecidos por células de pacientes infectados pelo HIV Figura 7. A co-imunização de HIVenv7 e pvaxgag não reduz a resposta contra peptídeos de Gag...43 Figura 8. A co-imunização de HIVenv7 e pvaxgag não reduz a resposta contra o pool de peptídeos de Gag...44 Figura 9. A co-imunização de HIVenv7 e HIVBr27 não reduz a resposta contra o pool de peptídeos da HIVBr Figura 10. Os peptídeos codificados por HIVenv7 são reconhecidos por células de pacientes infectados pelo HIV

15 VII RESUMO Almeida RR. Reconhecimento entre clados e efeito supressor induzido por vacinas de DNA codificando peptídeos conservados e promíscuos do grupo M do HIV-1 [tese]. São Paulo: Faculdade de Medicina, Universidade de São Paulo; A busca por uma construção vacinal contra o HIV-1 é urgente. Os linfócitos T CD4 + têm assumido um papel de destaque no campo de vacinas por participar no controle da replicação do HIV-1, seja auxiliando as funções efetoras de linfócitos T CD8 + e a produção de anticorpos por linfócitos B ou mesmo agindo de forma citotóxica sobre macrófagos infectados. A utilização de sequências consenso do grupo M do HIV-1 é apontada como uma das maneiras de se contornar os problemas relacionados à diversidade viral. Além disso, é preciso construir vacinas que apresentem potencial de induzir respostas imunes com grande cobertura populacional. Com o intuito de induzir respostas amplas de linfócitos T CD4 + contra diversos subtipos do HIV-1 em uma população geneticamente diversa para moléculas HLA-DR, identificamos em nosso trabalho prévio 34 peptídeos promíscuos (previstos de se ligarem a múltiplas moléculas HLA-DR) e conservados da sequência consenso do grupo M do HIV-1. Desenvolvemos uma vacina de DNA codificando 7 peptídeos de Env (HIVenv7) e outra vacina (HIVBr27) codificando os demais 27 peptídeos. A vacina HIVBr27 foi imunogênica em camundongos BALB/c, induzindo uma resposta ampla e polifuncional de linfócitos T CD4 + e CD8 +. A vacina HIVenv7 foi pouco imunogênica e mostrou-se capaz de suprimir a resposta induzida pela HIVBr27 em regime de co-imunização. No presente trabalho demonstramos que a imunização com HIVBr27 induz uma resposta imune celular mediada por linfócitos T CD4 + e CD8 + contra peptídeos de diferentes subtipos do HIV-1. Além disso, a imunização com HIVBr27 mostrou-se parcialmente protetora contra a infecção pelo vírus Vaccinia recombinante codificando as proteínas Gag e Pol do HIV-1. Ensaios in vitro demonstraram que os peptídeos codificados pela HIVBr27 se ligam a múltiplas moléculas HLA de classe II e são reconhecidos por células de pacientes infectados pelo HIV-1. Demonstramos também que a vacina HIVenv7 não possui propriedades imunossupressoras consistentes, contrariando os resultados obtidos previamente. Os peptídeos codificados pela HIVenv7 se ligaram a múltiplas moléculas HLA de classe II, mas apresentaram baixa frequência de reconhecimento por células de pacientes infectados pelo HIV-1. Acreditamos que a vacina HIVBr27 possui potencial de induzir uma resposta imune de grande cobertura populacional e direcionada a diferentes variantes do HIV-1. Por outro lado, a vacina HIVenv7 se mostrou pouco imunogênica e não deve ser utilizada em estudos futuros. Descritores: HIV-1, Vacinas de DNA, Linfócitos T CD4-positivos, Sequência consenso, Imunidade cruzada, Gene de classe II do complexo de histocompatibilidade (MHC), Imunossupressão, Camundongos, Humanos, Infecções pelo HIV

16 VIII ABSTRACT Almeida RR. Cross-clade immunity and immunosuppressive effects of DNA vaccines encoding conserved and promiscuous HIV-1 M-group peptides [thesis]. São Paulo: Faculdade de Medicina, Universidade de São Paulo ; The search for an HIV-1 vaccine construct is urgent. The CD4 + T cells have assumed a prominent role in the vaccine field participating in the control of HIV-1 replication either by helping CD8 + T cell effector function and B cell-mediated antibody production or by acting as citotoxic cells on infected macrophages. The use of HIV-1 M-group consensus sequences is pointed as an alternative to overcome viral diversity. Besides, it is necessary to construct vaccines that would potentially induce immune responses with broad population coverage. Intending to induce a broad CD4 + T-cell immune response against different HIV-1 subtypes in a population bearing diverse HLA-DR molecules we have previously identified 34 promiscuous peptides (potentially binding to multiple HLA-DR molecules) and conserved within the HIV-1 M-group consensus sequence. We construct a DNA vaccine encoding 7 Env peptides (HIVenv7) and another vaccine (HIVBr27) encoding 27 peptides. The HIVBr27 vaccine was immunogenic in BALB/c mice, inducing a broad and polyfunctional CD4 + and CD8 + T-cell response. The HIVenv7 vaccine was much less immunogenic and suppressed HIVBr27-induced immune responses when co-immunized. Here, we have shown that HIVBr27 immunization leads to a cross-clade CD4 + and CD8 + T-cell immune response. Besides, HIVBr27 immunization has partially protected mice challenged with a recombinant Vaccinia virus encoding HIV-1 Gag e Pol. In vitro assays have shown that HIVBr27- encoded peptides bind to multiple HLA class II molecules and are recognized by HIV- 1-infected patients. We have also shown that HIVenv7 has no consistent immunosuppressive properties, contradicting our previous results. The HIVenv7- encoded peptides bound to multiple HLA class II molecules but were recognized by a low number of HIV-1-infected patients. We believe that our vaccine HIVBr27 has potential to induce an immune response with broad population coverage, towards different HIV-1 variants. On the other hand, the HIVenv7 vaccine was poorly immunogenic and should not be used in future studies. Descriptors: HIV-1, DNA vaccines, CD4-positive lymphocytes, Consensus sequence, Cross-clade immunity, Genes, MHC class II, Immunosuppression, Mice, Humans, HIV infections

17 1 1. INTRODUÇÃO 1.1. AIDS A Síndrome da Imunodeficiência Adquirida (AIDS) é causada por uma infecção crônica com o Vírus da Imunodeficiência Humana (HIV). O início oficial da epidemia é marcado pelo relato do Centro para Controle e Prevenção de Doenças (CDC) dos Estados Unidos em 1981, que reportou a presença de pneumonia causada por Pneumocystis carinii em cinco homens homossexuais (1). A confirmação de que a AIDS era causada por um agente infeccioso ocorreu entre 1983 e 1984 após o isolamento de um retrovírus proveniente do sangue de pacientes com a doença (2, 3). O HIV é um retrovírus que infecta principalmente linfócitos T CD4 +, podendo também infectar outras células, incluindo monócitos. O vírus infecta as células alvo utilizando a molécula CD4 como seu receptor principal e as moléculas CCR5 e CXCR4 como co-receptores. Durante as primeiras semanas de infecção os indivíduos podem apresentar sintomas semelhantes ao da gripe, com a presença de erupções cutâneas. É observada uma deterioração gradual das funções imunológicas dos indivíduos infectados ao longo do curso da infecção, à medida que os linfócitos T CD4 + são destruídos. Indivíduos com contagem de linfócitos T CD4 + abaixo de 200 células/mm 3 se tornam extremamente vulneráveis a doenças oportunistas e a cânceres (4) A infecção pelo HIV ocorre principalmente pela rota sexual, sendo as mulheres mais propensas a se infectar durante o intercurso heterossexual. A transmissão também pode ocorrer pelo contato direto com sangue ou hemoderivados, no momento de uma transfusão sanguínea ou pela utilização de seringas contaminadas. Outra forma de transmissão que pode ocorrer é a vertical, da mãe para o recém-nascido (5). O

18 2 tratamento para infecção com HIV é sugerido para indivíduos com contagem de linfócitos T CD4 + entre 350 e 500 células/mm 3 e feito com o auxílio de antirretrovirais (ARV). Estes variam de inibidores de enzimas virais, como a transcriptase reversa, protease e integrase até inibidores de fusão, como os antagonistas de CCR5 (6). Embora o tratamento tenha assegurado maior qualidade de vida e longevidade aos pacientes, existem problemas relacionados ao desenvolvimento de resistência por seleção de vírus mutantes e a morbidade associada aos efeitos colaterais das drogas. Os países mais afetados pela AIDS são subdesenvolvidos ou em desenvolvimento, sendo, então, pouco capazes de arcar com as despesas na manutenção das drogas antirretrovirais à disposição da população (7) A profilaxia pré-exposição (PrEP) é um exemplo de tratamento com antirretrovirais que vem mostrando sucesso significativo. Essa forma de terapia é utilizada principalmente em casais sorodiscordantes e pessoas que vivem sob alto risco de infecção, como usuários de drogas injetáveis. Segundo a Organização Mundial da Saúde (OMS), é aconselhável a utilização de uma dose diária da droga Tenofovir sozinha ou em combinação com Emtricitabina (6). Alguns estudos realizados com o intuito de avaliar a eficácia de PrEP demonstraram uma redução na aquisição do vírus entre 44% e 75%, dependendo do grupo de estudo avaliado (8, 9). Em outro estudo, entretanto, não se observou proteção significativa de PrEP em mulheres africanas soronegativas. Foram relatados ainda problemas relacionados a efeitos colaterais do tratamento, como redução da densidade óssea (10). Portanto, ainda se faz necessário o desenvolvimento de vacinas e outras formas de combate ao vírus para que a pandemia de AIDS seja de fato controlada. Outra forma de combate à infecção pelo HIV frequentemente utilizada na clínica é a profilaxia pós-exposição. Nesse caso, drogas antirretrovirais são

19 3 administradas a indivíduos que passaram por exposições de risco. Atualmente, recomenda-se a utilização das drogas em sistema de profilaxia pós-exposição por um período de 28 dias, sendo importante receber a primeira dose nas primeiras 72 horas após exposição. Cada país adota sua própria combinação de drogas a ser utilizada, baseando-se nos medicamentos estabelecidos como tratamento inicial para indivíduos infectados (6) Epidemiologia O número de indivíduos infectados pelo HIV-1 desde o início da pandemia já ultrapassa 60 milhões e o número de mortos por AIDS chega aos 25 milhões. Isto coloca a infecção pelo HIV-1 como uma das mais importantes nas últimas décadas e mobiliza grandes esforços para sua contenção. Segundo dados recentes do Programa Conjunto das Nações Unidas sobre HIV/AIDS, aproximadamente 35,3 milhões de pessoas vivem com HIV, sendo 69% dessas na região da África sub-sahariana (11). É estimado que 0,8% dos adultos entre 15 e 49 anos vivam com HIV, embora a epidemia varie consideravelmente entre países e regiões. O Brasil apresenta-se como um dos países mais afetados pela pandemia na América Latina, com mais de 500 mil pessoas vivendo com HIV/AIDS. Até junho de 2009, 183 mil óbitos haviam sido constatados (12). O número de novas infecções pelo HIV vem diminuindo ao longo do tempo, sendo observada uma redução de 20% em 2011 quando comparado a Da mesma maneira, a mortalidade relacionada à AIDS também apresentou uma queda, com uma redução de 24% em 2011 quando comparado a A epidemia HIV/AIDS no Brasil tem mudado nos últimos anos devido a alguns fatores, como a modificação do

20 4 comportamento nos seguimentos populacionais de alto risco e a implementação de medidas preventivas. Entretanto, as características epidemiológicas variam entre regiões do país. Observa-se uma redução nos casos de infecção pelo HIV no sudeste e um aumento nas regiões norte e nordeste (13) Origem e diversidade genética do HIV A pandemia de AIDS iniciou-se na década de 80. Entretanto, amostras de sangue coletadas na África em 1959 já possuíam rastros da infecção pelo HIV (14), um retrovírus derivado do Vírus da Imunodeficiência Símia (SIV) (15). O primeiro contato do homem com o vírus possivelmente ocorreu por contaminação direta com sangue de primatas não humanos, no momento da caça (16). O HIV pode ser geneticamente diferenciado em dois tipos: HIV-1 e HIV-2. O primeiro é similar ao vírus SIVcpz, isolado de chimpanzés na África equatorial ocidental. Esta região é marcada por altos índices de infecção e foi considerada o epicentro da pandemia. O aparecimento posterior de infecções pelo HIV nos Estados Unidos, Europa e outros países foi também relacionado ao mesmo vírus que surgiu na África. O HIV-2 é endêmico da África ocidental e possui como ancestral o SIVsm, isolado de macacos mangabeus fuligentos nessa região. Os dois tipos virais possivelmente surgiram em decorrência de várias transmissões independentes entre primatas não humanos e humanos e acabaram se fixando na população humana (17). O HIV-1 é o principal responsável pela pandemia de AIDS e pode ser representado atualmente pelos grupos M, N, O e P. O grupo M é o grande responsável pela pandemia global, o grupo O causa algumas centenas de infecções na África centrooeste, o grupo N concentra-se em uma pequena população de Camarões, assim como o

21 5 grupo P, recentemente descrito em dois indivíduos nesse país (18-20). O grupo M, que é relacionado a mais de 90% dos casos de infecção, pode ser dividido em pelo menos 10 subtipos, sendo eles A, B, C, D, F, G, H, I, J e K, além de várias formas recombinantes. As variantes do HIV-1 formadas a partir da recombinação entre subtipos virais, sequenciadas e encontradas em 3 ou mais indivíduos epidemiologicamente não relacionados são determinadas CRFs. Quando não se atende a esses critérios, essas formas recombinantes são denominadas URFs (formas recombinantes únicas). A diversidade genética entre subtipos varia de 17 a 35%, podendo chegar a 42% e dentro de um mesmo subtipo a variação é de 8 a 17%, chegando a 30% em alguns casos (21). Isto se deve principalmente à grande quantidade de erros cometidos pela enzima transcriptase reversa (RT) na transcrição do RNA viral em DNA, um passo essencial no ciclo de replicação de um retrovírus. Além disso, outros fatores podem contribuir para diversificação genética, como a alta taxa de replicação e a propensão viral para recombinação genômica. O vírus pode, ainda, ter sua variabilidade genética aumentada devido a pressões ambientais, como as terapias farmacológicas e fatores relacionados ao hospedeiro, como as deaminases da família APOBEC (Apolipoprotein B (APOB) mrna-editing, catalytic polypeptide) (22-24). De acordo com os dados mais recentes sobre a epidemiologia molecular global do HIV-1, o subtipo C é responsável por 48% das infecções enquanto os subtipos A e B são responsáveis por 12% e 11%, respectivamente. As formas recombinantes CRF02_AG e CRF01_AE são encontradas em 8 e 5% dos infectados enquanto os subtipos G e D estão presentes em 5 e 2% dos mesmos. Os subtipos F, H, J e K são responsáveis por 1% das infecções no mundo enquanto outras CRFs e URFs representam os 8% restantes. Em conjunto, CRFs e URFs representam 20% das infecções globais (25). O subtipo A é o mais prevalente na Europa Oriental e Ásia

22 6 Central. O subtipo B é o mais encontrado nas Américas, na Europa Central e Ocidental, assim como no Oriente Médio. O subtipo C é o mais frequente no sul da África e Índia. A África Central é marcada pela distribuição de diversas variantes virais (Figura 1). Figura 1. Distribuição global de subtipos e formas recombinantes do grupo M do HIV-1. Os gráficos de pizza representam a distribuição da diversidade viral documentada entre os anos de 2004 e O tamanho das pizzas representa a quantidade relativa de pessoas vivendo com HIV-1 em cada região. CRFs (formas recombinantes); URFs (formas recombinantes únicas). Adaptado de (21). A fim de contornar o problema relacionado à diversidade genética do HIV-1, modelos evolucionários têm sido desenvolvidos para gerar sequências consenso e ancestrais. Enquanto uma sequência consenso é criada determinando-se o aminoácido mais comum em cada posição após um alinhamento entre sequências, uma sequência ancestral leva em consideração, por exemplo, os critérios de máxima verossimilhança

23 7 adotados em construções de árvores filogenéticas. Tanto as sequências consenso quanto ancestrais são vantajosas no desenvolvimento de vacinas contra o HIV-1 por apresentarem similaridade às diversas formas circulantes do vírus e possuírem potencial de induzir respostas imunológicas contra as mesmas (26) Ciclo replicativo do HIV A infecção pelo HIV tem início com a interação entre proteínas virais e receptores na superfície das células alvo. O envelope do HIV é um complexo proteico trimérico, composto das proteínas gp120 e gp41. A primeira se liga à glicoproteína CD4, uma molécula presente na superfície de monócitos/macrófagos, eosinófilos, células dendríticas, micróglia e 60% dos linfócitos T circulantes. Esta ligação causa mudanças conformacionais na gp120, expondo um domínio protéico que se liga especificamente aos receptores de quimiocinas CCR5 ou CXCR4. Após a ligação da gp120 ao CD4, a porção N-terminal da gp41 penetra na membrana celular. Isto leva a uma aproximação da partícula viral e a célula, fusionando-as (27, 28). A expressão diferencial dos receptores de quimiocinas CCR5 e CXCR4 nas células alvo tem sido apontada como o principal determinante do tropismo viral. O CXCR4 é expresso em diversas células, incluindo linfócitos, enquanto o CCR5 está presente em monócitos/macrófagos, células dendríticas e linfócitos T ativados. Os vírus que infectam células CCR5 + são denominados R5 e são mais frequentes em estágios iniciais da infecção. Os vírus que infectam células CXCR4 + são denominados X4, sendo encontrados em momentos mais tardios da infecção (29). Após a fusão do vírus à membrana celular, o core proteico libera o RNA viral no citoplasma. Esse é convertido em DNA pela transcriptase reversa, em um processo que

24 8 envolve a formação de um híbrido RNA-DNA, a quebra do mesmo e a síntese do DNA de fita dupla. A ausência de atividade revisora na transcriptase reversa torna-se, nesse momento, fundamental para geração de mutações e, consequententemente, diversidade genética do HIV (30). O DNA proviral recém-formado é então integrado no genôma da célula alvo, que deve estar em um estado ativado. Monócitos/macrófagos, micróglia e linfócitos T CD4 + em estado quiescente, após a infecção, tornam-se importantes reservatórios virais (31). A transcrição do DNA proviral em RNA mensageiro ocorre em células ativadas. O processo de tradução resulta primeiramente nas proteínas regulatórias Tat e Rev. A primeira é responsável por se ligar ao RNA viral no núcleo e dar início à transcrição de RNAs codificadores das enzimas e proteínas estruturais do vírus. A proteína Rev auxilia na transcrição desses RNAs e na expressão protéica, promovendo, assim, a formação de partículas virais maduras. As proteínas codificadas pelos genes pol e gag formam o core da partícula viral em maturação. O gene env codifica a proteína gp160, que posteriormente sofre clivagem e dá origem às proteínas do envelope, gp120 e gp41. A formação de uma nova partícula viral ocorre em um processo coordenado. Duas fitas de RNA se associam com as enzimas replicativas, enquanto as proteínas que compõem o core as envolvem, formando o capsídeo viral. A partícula imatura então migra para a superfície celular. As moléculas precursoras são clivadas pela protease, gerando uma nova partícula viral, que brota da membrana celular hospedeira (32) (Figura 2).

25 9 HIV-1 gp120 CD4 DNA celular DNA linear Co-receptor RNA DNA integrado RNAm Síntese proteica, processamento e formação da partícula viral Fusão RNA Brotamento Partícula viral madura Figura 2. Ciclo replicativo do HIV. A partícula viral se liga à célula hospedeira pelo contato da glicoproteína gp120 com a molécula CD4. Posteriormente uma mudança conformacional no envelope viral promove a ligação da gp41 ao correceptor (CCR5 ou CXCR4). O RNA viral é liberado no citoplasma, transcrito de forma reversa em DNA e esse é integrado no genoma hospedeiro. Novas moléculas de RNA são formadas, assim como novas proteínas virais. A protease cliva precursores protéicos em suas formas derivadas, o RNA viral é envolto em proteínas do vírus, dando origem ao capsídeo. O envelope é formado e novas partículas virais brotam da célula hospedeira. Adaptado de VSB associates Patogênese da infecção pelo HIV A infecção pelo HIV ainda é cercada por algumas incertezas. Não se sabe ao certo se o vírus é transmitido como partículas livres ou associado a células do muco cervicovaginal ou do sêmen. Sabe-se, entretanto, que o SIV pode ser transmitido sob as duas formas (33). Os vírus que alcançam e cruzam o epitélio mucoso possivelmente o fazem por transcitose ou por contato direto com células dendríticas intraepiteliais.

26 10 Mucosas danificadas por trauma físico ou infecções genitais permitem que os vírus cruzem mais facilmente a barreira epitelial (34, 35). A fase de eclipse da infecção pelo HIV ocorre dentro de um período de 10 dias após a transmissão. Neste momento o RNA viral não é detectável no plasma. É sugerido que o estabelecimento da infecção ocorra por um único foco de linfócitos T CD4 + infectados, caracterizando um efeito fundador. O recrutamento de células T susceptíveis à infecção, em resposta à precoce resposta imunológica inata local, permite que a replicação viral se mantenha (36). A taxa de erros cometidos pela trasncriptase reversa e a ação antiviral desempenhada por proteínas APOBEC podem resultar na produção de vírus defeituosos e culminar no fracasso de muitos focos de infecção (37). Partículas virais e/ou células infectadas chegam ao linfonodos drenantes ao fim da fase de eclipse, onde ocorre então a infecção de linfócitos T CD4 + CCR5 + ativados. Células dendríticas podem internalizar partículas virais e levá-las a linfócitos T ativados, exacerbando ainda mais o processo (38). Linfócitos B também podem estar envolvidos nesta fase inicial de espalhamento da infecção, devido à ligação de vírus a receptores do complemento (CD21) (39). O tecido linfóide associado à mucosa intestinal (GALT) torna-se então o alvo principal da infecção pelo HIV. Aproximadamente 20% dos linfócitos T CD4 + neste local são infectadas e até 60% das células não infectadas se ativam e morrem localmente por apoptose, liberando partículas apoptóticas que podem suprimir o sistema imunológico (40). Até 80% dos linfócitos T CD4 + podem ser depletados no GALT nas primeiras 3 semanas de infecção (41). O pico de viremia, caracterizado por intensa replicação viral no GALT e outros tecidos linfóides, alcança mais de um milhão de cópias de RNA por mililitro (ml) de sangue entre 21 e 28 dias após a infecção pelo HIV. O número de linfócitos T CD4 + é baixo durante esse período, posteriormente retornando a níveis próximos do normal no

27 11 sangue, mas não no GALT (41). A soro-conversão ocorre entre três e cinco semanas após o contato com o vírus. A presença de infecção na ausência de níveis dectectáveis de anticorpos é denominada janela imunológica (42). A fase seguinte é caracterizada pela queda da carga viral, que ocorre durante um período de 12 a 20 semanas após o pico de viremia, até chegar a um nível mais estável (set point) (43). O set point é caracterizado por ausência de sintomas e mantido por um balanço entre a replicação viral e a resposta imunológica. Neste momento observa-se o aparecimento de variantes virais selecionadas pela pressão imunológica, que havia surgido logo antes do estabelecimento do pico de viremia (44, 45). Alguns indivíduos, denominados controladores de elite, conseguem controlar eficientemente a viremia sem tratamento e permanecem por longos períodos com a carga viral indetectável (46). A ativação de células da resposta imunológica inata, assim como linfócitos T e B, é um marco da infecção pelo HIV e isto não ocorre apenas em células específicas para o vírus. De fato, é observada uma correlação positiva entre marcadores de ativação em linfócitos T CD8 + e progressão para AIDS, assim como uma extensiva ativação e apoptose de linfócitos T e B (40, 47). Diversos eventos são relacionados à ativação imunológica, ainda que isso não esteja claramente definido. Dentre eles são destacados a infecção viral direta das células do sistema imunológico, produção de citocinas proinflamatórias por células da resposta inata, translocação microbiana, perda de linfócitos T reguladores e co-infecção crônica por micobactérias e vírus (48). Outro fato que também caracteriza a infecção pelo HIV é a disfunção progressiva do sistema imunológico, quer seja contra antígenos do vírus, ou contra antígenos de outros patógenos (49-52). A meia vida dos linfócitos T CD4 + e CD8 + é reduzida, a migração dessas células se torna anormal, ocorre exaustão e redução da quantidade de linfócitos T de memória (50, 53).

28 12 Após 10 anos de infecção crônica, aproximadamente 50% dos indivíduos sem tratamento vão acabar desenvolvendo sintomas associados à queda no número de linfócitos T CD4 +. A queda no número dessas células leva ao aparecimento de doenças oportunistas, como pneumonia por Pneumocystis carinii, sarcoma de kaposi, criptosporidose, toxoplasmose e micobacteriose semelhante a tuberculose. Os indivíduos podem aprensentar inchaço difuso dos linfonodos, perda de peso, febre e distúrbios gastro-intestinais. O sistema nervoso também é afetado, com a presença de encefalopatia progressiva e demência. Anemia e linfopenia são frequentemente dectáveis, além do aumento no risco de se desenvolver doenças cardiovasculares (54, 55) Resposta imunológica contra o HIV As células matadoras naturais (NK), as células NKT, as células dendríticas plasmocitóides (pdcs) e as mielóides (mdcs) são responsáveis por funções efetoras e reguladoras da resposta imune inata envolvida no controle da infecção pelo HIV (56). Células dendríticas epidermais (células de Langerhans) possuem em sua superfície lectinas do tipo C (langerinas), que se ligam ao HIV e promovem sua internalização para os grânulos de Birbeck, nos quais o vírus é rapidamente degradado. Sugere-se que esse processo contribua como uma barreira à infecção (57). Entretanto, a ligação da gp120 do HIV à molécula DC-SIGN na superfície de células dendríticas promove sua internalização e localização em endosomas primários, onde o vírus não é degradado. Esse processo pode estar envolvido no espalhamento da infecção nos linfonodos, nos quais as células dendríticas entram em contato com linfócitos T CD4 +. Acredita-se que haja tranferência de vírus para esses linfócitos via sinapses virológias (pontos

29 13 polarizados de contato entre células onde corre transmissão viral) e fusão célula-célula com dendríticas (58). Os receptores do tipo Toll (TLR) são um importante grupo de receptores que reconhecem padrões moleculares (PRR) e estão diretamente envolvidos no reconhecimento e resposta contra o HIV. A estimulação de TLR7/8 induz a produção de diversas citocinas com papel antiviral, sendo o IFN-α uma das principais. De fato, a infecção pelo HIV-1 ou pelo SIV são marcadas por uma alta produção dessa citocina (59, 60). O IFN-α é capaz de ativar tanto células da resposta imune adaptativa quanto inata, incluindo a atividade citolítica de células NK (61). Entretanto, uma estimulação persistente de sua produção na infecção crônia pode contribuir para a patologia associada ao HIV (62). As células NK e NKT podem controlar a replicação viral por atividade citotóxica direta em células infectadas e pela produção de citocinas e quimiocinas. Além disso, essas células podem interagir com células dendríticas e influenciar a resposta dos linfócitos T. Durante a infecção aguda pelo HIV observa-se ativação e proliferação de células NK e NKT (48). A resposta adaptativa contra o HIV tem seu início marcado pelo aparecimento de linfócitos T específicos contra o vírus pouco antes do pico de viremia ser estabelecido e chega ao seu auge entre uma e duas semanas após esse período. Este momento é também marcado pela redução progressiva da carga viral (48). Uma seleção de mutações ocorre devido à pressão imunológica exercida pelos linfócitos T CD8 + e o set point viral é estabelecido (63) (Figura 3). A resposta celular precoce contra o HIV normalmente é direcionada às proteínas do envelope e a Nef. Proteínas mais conservadas como Pol e a subunidade p24 de Gag se tornam alvo da resposta celular em momentos mais tardios e isso pode representar um fato importante no controle da viremia (64, 65). Respostas contra epitopos imunodominantes presentes em proteínas

30 Número de células / título Viremia (cópias por ml de plasma) 14 conservadas provavelmente resultam em set point virais mais baixos (66). Diferentemente da resposta celular inicial contra o HIV, que foca em poucos epitopos virais, a resposta tardia é ampla e frequentemente ocorre contra mais de 10 epitopos (67). Aguda Assintomática, mas progressiva AIDS Risco de transmissão Diversidade viral Anticorpos anti-hiv Linfócitos T CD8 + citotóxicos Linfócitos T CD4 + Figura 3. Infecção pelo HIV-1 e resposta imunológica. Viremia no plasma (superior), e dinâmica de alterações no compartimento de linfócitos T CD4 + (inferior). A infecção aguda é caracterizada por uma elevada viremia plasmática (linha vermelha superior), queda acentuada de linfócitos CD4 + (linha verde inferior) e níveis baixos de anticorpos específicos (linha alaranjada tracejada inferior). A viremia decai com o aparecimento de linfócitos CD8 + citotóxicas (CTL) (linha azul tracejada inferior) e o set point viral é atingido (linha vermelha superior). O set point viral pode diferir entre os indivíduos (ex. linha vermelha tracejada superior). O risco de transmissão é maior durante o pico de viremia, cai durante a fase assintomática e volta a aumentar após o desenvolvimento de AIDS (círculos preenchidos superior). A diversidade viral aumenta ao fim da fase assintomática e início da AIDS (círculos preenchidos superior). A depleção de linfócitos T CD4 + no tecido linfóide associado ao intestino (GALT) é alta e constante ao longo do curso da infecção (círculos preenchidos inferior). Adaptado de (68).

31 15 Os linfócitos T CD8 + podem controlar a replicação viral por outros mecanismos além da resposta citotóxica contra células infectadas, sendo um deles a secreção de quimiocinas β, como MIP-1α, MIP- 1β e RANTES. Essas quimiocinas se ligam aos coreceptores do vírus na superfície dos linfócitos T CD4 + e impedem que ocorra a infecção. A liberação das quimiocinas é um processo antígeno-específico e pode auxiliar no desenvolvimento da atividade citotóxica dos linfócitos citolíticos (69). Acredita-se que indivíduos com respostas de linfócitos T CD8 + restritas a diversos HLAs (antígeno leucocitário humano) de classe I apresentam vantagens no controle da replicação viral (70). Os HLAs de classe I mais frequentemente associados ao controle do HIV-1 são HLA-B27 e HLA-B57 (71, 72). Sugere-se ainda que os HLAs B27 e B57 estão relacionados a uma resposta mais forte de células NK, via KIR3DL1 (73, 74). Os linfócitos T CD4 + HIV-específicos também participam no desenvolvimento de uma resposta imunológica contra a infecção e isto se dá principalmente pela produção de citocinas que auxiliam na indução e manutenção da atividade dos linfócitos citolíticos, bem como na estimulação da produção de anticorpos por linfócitos B (75). A depleção progressiva dos linfócitos T CD4 + associa-se com um declínio na atividade de linfócitos citotóxicos. Isto pode, em parte, explicar a perda do controle da carga viral em um indivíduo cronicamente infectado que ainda apresenta linfócitos T CD8 + vírusespecíficas (69). Indivíduos que conseguem controlar a viremia sem a ajuda da terapia antiretroviral apresentam uma resposta policlonal, persistente e vigorosa de linfócitos T CD4 + vírus-específicos, resultando na produção de interferon gama (IFN-γ) e quimiocinas β (76). A proliferação dos linfócitos T CD4 + vírus-específicos foi, ainda, correlacionada a uma forte resposta de linfócitos citotóxicos vírus-específicas e ao conseqüente controle da viremia (77). Respostas de linfócitos T CD4 + específicos contra

32 16 Gag na fase inicial da infecção foram relacionadas a menores set points virais (78) e maior contagem de linfócitos T CD4 + em pacientes cronicamente infectados pelo subtipo C do HIV-1 (79). Além disso, foi observdado que indivíduos infectados caracterizados como progressores lentos apresentam respostas potentes de linfócitos T CD4 + contra Gag (80) Acredita-se atualmente que linfócitos T CD4 + apresentam funções efetoras diretas no controle da replicação do HIV. Entretanto, a expresssão limitada de HLAs de classe II na superfície dessas células, quando infectadas, sugere que o controle da disseminação viral nas mesmas por linfócitos T CD4 + efetores seja prejudicada, cabendo aos linfócitos T CD8 + essa função. É possível que os linfócitos T CD4 + efetores contribuam para o controle da replicação viral em macrófagos e células dendríticas, por apresentarem grande expressão de HLAs de classe II. Os linfócitos T CD8 +, por outro lado, são ineficientes em controlar a replicação viral nessas células (81). Neste contexto, linfócitos T CD4 + expressando granzima A durante as fases iniciais da infecção foram associados a um menor set point viral e a uma progressão mais lenta para AIDS (82). A produção de anticorpos contra o HIV é geralmente expressiva e direcionada às proteínas estruturais do vírus. Apesar disso, apenas uma pequena parte desses anticorpos possuem atividade neutralizante e podem, então, participar na inibição da infecção viral. Epitopos das proteínas do envelope viral que se ligam aos receptores da célula a ser infectada são os principais alvos dos anticorpos neutralizantes. A maioria dos anticorpos neutraliza virions livres, antes que ocorra a infecção de uma célula alvo (83). Entretanto, o HIV-1 pode ser transmitido entre células próximas por sinapse virológica. Este processo foi documentado na transmissão do vírus entre células T CD4 + e mesmo entre células T CD4 + e células dendríticas. As partículas virais transmitidas

33 17 dessa forma são então menos acessíveis aos anticorpos neutralizantes (84). Vacinas indutoras de anticorpos amplamente neutralizantes são previstas como a melhor forma de se evitar a infecção pelo HIV. Entretanto, nenhuma vacina desenvolvida até o momento foi capaz de desempenhar tal função. Portanto, estratégias que envolvam tanto a resposta imune humoral quanto celular, inclusive aquela mediada por linfócitos T CD4 +, devem ser abordadas para o desenvolvimento de uma vacina eficaz contra o HIV Estratégias vacinais contra o HIV O desenvolvimento de vacinas contra o HIV aborda diversos conceitos genéticos e imunológicos, tanto do hospedeiro quanto do vírus, e pode ser dividido em estratégias vacinais tradicionais e recentes. As estratégias tradicionais envolvem vírus vivos atenuados, vírus mortos e subunidades proteicas. Embora este conjunto de tecnologias tenha demonstrado grande sucesso em vacinas contra outros vírus, elas ainda desempenham uma utilidade limitada contra o HIV. A imunização com vírus atenuados fornece uma proteção substancial contra o SIV em modelos envolvendo macacos resos (85, 86). Entretanto, preocupações quanto à segurança dessa estratégia a tornam improvável de ser testada em humanos (87, 88). Vírus mortos e subunidades protéicas possuem uma capacidade baixa de induzir anticorpos neutralizantes, assim como linfócitos T CD8 +, sendo pouco eficazes contra o HIV (89). As estratégias vacinais recentes incluem DNA plasmidial e vetores virais recombinantes, construídos para expressar antígenos do HIV. Os plasmídeos são bastante simples de usar e muito versáteis, porém as respostas imunológicas induzidas são geralmente fracas. Múltiplas doses, adjuvantes, ou mesmo novas tecnologias como

34 18 a eletroporação in vivo, são requeridos para que haja uma efetiva imunização de primatas não humanos e humanos (90-93). A maioria dos testes clínicos em andamento, entretanto, utiliza vetores virais administrados sozinhos ou como reforço de imunizações prévias com plasmídeos (89). Embora vacinas indutoras de imunidade celular não sejam capazes de bloquear a infecção viral, modelos animais têm demonstrado que essa abordagem é eficiente no controle da replicação do vírus (94, 95). Portanto, a expectativa é de que tais vacinas sejam capazes de controlar a viremia também no contexto humano, prevenir a destruição acentuada de linfócitos T CD4 + de memória e reduzir a transmissão do vírus (96). De fato, uma baixa viremia (menor que cópias de RNA/ml de sangue) está associada à progressão mais lenta para AIDS, assim como a uma menor taxa de transmissão (97-99). Os ensaios clínicos de eficácia de vacinas contra o HIV em andamento ou encerrados são fonte de valiosas informações para o desenvolvimento de novas estratégias vacinais. A vacina AIDSVAX, desenhada para indução de anticorpos contra a proteína gp120, foi utilizada em dois estudos de eficácia de fase III, conduzidos nos Estados Unidos e Tailândia e não foi observada proteção contra a infecção (100, 101). O ensaio clínico de fase IIb (STEP), conduzido pela indústria farmacêutica Merck, baseou-se em uma vacina trivalente composta por adenovírus 5 codificando para as proteínas Gag, Pol e Nef do HIV-1. Este ensaio visava à indução de uma resposta imunológica celular e foi encerrado em 2007 por ausência de eficácia. Foram observadas frequências similiares de resposta de linfócitos T CD8 + específicos contra Gag tanto em participantes que receberam a vacina como no gupo controle (40% vs 42%, respectivamente) (102). A resposta de linfócitos T CD8+ foi direcionada a apenas um epitopo da vacina, em média, sendo observada resposta para epitopos tanto de

35 19 regiões conservadas quanto não conservadas das proteínas (103). Não houve proteção contra infecção e nem mesmo redução da viremia nos indivíduos vacinados e infectados. Esssa falta de eficiência tem sido relacionada à baixa amplitude de resposta induzida (104, 105). Acredita-se que a amplitude de resposta induzida contra o SIV/HIV seja um dos fatores determinantes no controle viral. De fato, alguns regimes vacinais indutores de respostas amplas promoveram um controle eficaz do vírus SIV em macacos resos. Um regime vacinal de prime/boost envolvendo plasmídeos e adenovírus 5, codificando 8 dos 9 genes do SIV (exceto env), testado em macacos resos, induziu reconhecimento de 12 epitopos provenientes das proteínas Gag, Pol e Nef e se mostrou bastante protetor (106). Em outra situação, a utilização do vírus CMV símio recombinante codificando diversos genes do SIV resultou em uma resposta ampla, mediada por linfócitos T CD4+ e CD8+ contra o SIV. Sugere-se que essa resposta foi capaz de eliminar o vírus SIV de macacos infectados (95). O ensaio RV144, conduzido na Tailândia, foi concluído recentemente. Este ensaio era composto pelas vacinas ALVAC e AIDSVAX e visava induzir uma resposta celular, bem como uma resposta humoral contra o HIV-1. Os indivíduos receberam quatro doses iniciais com o vetor recombinante Canarypox codificando gp120, Gag e protease (ALVAC). Em seguida, foram administradas duas doses auxiliares da proteína gp120 (AIDSVAX). Os indivíduos vacinados apresentaram uma redução de 30% na taxa de infecção, mas não houve efeito sobre a carga viral (107). As respostas imunológicas mais consistentes entre os participantes que receberam as vacinas foram proliferação de linfócitos T CD4 + e anticorpos contra gp120, além de citotoxicidade celular dependente de anticorpo (ADCC) (108). Acredita-se que anticorpos contra as

36 20 regiões V1V2 possam ter contribuído para a proteção, enquanto altos níveis de IgA anti- Env podem ter prejudicado (109). As vacinas indutoras de resposta celular contra o HIV-1 desenvolvidas até o momento são em sua maioria destinadas a induzir respostas mediadas por linfócitos T CD8 + (110). Entretanto, a participação dos linfócitos T CD4 + na imunidade contra o HIV-1, evidenciada por estudos com pacientes infectados, e modelos de vacinas experimentais têm corroborado com a importância do desenvolvimento de uma vacina indutora de tal subtipo celular. De fato, foi observado que a vacinação de macacos resos com o vírus atenuado SIVmac239Δnef induziu alta frequência de linfócitos T CD4 + efetores e resultou no controle da viremia (111). Em outro estudo com macacos resos, o citomegalovírus símio RhCMV foi utilizado como vetor, codificando as ptroteínas Gag, Rev-Tat-Nef e Env do SIV. Neste caso, foi observado uma forte indução de linfócitos T CD4 + de memória efetora e os macacos foram capazes de controlar a viremia, na ausência de anticorpos neutralizantes. Quatro macacos vacinados controlaram a infecção do vírus na mucosa e não demonstraram disseminação sistêmica do mesmo (94). Acredita-se que um papel efetor direto de linfócitos T CD4 + contribua para essa proteção, como observado em um estudo no qual linfócitos T CD4 + específicos contra Nef e Gag eram capazes de eliminar macrófagos infectados pelo SIV (112). As vacinas testadas até o momento são, em sua maioria, compostas por genes ou proteínas inteiras do HIV-1. Dessa forma, as mutações de escape induzidas pela pressão do sistema imunológico permanecem no imunógeno, o que pode estar relacionado ao baixo potencial de proteção das mesmas (102, 107). Além disso, a imunodominância observada em regimes de imunização com proteínas inteiras é sugerida como redutora da amplitude de resposta, uma vez que a resposta imunológica acaba sendo direcionada

37 21 a apenas alguns epitopos imunodominantes ( ). As vacinas multiepitópicas podem combinar múltiplos epitopos de linfócitos T alinhados e focar a resposta imune nos mesmos. Nessa estratégia vacinal cada epitopo isoladamente pode ser imunogênico e algumas observações já foram feitas sobre a capacidade dessas vacinas em induzir respostas amplas e potentes ( ). A grande diversidade genética do HIV-1 exige o desenvolvimento de vacinas que sejam capazes de induzir uma imunidade protetora ampla, contra as diversas variantes circulantes do vírus. As vacinas de mosaico, baseadas em algoritmos que recombinam sequências do HIV-1, são exemplos de estratégias que têm como objetivo melhorar a cobertura vacinal (119). Estudos experimentais realizados em macacos resos sugerem que essas estratégias podem ser eficientes para induzir respostas celulares amplas, em diferentes contextos de imunização (120, 121). A utilização de regiões conservadas entre vários subtipos do vírus, assim como sequências consenso do grupo M do HIV-1 também têm sido testadas por alguns grupos a fim de contornar o problema da diversidade viral (122, 123). As vacinas compostas por regiões de múltiplos subtipos virais também têm sido utilizadas visando contornar o mesmo problema (92, 124, 125). Até o momento, entretanto, nenhuma vacina foi desenhada para induzir, em grande cobertura populacional, uma resposta ampla de linfócitos T CD4 + contra peptídeos conservados entre os vários subtipos circulantes do HIV-1. Tendo em vista a importância da indução de uma resposta mediada por linfócitos T CD4 + no desenvolvimento de uma vacina contra o HIV-1, assim como os benefícios previamente descritos sobre a inclusão de múltiplos epitopos em uma vacina, a utilização de algoritmos de predição de epitopos para linfócitos T CD4 + pode ser uma ferramenta interessante no desenvolvimento de estratégias vacinais contra o HIV-1. O algoritmo TEPITOPE, estruturado para predição de epitopos para linfócitos T CD4 +, se

38 22 baseia em matrizes matemáticas, determinadas a partir de dados quantitativos de ligação entre todos os resíduos de aminoácidos naturais e os bolsões de interação presentes em moléculas HLA-DR. Uma pontuação de ligação peptídeo-hla-dr é gerada para cada região de 9 aminoácidos da proteína varrida com o algoritmo. Peptídeos que possuem alta pontuação de interação são sinalizados como potenciais ligadores a moléculas de HLA-DR descritas no algoritmo (126). O nosso grupo tem utilizado o algoritmo TEPITOPE para construção de vacinas contra o HIV-1 e em um trabalho prévio foram identificados 18 peptídeos na sequência consenso do subtipo B do HIV-1. A capacidade de ligação dos peptídeos a múltiplas moléculas HLA-DR foi confirmada por testes in vitro. Observou-se que os peptídeos foram reconhecidos por células mononucleares do sangue de mais de 90% dos pacientes infectados pelo HIV-1 avaliados no estudo. Em média, cada paciente reconheceu 5 peptídeos, considerando a produção de IFN-γ tanto por linfócitos T CD4 + quanto CD8 +. (127). Uma vacina de DNA (HIVBr18) codificando esses peptídeos mostrou-se bastante imunogênica em camundongos transgênicos para moléculas HLA de classe II, apresentando grande amplitude de resposta (16 dos 18 peptídeos reconhecidos) e grande cobertura vacinal, sendo observadas respostas imunes contra os peptídeos codificados pela vacina em todas as linhagens de camundongos utilizadas (128). A vacina HIVBr18 mostrou-se também capaz de induzir uma resposta ampla, polifuncional e de longa duração em camundongos BALB/c (129). Seguindo a linha de raciocínio do grupo, desenvolvemos em trabalho recente duas vacinas de DNA codificando peptídeos conservados do grupo M do HIV-1 (130). Uma vacina foi desenhada para codificar 7 peptídeos do envelope viral (HIVenv7) enquanto a outra foi desenhada para codificar 27 peptídeos, de 7 proteínas virais, exceto Env e Tat (HIVBr27). A fim de induzir uma resposta imune de grande cobertura

39 23 vacinal, utilizamos o algoritmo TEPITOPE para selecionar peptídeos potencialmente ligadores a múltiplas moléculas HLA-DR (126). Nossos resultados prévios demonstraram que a vacina HIVenv7 foi pouco imunogênica em camundongos BALB/c, sendo observada uma resposta fraca, contra apenas um peptídeo codificado pela vacina. Observou-se ainda que a co-imunuização de HIVenv7 com HIVBr27 reduzia a resposta da segunda, sugerindo uma resposta imunossupressora induzida pela primeira. No presente trabalho, avaliamos a ligação dos peptídeos codificados por HIVenv7 a moléculas HLA de classe II e confirmamos que os mesmos se ligam de fato a múltiplas moléculas HLA-DR, assim como a moléculas HLA-DP e HLA-DQ e moléculas de classe II murinas. Demonstramos também que a frequência de reconhecimento desses peptídeos por indivíduos infectados pelo HIV-1 é bastante baixa. No presente estudo buscamos também avaliar a possível imunosupressão induzida por HIVenv7 e observamos que essa vacina não apresenta propriedades imunossupressoras consistentes. Em nosso trabalho prévio demonstramos que a vacina HIVBr27 induz uma resposta imune celular ampla e polifuncional, mediada tanto por linfócitos T CD4 + e CD8 +. No presente estudo mostramos que essa resposta imune induzida pela HIVBr27 também se estende a peptídeos de diferentes subtipos virais. Além disso, mostramos que os peptídeos codificados pela HIVBr27 se ligam a múltiplas moléculas HLA-DR, assim como a moléculas HLA-DP e HLA-DQ e moléculas de classe II murinas. Os peptídeos foram antigênicos no contexto da infecção natural pelo HIV-1, sendo reconhecidos por pacientes infectados por diferentes variantes do vírus. Os dados provenientes do nosso estudo prévio foram compilados aos dados obtidos no presente estudo e publicados (131).

40 24 2. OBJETIVOS 2.1. Objetivo geral O objetivo geral desse trabalho é avaliar as propriedas imunológicas das vacinas HIVBr27 e HIVenv7, bem como de seus peptídeos codificados Objetivos específicos Avaliar a capacidade da vacina HIVBr27 em induzir respostas de células T CD4 + com reatividade cruzada entre subtipos do HIV Avaliar a capacidade de ligação dos peptídeos codificados pelas vacinas HIVBr27 e HIVenv7 a moléculas HLA de classe II e MHC de classe II murinas Avaliar a frequência de reconhecimento dos peptídeos codificados pelas vacinas HIVBr27 e HIVenv7 por pacientes infectados pelo HIV Avaliar a imunossupressão induzida pela vacina HIVenv Avaliar a capacidade protetora da vacina HIVBr27

41 25 3. METODOLOGIA 3.1. Transformação de bactérias DH5α com plasmídeos vacinais Para a transformação, aproximadamente 100 ng do DNA plasmidial foram adicionados em tubos de microcentrífuga contendo alíquotas de 100 µl de bactérias DH5α competentes. Após incubação em gelo por 10 minutos, os tubos foram colocados a 42 o C por 30 segundos e recolocados em gelo por 5 minutos. Em seguida, foram acrescentados 400 µl de meio SOC e os tubos foram incubados a 37 o C por 1 hora, sob agitação. O volume total da mistura foi semeado em placas de LB sólido contendo 50 µg/ml de Kanamicina (Sigma) e estas foram incubadas a 37 o C durante 14-16h para seleção dos clones resistentes ao antibiótico Extração e purificação do DNA plasmidial A purificação em larga escala dos plasmídeos HIVBr27, HIVenv7, HIVenv7 modificada, pvax-gag, bem como do vetor vazio pvax1, foi feita utilizando-se o kit Endofree Giga Plasmid Purification (Qiagen), conforme instrução do fabricante. A cultura das bactérias transformadas com cada plasmídeo foi incubada a 37 C sob agitação (250rpm) durante 16 horas e posteriormente centrifugada. O sobrenadante foi desprezado e o pellet de bactérias foi lisado em tampão alcalino (NaOH 200 mm; SDS 1 %). Em seguida a solução foi neutralizada (acetato de potássio 3,0 M ph 5,5). A solução com as bactérias lisadas foi então transferida para um filtro cedido pelo kit e mantida em repouso por 10 minutos à temperatura ambiente. Após esse período o líquido foi filtrado à vácuo, lavado com 50 ml do tampão FWB2 (acetato de potássio1m, ph 5,0) e mantido no gelo por 30 minutos após adição de 30mL de tampão

42 26 para remoção de endotoxinas. O filtrado foi aplicado em uma coluna de cefarose (Qiagen). Os DNAs plasmidiais foram eluídos com 100 ml de tampão QN (NaCI,25 M; MOPS 50 mm ph 7,0; etanol 15 %). Os DNAs eluídos foram precipitados com 70mL de isopropanol, centrifugados, lavados com 10mL de etanol 70% (em água livre de endotoxinas) e centrifugados novamente. Finalmente os pellets foram ressuspensos em 2,0 ml de água estéril livre de endotoxinas Peptídeos Os peptídeos da sequência consenso do grupo M do HIV-1 codificados por HIVBr27 e HIVenv7 foram sintetizados em fase sólida pela empresa GL Biochem (Shanghai) Ltda, utilizando-se a estratégia de Fmoc. A pureza e qualidade dos peptídeos foram determinadas por cromatografia líquida de alto desempenho e por espectrometria de massa. A pureza e qualidade dos peptídeos foram determinadas como descrito acima, resultando em valores frequentemente superiores a 80%. Os peptídeos correspondentes às variantes dos peptídeos codificados por HIVBr27, provenientes de sequências dos subtipos do HIV-1, foram sintetizados e avaliados da mesma forma anteriormente descrita. Os peptídeos cobrindo a seqüência da proteína Gag do consenso B foram provenientes do Programa de Reagentes do NIH ( Os conjuntos de peptídeos compreenderam peptídeos de 15 aminoácidos de comprimento com sobreposição de 11 aminoácidos. A maioria deles foram descritos com grau de pureza superior a 80%. Os mesmos foram solubilizados em DMSO (dimetilsufóxido) na concentração de 10mg/mL para comporem os pools de peptídeos.

43 Teste de ligação peptídeos-hla de classe II O teste de ligação entre os peptídeos codificados por HIVBr27 a moléculas HLA-DR, -DP, -DQ e moléculas murinas IAb e IAd foi feito por incubação das moléculas purificadas (5-500 nm) com concentrações variadas dos peptídeos testes não marcados e 1-10nM dos peptídeos identificados marcados com 125 I, por 48h Camundongos e imunizações Camundongos isogênicos fêmeas de 6 a 8 semanas de idade das linhagens BALB/c (H-2 d ) e C57BL/6 (H-2 b ) foram utilizados para os experimentos de imunização. Os animais foram mantidos e manipulados em condições SPF no biotério do Laboratório de Imunologia do InCor, situado no prédio do Instituto de Medicina Tropical da Faculdade de Medicina da Universidade de São Paulo (IMT/FMUSP). Os experimentos envolvendo os animais foram realizados de acordo com o guia do comitê ético da Universidade de São Paulo. Os animais receberam, de acordo com cada protocolo experimental, quantidades e concentrações de DNA diferentes. Em síntese, os animais imunizados com um plasmídeo receberam 100 µg de DNA por dose, em volume de 100 µl, divididos em 50 µl por quadríceps. Os animais co-imunizados receberam 100 µg de DNA (resultado da soma de 50µg de cada plasmídeo) em volume final de 100 µl, divididos em 50 µl por quadríceps. Os animais foram sacrificados, independente do protocolo experimental, em câmara de CO 2 duas semanas após a última imunização.

44 Suspensão de células esplênicas Camundongos imunizados foram sacrificados 2 semanas após a última dose e os baços foram retirados. As células esplênicas foram obtidas após maceração do órgão, lavadas com 10 ml de meio RPMI (Gibco) e centrifugadas a 1700 RPM por 6 minutos a 4 C. Em seguida, as células foram tratadas com tampão hemolítico ACK (NH4Cl 0,15 M, KHCO3 1 mm, Na2EDTA 0,1 mm) e lavadas duas vezes com meio RPMI (10 ml por lavagem). Ao final das lavagens, as células foram ressuspensas em 1 ml de meio RPMI completo [meio RPMI suplementado com L glutamina 2 mm (Sigma), solução de aminoácidos não essenciais (1% vol/vol) (Gibco), piruvato de sódio 1 mm, 2- Mercaptoetanol 5x10-5 M (Sigma), 10% vol/vol de soro fetal bovino (Gibco) e os antibióticos Gentamicina (40 mg/ml) e Peflacin (20 mg/ml)] Proliferação celular contra peptídeos do HIV-1 Esplenócitos recém isolados (concentração de 100x10 6 células/ml) foram ressuspensos em PBS pré-aquecido e marcados com 1,25 µm de CFSE durante 10 minutos a 37 o C. Em seguida, o material foi centrifugado a 1700rpm por 6 minutos e a reação interrompida pela adição de 5mL de meio RPMI contendo 10% de soro fetal bovino. As células foram lavadas e ressuspensas em meio RPMI completo e contadas com o auxílio de uma câmara de Neubauer. As células foram cultivadas em placas de 96 poços de fundo U (Nunc) na concentração de 1,5x10 6 /ml em volume final de 200µL de RMPI completo por poço, na presença ou ausência de peptídeos sintéticos derivados do HIV-1 (5 µm), em estufa a 37 o C e 5% de CO 2 por 5 dias. Os controles positivos foram estimulados com 2 µg/ml de Concanavalina A (Sigma). As células foram marcadas em

45 29 placa de fundo V (Nunc) em volume final de 25 µl de MACS buffer, contendo os anticorpos anti-cd3-pe (BD Pharmingen), anti-cd4-percp (BD Pharmingen) e anti- CD8-APC (BD Pharmingen), ao abrigo da luz por 40 minutos a 4 C. As células foram lavadas e ressuspensas em volume final de 200 µl de MACS buffer. As amostras foram adquiridas usando-se o citômetro FACScanto (BD Biosciences) e analisadas com o auxílio do software FlowJo (versão 8.7.1, Tree Star). Para se avaliar a proliferação celular, foram adquiridos 50x10 3 eventos no gate de linfócitos. A porcentagem proliferativa de linfócitos T CD4 + e T CD8 + (células CFSE baixo ) foi determinada na população celular CD3 + e definida como positiva quando superior ao cutoff, em pelo menos 75% dos experimentos realizados. O cutoff foi determinado pela média mais 3 desvios padrões da proliferação obtida dos esplenócitos provenientes de camundongos imunizados com pvax1 vazio, estimulados com cada peptídeo individual ELISPOT de IFN-γ Os ensaios de ELISPOT foram realizados utilizando-se os sets anti-mouse IFN-γ (BD) e anti-human IFN-γ (BD). O protocolo foi realizado de acordo com as instruções do fabricante. Em síntese, o anticorpo de captura foi incubado na concentração de 5 µg/ml em volume final de 100 µl por 14h à 4ºC. As placas foram lavadas e bloqueadas com 200 µl de RPMI contendo 10% de soro fetal bovino (SFB) por 2 horas à temperatura ambiente. Após esse período, foram adicionados os estímulos: meio RPMI, como controle negativo, ConA (2,5 µg/poço) ou PMA (100 ng/ml) e Ionomicina (500 ng/ml), como controle positivo para camundongos e humanos, respectivamente, assim como peptídeos do HIV-1 (5 µm) em um volume final de 100 µl. Foram também adicionadas as suspensões celulares na concentração de 3 x 10 5 células/poço para

46 30 camundongos e 1 x 10 5 células/poço para humanos, em volume final de 100 µl. As placas foram novamente incubadas a 37 o C na estufa a 5% de CO 2 por 14h. Posteriormente as placas foram lavadas e o anticorpo de detecção (anti-ifn-γ biotinilado) foi adicionado aos poços (volume 100 µl/poço) na concentração final de 2 µg/ml e incubado novamente durante 2hs à temperatura ambiente. As placas foram novamente lavadas e foi adicionado o conjugado enzimático (streptavidin-hrp) na concentração final de 1x (100 µl/poço), por 1h à temperatura ambiente. Para revelação as placas foram lavadas e foram adicionados 100 µl da solução final de substrato/poço AEC (3-amino-9-ethylcarbazole - BD). A formação de spots foi monitorada de 5 a 60 minutos, levando em consideração o aparecimento dos mesmos em poços correspondentes ao controle negativo. A reação foi interrompida com água deionizada. A contagem dos spots foi realizada no Leitor de ELISPOT AID ELISPOT reader e os resultados foram expressos em número de spots por 10 6 células. Foram considerados positivos os estímulos cujo número de spots/10 6 células foi superior ao cutoff do experimento em questão. O cutoff para camundongos foi definido como sendo a média de spots mais 3 desvios padrões, obtidos na análise do grupo pvax1 vazio, estimulado com os peptídeos. O cutoff para humanos foi definido como 50 spots/10 6 células e 3x superior à média de spots das células sem estímulo.

47 Desafio com vírus Vaccinia recombinante Camundongos imunizados com HIVBr27 ou pvax1 vazio foram desafiados por injeção intraperitoneal de 1x10 7 UFPs de vírus Vaccinia recombinante, codificando as proteínas Gag e Pol do HIV-1 (subtipo B) ( Os vírus foram diluídos em salina e injetados na concentração desejada duas semanas após a última dose de imunização. Os animais foram mantidos infectados por 4 dias. Após esse período os animais foram sacrificados para obtenção dos ovários. Esses foram macerados e a solução resultante sofreu diluições seriadas para posterior incubação com células vero em placas de cultura de 6 poços. A determinação das placas virais foi feita por coloração com cristal violeta.

48 32 4. RESULTADOS 4.1. A imunização com HIVBr27 induz resposta imune celular contra peptídeos de diferentes subtipos do HIV-1. A vacina de DNA HIVBr27 foi previamente construída para induzir uma resposta imune mediada principalmente por linfócitos T CD4 +, contra regiões conservadas do grupo M do HIV-1. Em nosso estudo prévio, demonstramos que a HIVBr27 induz uma resposta imune celular ampla e polifuncional contra peptídeos do grupo M do HIV-1, mediada tanto por linfócitos T CD4 + quanto CD8 +. No presente trabalho pretendíamos avaliar se a resposta imune celular induzida por HIVBr27 também se estendia a peptídeos de diferentes subtipos do HIV-1. Para isso, sintetizamos variantes dos peptídeos mais imunogênicos da vacina: RT( ), RT( ), int( ) e p24( ). Essas variantes foram determinadas a partir do banco de dados Los Alamos e correspondem às sequências virais circulantes mais frequentes (Tabela 1). Observamos que a imunização com HIVBr27 induziu frequências semelhantes de linfócitos produtores de IFN-γ contra os peptídeos vacinais RT( ), int( ) e p24( ) e seus varitantes, enquanto que a frequência de resposta contra os variantes B e D do peptídeo RT( ) foi reduzida (Figura 4A). O mesmo padrão de resposta foi observado quando avaliamos a proliferação de linfócitos T CD4 + e CD8 + contra os peptídeos (Figura 4B e 4C). Além disso, avaliamos in silico as propriedas de ligação dos peptídeos variantes a moléculas HLA-DR, como feito anteriormente para os peptídeos vacinais, e observamos que os mesmos apresentaram propriedades promíscuas (ligação a várias moléculas HLA-DR), de acordo com o algoritmo TEPITOPE (Tabela 1). Esses resultados em conjnto sugerem que o nosso desenho

49 33 vacinal atingiu um de seus objetivos principais, induzindo uma resposta imune celular contra diferentes variantes do HIV-1. Peptídeo Sequência Variante Ligação a HLA-DR (TEPITOPE) RT( ) EWEFVNTPPLVKLWY consenso M 72% DWEFVNTPPLVKLWY subtipo A1 72% EWEFVSTPPLVKLWY subtipo B 72% EWEFINTPPLVKLWY subtipo C 64% EWEYVNTPPLVKLWY subtipo D 60% RT( ) KEKVYLSWVPAHKGIGGNE consenso M 76% KDKVYLSWVPAHKGIGGNE subtipo A1 76% KEKVYLAWVPAHKGIGGNE subtipo B 76% KERVYLSWVPAHKGIGGNE subtipo C 76% KEKIYLSWVPAHKGIGGNE subtipo F1 76% KEKVYLTWVPAHKGIGGNE subtipo G 68% integrase( ) QITKIQNFRVYYRDSRDPIW consenso M 84% QITKIQNFRVYYRDSRDPLW subtipo A1 84% QITKIQNFRVYYRDNRDPLW subtipo B 84% QIIKIQNFRVYYRDSRDPIW subtipo C 88% QIIKIQNFRVYYRDSRDPVW subtipo D 84% QITKIQNFRVYYRDSRDPVW subtipo F1 88% QITKIQNFRVYFRDSRDPIW subtipo G 84% p24( ) GEIYKRWIILGLNKIVRMY consenso M 100% GDIYKRWIILGLNKIVRMY subtipo A1 100% GEIYKRWIIMGLNKIVRMY subtipo B 100% Tabela 1. Peptídeos variantes do HIV-1 e previsão de ligação a moléculas HLA-DR. As sequências variantes dos 4 peptídeos vacinais mais imunogênicos foram obtidas em ( As mutações nas sequências variantes em relação aos peptídeos vacinais do consenso M são mostradas em vermelho. A previsão de ligação das sequências variantes a moléculas HLA-DR foi obtida utilizando-se o algoritmo TEPITOPE e está representada como a porcentagem de moléculas ligadas a cada variante, dentre as 25 constantes na matriz do algoritmo.

50 UFS/10 6 células 34 A B % Células T CD4 + CFSE baixo M A1 B M A1 B C D M A1 B C F1 G M A1 B C D F1 G M A1 B 0 M A1 B C D 0 M A1 B C F1 G 0 M A1 B C D F1 G C % Células T CD8 + CFSE baixo M A1 B 0 M A1 B C D 0 M A1 B C F1 G 0 M A1 B C D F1 G p24( ) RT( ) RT( ) int( ) Figura 4. A imunização com HIVBr27 induz resposta imune celular contra diferentes subtipos do HIV-1. Duas semanas após imunização com HIVBr27 ou vetor pvax1 vazio, o pool de esplenócitos de 6 animais de cada grupo foi cultivado na presença ou ausência de peptídeos vacinais do consenso M (5µM) ou seus variantes (5µM), representados pelas barras pretas e coloridas, respectivamente. A frequência de linfócitos produtores de IFN-γ (A) foi determinada por ELISPOT. A proliferação de linfócitos T CD4 + (B) e CD8 + (C) foi determinada pelo ensaio de CFSE. As linhas tracejadas representam os cutoff de respostas, que foram calculados como a média mais 3 desvios padrões da resposta inespecífica obitda com esplenócitos provenientes de animais imunizados com pvax1. Os dados são mostrados como média e desvio padrão de 2 experimentos independentes.

51 titulação viral (UFP) A imunização com a vacina HIVBr27 protege parcialmente contra infecção pelo vírus Vaccinia recombinante codificando Gag e Pol do HIV-1 A fim de avaliar se a resposta imune induzida por HIVBr27 poderia proteger contra uma infecção viral, desafiamos camundongos com uma injeção intraperitoneal do vírus Vaccinia recombinante codificando Gag e Pol do HIV-1, subtipo B, duas semanas após a última imunização. Avaliamos a carga viral nos ovários dos animais 4 dias após a infecção. Os vírus provenientes dos ovários foram titulados em células vero e contados como unidades formadoras de placa. Observamos que a imunização com HIVBr27 reduziu em 3 vezes a quantidade de vírus recuperados dos ovários dos animais desafiados (Figura 5). Esse resultado sugere que a vacina HIVBr27 induz uma resposta antiviral, mesmo que parcialmente protetora p=0, pvax HIVBr27 Figura 5. A imunização com HIVBr27 protege parcialmente contra desafio com vírus Vaccinia recombinante codificando Gag e Pol do HIV-1. Camundongos BALB/c (6 animais por grupo) foram imunizados em regime de 3 doses de pvax1 ou HIVBr27 e desafiados duas semanas após a última imunização. Os animais receberam uma dose intraperitoneal de 1x10 7 vírus Vaccinia recombinante codificando Gag e Pol do HIV-1, subtipo B. Os animais foram sacrificados e os ovários retirados para titulação viral 4 dias após infecção. Os dados são apresentados como média e desvio padrão da titulação viral, determinada por unidades formadoras de placas. Os animais imunizados e desafiados são representados por símbolos.

52 Os peptídeos codificados por HIVBr27 se ligam a múltiplas moléculas HLA de classe II Com o intuito de construir uma vacina capaz de induzir resposta imune celular em uma população geneticamente distinta (grande cobertura vacinal), utilizamos o algoritmo TEPITOPE para selecionar peptídeos potencialmente promíscuous (capazes de se ligarem a múltiplas moléculas HLA-DR). No presente estudo tínhamos como objetivo confirmar a real capacidade de ligação desses peptídeos a moléculas HLA de classe II. Para isso, incubamos os 27 peptídeos vacinais com diversas moléculas HLA- DR purificadas, assim como com moléculas HLA-DP e HLA-DQ. Observamos que cada peptídeo vacinal se ligou em média a 12 das 17 moléculas HLA-DR testadas, as quais se ligaram em média a 19 dos 27 peptídeos vacinais (Tabela 2). Os peptídeos vacinais se ligaram em média a 50% e 30% das moléculas HLA-DP e HLA-DQ, respectivamente. Essas por sua vez se ligaram em média a 15 e 7 peptídeos, respectivamente (Tabelas 3 e 4). Além disso, avaliamos a ligação dos peptídeos vacinais a moléculas MHC de classe II de camundongos BALB/c e C57BL/6. Observamos que 21 e 6 peptídeos se ligaram a IA d e IA b, respectivamente (Tabela 5). Assim, concluímos que o algoritmo TEPITOPE foi eficaz na seleção de peptídeos promíscuos e acreditamos que nossa vacina seria capaz de induzir uma resposta imune celular de grande cobertura populacional.

53 37 Capacidade de ligação - IC 50 (nm) Ligação a HLA-DR Peptídeo DRB1*01:01 DRB1*03:01 DRB1*04:01 DRB1*04:05 DRB1*07:01 DRB1*08:02 DRB1*09:01 DRB1*10:01 DRB1*11:01 DRB1*12:01 DRB1*13:02 DRB1*15:01 DRB1*16:02 DRB3*01:01 DRB3*02:02 DRB4*01:01 DRB5*01:01 protease(53-75) ,5 protease(79-95) ,3 RT( ) , ,7 RT( ) , ,6 RT( ) ,6 RT( ) , ,4 RT( ) 5, , ,1 RT( ) , ,2 integrase(28-43) ,2 integrase(69-85) 0, , , ,5 integrase(96-113) 4, , ,7 integrase( ) 0, , , ,3 7, , ,2 integrase( ) ,9 p17(72-90) ,6 p17( )/p24(1-18) 4, ,5 p24(33-48) 1, , , , , ,6 p24( ) ,1 p24( ) 2, ,4 3,1 36 6, , ,4 p24( ) ,4 vif(1-15) ,3 vif( ) 0, ,4 2,3 4, , ,1 3, , ,2 rev(9-27) 7, , , , ,1 vpr(29-42) ,9 vpr(58-80) ,5 vpu(13-26) ,8 nef(67-87) ,8 nef( ) , ,9 Peptídeos ligados a HLA-DR Moléculas ligadas Frequência (%) Tabela 2. Os peptídeos codificados por HIVBr27 se ligam a múltiplas moléculas HLA-DR. Os peptídeos vacinais foram incubados com moléculas HLA-DR purificadas (5-500 nm) na presença de peptídeos competidores marcados com I 125 (1-10nM) por 48 horas. As ligações que ocorreram com concentrações de peptídeos vacinais menores que 1000 nm foram consideradas positivas e estão marcadas em cinza. Os traços representam ligações com IC 50 (concentração necessária de peptídeo teste para deslocar 50% dos peptídeos marcados) superior a nm.

54 38 Capacidade de ligação /IC 50 (nm) Peptídeos DPA1*02:01 DPA1*01:03 DPA1*01:03 DPA1*01:03 DPA1*03:01 DPA1*02:01 DPA1*02:01 DPA1*01:03 Frequência Moléculas ligadas DPB1*01:01 DPB1*02:01 DPB1*03:01 DPB1*04:01 DPB1*04:02 DPB1*05:01 DPB1*14:01 DPB1*20:01 (%) protease(53-75) , ,5 protease(79-95) , RT( ) ,5 RT( ) ,5 RT( ) ,5 RT( ) , RT( ) ,5 RT( ) ,0 5 62,5 integrase(28-43) ,5 integrase(69-85) , ,5 integrase(96-113) integrase( ) , ,5 integrase( ) p17(72-90) p17( )/p24(1-18) p24(33-48) p24( ) ,5 p24( ) , , p24( ) , ,5 vif(1-15) vif( ) , rev(9-27) 461 7, , vpr(29-42) ,5 vpr(58-80) ,5 vpu(13-26) nef(67-87) ,5 nef( ) Peptídeos ligados a HLA-DP Tabela 3. Os peptídeos codificados por HIVBr27 se ligam a múltiplas moléculas HLA-DP. Os peptídeos vacinais foram incubados com moléculas HLA-DP purificadas (5-500 nm) na presença de peptídeos competidores marcados com I 125 (1-10nM) por 48 horas. As ligações que ocorreram com concentrações de peptídeos vacinais menores que 1000 nm foram consideradas positivas e estão marcadas em cinza. Os traços representam ligações com IC 50 (concentração necessária de peptídeo teste para deslocar 50% dos peptídeos marcados) superior a nm.

55 39 Capacidade de ligação /IC 50 (nm) Peptídeos DQA1*05:01 DQA1*02:01 DQA1*03:01 DQA1*05:01 DQA1*03:01 DQA1*04:01 DQA1*01:01 DQA1*01:02 DQA1*01:04 DQA1*01:02 DQA1*01:03 DQB1*02:01 DQB1*02:02 DQB1*03:01 DQB1*03:01 DQB1*03:02 DQB1*04:02 DQB1*05:01 DQB1*05:02 DQB1*05:03 DQB1*06:02 DQB1*06:03 Moléculas ligadas Frequência (%) protease(53-75) ,3 protease(79-95) ,0 RT( ) ,0 RT( ) ,4 RT( ) ,2 RT( ) ,1 RT( ) ,7 RT( ) ,4 integrase(28-43) ,1 integrase(69-85) ,5 integrase(96-113) ,4 integrase( ) ,4 integrase( ) ,1 p17(72-90) ,2 p17( )/p24(1-18) ,5 p24(33-48) ,5 6 54,5 p24( ) ,7 3 27,3 p24( ) ,5 p24( ) ,3 vif(1-15) ,3 vif( ) ,3 rev(9-27) ,5 vpr(29-42) ,0 vpr(58-80) ,4 vpu(13-26) ,0 nef(67-87) ,1 nef( ) ,1 Peptídeos ligados a HLA-DQ Tabela 4. Os peptídeos codificados por HIVBr27 se ligam a múltiplas moléculas HLA-DQ. Os peptídeos vacinais foram incubados com moléculas HLA-DQ purificadas (5-500 nm) na presença de peptídeos competidores marcados com I 125 (1-10nM) por 48 horas. As ligações que ocorreram com concentrações de peptídeos vacinais menores que 1000 nm foram consideradas positivas e estão marcadas em cinza. Os traços representam ligações com IC 50 (concentração necessária de peptídeo teste para deslocar 50% dos peptídeos marcados) superior a nm.

56 40 Capacidade de ligação /IC 50 (nm) Peptídeos IA b IA d Moléculas ligadas protease(53-75) protease(79-95) RT( ) RT( ) RT( ) RT( ) RT( ) RT( ) integrase(28-43) integrase(69-85) integrase(96-113) integrase( ) integrase( ) p17(72-90) p17( )/p24(1-18) p24(33-48) p24( ) p24( ) p24( ) vif(1-15) vif( ) 198 7,4 2 rev(9-27) vpr(29-42) vpr(58-80) vpu(13-26) nef(67-87) nef( ) Peptídeos ligados a IA b e IA d 6 21 Tabela 5. Os peptídeos codificados por HIVBr27 se ligam a moléculas IA b e IA d. Os peptídeos vacinais foram incubados com moléculas IA b e IA d purificadas (5-500 nm) na presença de peptídeos competidores marcados com I 125 (1-10nM) por 48 horas. As ligações que ocorreram com concentrações de peptídeos vacinais menores que 1000 nm foram consideradas positivas e estão marcadas em cinza. Os traços representam ligações com IC 50 (concentração necessária de peptídeo teste para deslocar 50% dos peptídeos marcados) superior a nm.

57 Os peptídeos codificados por HIVBr27 são reconhecidos por células de pacientes infectados com HIV-1 Uma vez confirmado que os peptídeos codificados por HIVBr27 se ligavam a múltiplas moléculas HLA de classe II, buscamos determinar se os mesmos seriam antigênicos no contexto da infecção por diferentes subtipos do HIV-1. Para isso, avaliamos a frequência de linfócitos produtores de IFN-γ em células mononucleares do sangue periférico (CMSP) de 25 pacientes infectados com diferentes subtipos virais (Tabela 6). Nós observamos que 7 dos 13 peptídeos de Pol e 4 dos 6 peptídeos de Gag foram reconhecidos pelo menos uma vez (Figura 6A e 6B). Cinco peptídeos dentre os 8 pertencentes ao conjunto de proteínas Vif, Rev, Vpr, Vpu e Nef foram também reconhecidos (Figura 6C). Observamos que 72% dos pacientes reconheceram pelo menos um peptídeo e que cada paciente reconheceu em média 2 peptídeos. Além disso, nossos dados mostram que 16 dos 27 peptídeos vacinais (60%) foram reconhecidos por pacientes infectados pelo HIV-1. Portanto, consideramos que a maioria dos peptídeos vacinais identificados com o algoritmo TEPITOPE se mostrou antigênica no contexto da infecção natural por HIV-1. Subtipo B Subtipo F Recombinante BC Recombinante BF Total n Gênero (% de homens) 93% (13/14) 100% (1/1) 100% (1/1) 67% (6/9) 84% (21/25) Idade média (anos)* 28 ± ± ± 9 Tempo pós recrutamento (dias) 526 ± ± ± 296 Média de contagem de CD4+ (cels/mm 3 ) 570 ± ± ± 198 Média de contagem de CD8+ (cels/mm 3 ) ± , ± ± 457 Carga viral média (cópias/ml) ± ,000 11, ± ± ART (% de pacientes que iniciaram) 29% (4/14) 0% (0/1) 0% (0/1) 78% (7/9) 44% (11/25) * No momento do recrutamento Tabela 6. Dados clínicos dos pacientes HIV +. Células de 25 pacientes infectados por diferentes subtipos do HIV-1 foram utilizados nesse estudo. Os dados clínicos dos pacientes estão sumarizados na tabela. ART, terapia anti-retroviral.

58 UFS/10 6 cells UFS/10 6 cells UFS/10 6 cells 42 Subtipo B Recombinante BC A) Recombinante BF Subtipo F protease(53-75) protease(79-95) RT( ) RT( ) RT( ) RT( ) RT( ) RT( ) integrase(28-43) integrase(69-85) integrase(96-113) integrase( ) integrase( ) B) p17(72-90) p17( )/p24(1-18) p24(33-48) p24( ) p24( ) p24( ) C) vif(1-15) vif( ) rev(9-27) vpr(29-42) vpr(58-80) vpu(13-26) nef(67-87) nef( )

59 43 Figura 6. Os peptídeos codificados por HIVBr27 são reconhecidos por células de pacientes infectados pelo HIV-1. Os 27 peptídeos vacinais foram incubados com células de pacientes infectados pelo HIV-1 (representados pelos símbolos) e a frequência de resposta foi determinada por ELISPOT de IFN-γ. Resposta contra peptídeos de Pol (A), Gag (B) e Vif, Rev, Vpr, Vpu e Nef (C) são mostradas. Pacientes infectados pelo subtipo B (n = 14), pacientes infectados pelo recombinante BC (n = 1), pacientes infectados pelo recombinante BF (n = 1) e pacientes infectados pelo subtipo F (n = 1). As linhas tracejadas representam o cutoff de resposta, correspondente a 50 spots/ 10 6 células. Apenas respostas positivas são mostradas A vacina HIVenv7 não apresenta propriedades imunossupressoras consistentes Em nosso estudo prévio desenvolvemos, além da vacina HIVBr27, uma vacina denominada HIVenv7, codificando 7 peptídeos do envelope do HIV-1. Ao coimunizarmos essa vacina juntamente com a vacina HIVBr27 observamos uma redução na resposta imune contra o pool de peptídeos da HIVBr27. Esse resultado sugeriu que a vacina HIVenv7 poderia apresentar propriedades imunossupressoras. No presente trabalho, pretendíamos aprofundar os estudos sobre a vacina HIVenv7. Para isso coimunizamos essa vacina com outra vacina de DNA, agora codificando a proteína Gag do HIV-1. Observamos que a co-imunização com HIVenv7 não reduziu significativamente a resposta contra pools de peptídeos de Gag em camundongos BALB/c (Figura 7). Co-imunizamos também camundongos C57BL/6 com a mesma formulação vacinal e não observamos redução significativa da resposta contra pools de peptídeos de Gag (Figura 8). Em nossos experimentos prévios utilizamos como controle a co-imunização de pvaxgag com o vetor pvax1 vazio. Para confirmar nossos resultados, fizemos um novo experimento de co-imunização de HIVenv7 com a vacina

60 UFS/10 6 células 44 HIVBr27. Nesse caso utilizamos como controle a co-imunização com uma vacina HIVenv7 modificada, codificando os mesmos 7 peptídeos do envelope, porém com os aminoácidos embaralhados. Não observamos redução significativa da resposta contra o pool de peptídeos da HIVBr27, quando essa foi co-imunizada com HIVenv7 (Figura 9). Esses resultados sugerem que o fenômeno de imunossupressão mediado por HIVenv7 observado em nosso estudo prévio pode ter ocorrido por problemas técnicos, não se repetindo em nossos experimentos atuais NS NS pvaxgag+pvax pvaxgag+hivenv Pool 4 Pool 5 Figura 7. A co-imunização de HIVenv7 e pvaxgag não reduz a resposta contra peptídeos de Gag. Camundongos BALB/c (6 animais por grupo) foram co-imunizados com pvaxgag+pvax1 ou pvaxgag+hivenv7 (50µg de cada plasmídeo) em regime de 3 doses. Duas semanas após a última dose, esplenócitos dos animais imunizados foram incubados com pools de peptídeos de Gag e a frequência de linfócitos produtores de IFN-γ foi determinada por ELISPOT. Os resultados apresentados correspondem à média mais desvio padrão de 3 experimentos independentes.

61 UFS/10 6 células UFS/10 6 células pvaxgag+pvax pvaxgag+hivenv *** NS NS NS NS 0 Pool 1 Pool 2 Pool 3 Pool 4 Pool 5 Pool 6 Pool 7 Pool 8 Pool 9 Pool 10 Figura 8. A co-imunização de HIVenv7 e pvaxgag não reduz a resposta contra o pool de peptídeos de Gag. Camundongos C57BL/6 (6 animais por grupo) foram co-imunizados com pvaxgag+pvax1 ou pvaxgag+hivenv7 (50µg de cada plasmídeo) em regime de 3 doses. Duas semanas após a última dose, esplenócitos dos animais imunizados foram incubados com pools de peptídeos de Gag e a frequência de linfócitos produtores de IFN-γ foi determinada por ELISPOT. Os resultados apresentados correspondem a um experimento representativo de 2. *** p<0, NS HIVBr27 + HIVenv7 HIVBr27 + HIVenv7 mod Figura 9. A co-imunização de HIVenv7 e HIVBr27 não reduz a resposta contra o pool de peptídeos da HIVBr27. Camundongos BALB/c (6 animais por grupo) foram co-imunizados com HIVBr27+HIVenv7 ou HIVBr27+HIVenv7 modificada (50µg de cada plasmídeo) em regime de 3 doses. Duas semanas após a última dose, esplenócitos dos animais imunizados foram incubados com o pool de peptídeos da HIVBr27 e a frequência de linfócitos produtores de IFN-γ foi determinada por ELISPOT. Os resultados apresentados correspondem a um experimento.

62 Os peptídeos codificados por HIVenv7 se ligam a múltiplas moléculas HLA de classe II Assim como feito com os peptídeos codificados pela HIVBr27, determinamos se os peptídeos codificados pela HIVenv7 se ligavam a moléculas HLA de classe II. Nós observamos que os peptídeos codificados por HIVenv7 se ligaram em média a 11 das 17 moléculas HLA-DR testadas e que cada molécula HLA-DR se ligou em média a 5 dos 7 peptídeos (Tabela 7). Os peptídeos se ligaram em média a 4 das 8 moléculas HLA-DP testadas e cada molécula HLA-DP se ligou em média a 4 dos 7 peptídeos (Tabela 8). Os peptídeos se ligaram em média a 6 das 11 moléculas HLA-DQ testadas e cada molécula HLA-DQ se ligou em média a 4 dos 7 peptídeos (Tabela 9). Nós observamos que a molécula IA b se ligou a 2 dos 7 peptídeos e que a molécula IA d se ligou a 4 dos 7 peptídeos (Tabela 10). Estes dados sugerem que a vacina HIVenv7 codifica peptídeos promíscuos, identificados de forma eficaz pelo algoritmo TEPITOPE.

63 47 Capacidade de ligação/ic 50 (nm) Ligação a HLA-DR Peptídeo DRB1*01:01 DRB1*03:01 DRB1*04:01 DRB1*04:05 DRB1*07:01 DRB1*08:02 DRB1*09:01 DRB1*10:01 DRB1*11:01 DRB1*12:01 DRB1*13:02 DRB1*15:01 DRB1*16:02 DRB3*01:01 DRB3*02:02 DRB4*01:01 DRB5*01:01 gp160( ) gp160( ) gp160( ) gp160( ) gp160( ) gp160( ) gp160( ) Moleculas Ligadas Frequência (%) Peptídeos ligados a HLA-DR (n) Tabela 7. Os peptídeos codificados por HIVenv7 se ligam a múltiplas moléculas HLA-DR. Os peptídeos vacinais foram incubados com moléculas HLA-DR purificadas (5-500 nm) na presença de peptídeos competidores marcados com I 125 (1-10nM) por 48 horas. As ligações que ocorreram com concentrações de peptídeos vacinais menores que 1000 nm foram consideradas positivas e estão marcadas em cinza. Os traços representam ligações com IC 50 (concentração necessária de peptídeo teste para deslocar 50% dos peptídeos marcados) superior a nm.

64 48 Capacidade de Ligação /IC 50 (nm) Peptídeo gp160( ) gp160( ) gp160( ) gp160( ) gp160( ) gp160( ) gp160( ) DPA1*02:01 DPA1*01:03 DPA1*01:03 DPA1*01:03 DPA1*03:01 DPA1*02:01 DPA1*02:01 DPA1*01:03 DPB1*01:01 DPB1*02:01 DPB1*03:01 DPB1*04:01 DPB1*04:02 DPB1*05:01 DPB1*14:01 DPB1*20:01 Moléculas ligadas Frequência (%) Peptídeos ligados a HLA-DP Tabela 8. Os peptídeos codificados por HIVenv7 se ligam a múltiplas moléculas HLA-DP. Os peptídeos vacinais foram incubados com moléculas HLA-DP purificadas (5-500 nm) na presença de peptídeos competidores marcados com I 125 (1-10nM) por 48 horas. As ligações que ocorreram com concentrações de peptídeos vacinais menores que 1000 nm foram consideradas positivas e estão marcadas em cinza. Os traços representam ligações com IC 50 (concentração necessária de peptídeo teste para deslocar 50% dos peptídeos marcados) superior a nm.

65 49 Capacidade de ligação /IC 50 (nm) DQA1*05:01 DQA1*02:01 DQA1*03:01 DQA1*05:01 DQA1*03:01 DQA1*04:01 DQA1*01:01 DQA1*01:02 DQA1*01:04 DQA1*01:02 DQA1*01:03 Moléculas Frequência Peptídeo ligadas (%) DQB1*02:01 DQB1*02:02 DQB1*03:01 DQB1*03:01 DQB1*03:02 DQB1*04:02 DQB1*05:01 DQB1*05:02 DQB1*05:03 DQB1*06:02 DQB1*06:03 gp160( ) gp160( ) gp160( ) gp160( ) gp160( ) gp160( ) gp160( ) Peptídeos ligados a HLA-DQ Tabela 9. Os peptídeos codificados por HIVenv7 se ligam a múltiplas moléculas HLA-DQ. Os peptídeos vacinais foram incubados com moléculas HLA-DQ purificadas (5-500 nm) na presença de peptídeos competidores marcados com I 125 (1-10nM) por 48 horas. As ligações que ocorreram com concentrações de peptídeos vacinais menores que 1000 nm foram consideradas positivas e estão marcadas em cinza. Os traços representam ligações com IC 50 (concentração necessária de peptídeo teste para deslocar 50% dos peptídeos marcados) superior a nm.

66 50 Capacidade de ligação/ic 50 (nm) Peptídeo IA b IA d Moléculas ligadas gp160( ) gp160( ) gp160( ) gp160( ) gp160( ) gp160( ) gp160( ) Peptídeos ligados a IA b, IA d 2 4 Tabela 10. Os peptídeos codificados por HIVenv7 se ligam a múltiplas moléculas IA b e IA d. Os peptídeos vacinais foram incubados com moléculas IA b e IA d purificadas (5-500 nm) na presença de peptídeos competidores marcados com I 125 (1-10nM) por 48 horas. As ligações que ocorreram com concentrações de peptídeos vacinais menores que 1000 nm foram consideradas positivas e estão marcadas em cinza. Os traços representam ligações com IC 50 (concentração necessária de peptídeo teste para deslocar 50% dos peptídeos marcados) superior a nm.

67 UFS/10 6 cells Os peptídeos codificados por HIVenv7 são reconhecidos por células de pacientes infectados com HIV-1 A fim de avaliar se os peptídeos codificados pela vacina HIVenv7 eram reconhecidos durante a infecção pelo HIV-1, nós fizemos ensaios de ELISPOT incubando os peptídeos com CMSP de 25 pacientes infectados pelo HIV-1, previamente mencionados nos resultados referentes à vacina HIVBr27. Nós observamos que 5 dos 7 peptídeos de Env codificados pela vacina HIVenv7 foram reconhecidos pelo menos uma vez e que 5 dos 25 pacientes apresentaram alguma resposta contra os peptídeos (Figura 10). Esses dados sugerem que os peptídeos codificados por HIVenv7 são pouco antigênicos no contexto da infecção pelo HIV Subtipo B Recombinante BF gp160( ) gp160( ) gp160( ) gp160( ) gp160( ) gp160( ) gp160( ) Figura 10. Os peptídeos codificados por HIVenv7 são reconhecidos por células de pacientes infectados pelo HIV-1. O ensaio de ELISPOT de IFN-γ foi realizado incubando-se CMSP de pacientes infectados pelo HIV-1 (representados pelos símbolos coloridos) ou controles saudáveis com os peptídeos codificados pela vacina HIVenv7. Pacientes infectados pelo subtipo B (n=14). Pacientes infectados pelo recombinante BC (n=1). Pacientes infectados pelo recombinante BF (n=9). Pacientes infectados pelo subtipo F (n=1). As linhas tracejadas representam o limiar do ELISPOT, que foi determinado como 50 spots/10 6 células. Apenas as respostas positivas são mostradas nos gráficos.

68 52 5. DISCUSSÃO Neste trabalho estendemos o estudo sobre as vacinas de DNA HIVBr27 e HIVenv7, previamente descritas em minha dissertação de mestrado. Demonstramos que a imunização com HIVBr27 induz uma resposta imune celular mediada por linfócitos T CD4 + e CD8 + contra peptídeos de diferentes subtipos do HIV-1. Além disso, a imunização com HIVBr27 mostrou-se parcialmente protetora contra a infecção pelo vírus Vaccinia recombinante codificando as proteínas Gag e Pol do HIV-1. Ensaios in vitro demonstraram que os peptídeos codificados pela HIVBr27 se ligam a múltiplas moléculas HLA de classe II e são reconhecidos por células de pacientes infectados pelo HIV-1. Demonstramos também que a vacina HIVenv7 não possui propriedades imunossupressoras consistentes, contrariando os resultados obtidos previamente. Os peptídeos codificados pela HIVenv7 se ligaram a múltiplas moléculas HLA de classe II, mas apresentaram baixa frequência de reconhecimento por células de pacientes infectados pelo HIV-1. Em nosso trabalho prévio demonstramos que a vacina HIVBr27 induzia uma resposta imune celular ampla e polifuncional contra peptídeos do HIV-1, mediada tanto por linfócitos T CD4 + quanto CD8 +. Aqui demonstramos que essa resposta imune celular também se direciona a peptídeos de diferentes subtipos virais, o que vai de encontro à nossa hipótese inicial. De fato, ao construir a vacina HIVBr27, utilizamos a sequência consenso do grupo M do HIV-1 com o objetivo de induzir uma resposta cruzada contra as diversas variantes do vírus, sugerida como uma das alternativas para a proteção contra a diversidade global do HIV-1 (132). Acreditamos que nossa estratégia vacinal trouxe melhorias significativas no que se diz respeito ao uso de sequências consenso do grupo M do HIV-1. Enquanto a HIVBr27 foi desenhada para codificar epitopos altamente conservados e promíscuos,

69 53 reconhecidos principalmente por linfócitos T CD4 + e provenientes de 7 proteínas virais diferentes, outros estudos se focaram apenas na indução de respostas cruzadas contra o envelope viral ( ). Apesar dos estudos mencionados apresentarem resultados importantes quanto à indução de respostas imunes celulares e humorais contra diferentes subtipos virais, acreditamos que o foco em apenas uma proteína viral poderia não ser suficiente para combater a infecção. Diferentes estratégias vacinais têm sido desenvolvidas para superar a diversidade genética do HIV-1. Uma vacina baseada em um vetor adenovírus 26 codificando mosaicos das proteínas Gag, Pol e Env mostrou-se mais imunogênica do que uma vacina similar codificando proteínas do consenso M do HIV-1 em macacos resos (120). Entretanto, é importante notar que as vacinas desse estudo foram baseadas em proteínas inteiras do HIV-1, ao contrário de nossa estratégia multiepitópica. Outro estudo, baseado em proteínas quiméricas compostas de regiões conservadas de Pol, Gag, Vif e Env dos subtipos A, B, C e D do HIV-1 (122), induziu respostas significativas de linfócitos T CD8 +, mas respostas bem limitadas de linfócitos T CD4 +, quando comparamos à nossa vacina HIVBr27. De fato, a nossa vacina foi a primeira construção especificamente desenhada para induzir respostas de linfócitos T CD4 + amplas e cruzadas entre os diversos subtipos virais. Acreditamos que nossa vacina pode conferir uma resposta antiviral diretamente desempenhada por linfócitos T CD4 +, assim como auxiliar vacinas indutoras de respostas mediadas por linfócitos T CD8 +. Acredita-se que vacinas multiepitópicas, como a HIVBr27, facilitam a emergência de peptídeos individualmente imunogênicos, contribuindo para a indução mais eficiente de respostas imunes celulares amplas ( ). Entretanto, esse tópico é ainda controverso. Enquanto a imunização de camundongos e macacos com vacinas de DNA codificando fragmentos da proteína Gag do HIV-1 se mostrou mais eficiente na

70 54 indução de respostas imunes amplas, em dois estudos independentes (139, 140), um terceiro estudo demonstrou que a imunização de macacos em regime de prime-boost com os vetores adenovírus 35 e 26, codificando a proteína Gag inteira, induziu uma resposta de maior magnitude e de amplitude semelhante quando comparada ao mesmo sistema vacinal codificando fragmentos da proteína (141). É possível que diferenças entre vacinas de DNA e vetores virais tenham contribuído para os resultados discrepantes. Acreditamos que um estudo envolvendo diferentes vetores e proteínas seria interessante para determinar a melhor maneira de se induzir respostas imunes celulares de grande amplitude. Um ponto importante da nossa construção vacinal é a presença de regiões conservadas do HIV-1. Além de promover uma resposta cruzada entre os subtipos, como discutido anteriormente, acredita-se que essas regiões sejam propícias a um menor escape imunológico por mutações e por isso, promovam uma maior proteção contra a infecção. Neste contexto, um estudo recente demonstrou que a imunização de indivíduos não infectados pelo HIV-1 com um regime vacinal de prime-boost composto por uma vacina de DNA, um adenovírus símio e o vírus Vaccinia Ankara modificado codificando regiões virais conservadas, induziu altos níveis de linfócitos T CD8 + citotóxicos contra porções subdominantes de Gag e Pol. Essas células foram capazes de reconhecer linfócitos T CD4 + autólogos e inibir em quase 6 logs a replicação viral in vitro (142). Em outro estudo, a imunização de indivíduos não infectados com adenovírus 35 codificando as proteínas Gag, transcriptase reversa, integrase, Nef e Env do subtipo A do HIV-1 induziu respostas tanto contra regiões variáveis quanto conservadas dessas proteínas. Entretanto, as respostas direcionadas contra regiões conservadas foram aquelas capazes de inibir de forma mais eficaz a replicação in vitro de vírus provenientes de múltipos clados do HIV-1 (143).

71 55 A fim de avaliar se a resposta imune induzida pela vacina HIVBr27 era de fato antiviral, desafiamos camundongos imunizados com o vírus Vaccinia modificado, codificando as proteínas Gag e Pol do HIV-1 (Vaccinia Gag-Pol). A inabilidade do HIV-1 em infectar camundongos torna difícil o estabelecimento de modelos de desafio. Entretanto, julgamos nossa escolha como suficiente para a prova de conceito pretendida. Observamos que a imunização com HIVBr27 levou à redução da carga viral em três vezes. Se comparada a um estudo anterior (144), essa proteção pode ser considerada pequena. No estudo mencionado, a imunização de camundongos com uma vacina de DNA codificando Gag p41 do HIV-1 fusionada a um anticorpo anti-dec205 (receptor de células dendríticas) protegeu de forma eficaz contra o desafio com vírus Vaccinia modificado codificando Gag p41 do HIV-1, reduzindo a carga viral em 2 a 4 logs, dependendo da dose vacinal. Entretanto, em um estudo mais recente também focado no direcionamento de antígenos a células dendríticas, foi observado que a imunização com uma vacina de DNA codificando Gag p24 do HIV-1 fusionada à proteína PD1 (programmed death 1) levou a uma proteção contra o vírus Vaccinia Gag-Pol semelhante à obtida com HIVBr27 (145). Portanto, não podemos subjulgar a proteção induzida pela HIVBr27. Acreditamos que uma formulação vacinal otimizada com a inclusão de um adjuvante potente, como feito nos estudos mencionados acima, poderia aumentar a capacidade protetora de nossa vacina. No momento da construção das vacinas HIVBr27 e HIVenv7 buscávamos selecionar peptídeos conservados do HIV-1 que fossem ligadores em potencial a múltiplas moléculas HLA-DR, com o intuito de induzir uma resposta imune de grande cobertura populacional. Nesse trabalho mostramos que os peptídeos selecionados pelo algoritmo TEPITOPE (126) se ligam de fato a múltiplas moléculas HLA-DR, sendo que os peptídeos codificados pela HIVBr27 se ligaram a 12 das 17 moléculas testadas e os

72 56 peptídeos codificados pela HIVenv7 se ligaram a 11 dessas moléculas. As moléculas HLA-DR se ligaram em média a 19 peptídeos da HIVBr27 e 5 peptídeos da HIVenv7. Além disso, observamos ligação dos peptídeos vacinais a múltiplas moléculas HLA-DP e HLA-DQ e a moléculas de classe II murinas. É importante notar que os peptídeos vacinais se ligaram a algumas moléculas HLA de classe II associadas a proteção contra AIDS, como HLA-DRB1*0101, -DRB3*0202, -DRB1*13 e -DQB1*06 (146, 147). Portanto, consideramos que os peptídeos vacinais possuem potencial de induzir respostas imunes de grande cobertura vacinal, muitas vezes restritas a moléculas HLA de classe II associadas à proteção contra a progressão da doença. Os peptídeos codificados pela vacina HIVBr27 se mostraram antigênicos no contexto da infecção natural pelo HIV-1. Observamos que 72% dos pacientes reconheceram pelo menos um peptídeo vacinal e que 16 dos 27 peptídeos foram reconhecidos. As respostas mostradas aqui foram similares às obtidas por nosso grupo, quando se avaliou o reconhecimento de peptídeos do subtipo B do HIV-1 por células de pacientes infectados clinicamente semelhantes aos participantes do presente estudo (127). Os peptídeos codificados por HIVenv7 apresentaram baixa frequência de reconhecimento, o que está em concordância com nossos resultados prévios de imunogenicidade dessa vacina em camundongos BALB/c, no qual as respostas foram bastante limitadas, com apenas um peptídeo sendo imunogênico. Assim como feito no presente trabalho, um estudo anterior avaliou o reconhecimento de peptídeos de Gag provenientes do consenso do grupo M do HIV-1 por pacientes infectados (148). Nesse caso, 34% dos peptídeos de 15 aminoácidos utilizados foram reconhecidos por pacientes infectados pelo subtipo B viral, enquanto nós observamos que 44% de nossos peptídeos foram reconhecidos por pacientes infectados pelo mesmo subtipo do vírus. Se considerarmos apenas os peptídeos de Gag

73 57 codificados pela HIVBr27, temos 50% dos mesmos sendo reconhecidos por pacientes infectados pelo subtipo B do HIV-1. Isso sugere que os peptídeos conservados e promíscuos identificados com o auxílio do algoritmo TEPITOPE apresentam alguma vantagem em seu reconhecimento por células provenientes de pacientes infectados quando comparados a peptídeos de 15 aminoácidos corriqueiramente utilizados em ensaios de identificação de resposta celular. No presente trabalho pretendíamos também estender os estudos sobre a vacina HIVenv7 no que se diz respeito às suas propriedades imunossupressoras. Em nosso trabalho prévio mostramos que a co-imunização de HIVenv7 e HIVBr27 levava à redução da resposta imune celular induzida por HIVBr27. Aqui demonstramos que a vacina HIVenv7 não apresenta tais propriedades, não sendo observada a redução de forma consistente da resposta imune induzida pela vacina pvaxgag quando coimunizada com HIVenv7. Ao analisar experimentos de co-imunização isolados, observamos em alguns casos uma redução da resposta de pvaxgag, porém, ao considerarmos todos os experimentos, os resultados acabaram não sendo significativos. Ainda para confirmar os dados obtidos em nosso trabalho prévio, co-imunizamos HIVBr27 com HIVenv7 novamente. Nesse caso, utilizamos como controle a coimunização de HIVBr27 com uma HIVenv7 modificada, codificando os mesmos peptídeos, porém com os aminoácidos embaralhados. Contrário ao observado anteriormente, em que o controle foi a co-imunização de HIVBr27 com pvax1 vazio, não houve redução da resposta induzida pela HIVBr27. As proteínas gp120 e gp41 do envelope do HIV-1, assim como alguns de seus peptídeos, foram descritos como imunossupressoras. De fato, foi observado que o prétratamento de clones de linfócitos T específicos para o toxóide de tétano com gp120 era capaz de inibir a produção de IL-2 e a proliferação dessas células frente ao estímulo

74 58 específico, fato esse relacionado à supressão da translocação de PKC e à redução de fosfatos de inositol e cálcio intracelular (149). A inibição da resposta in vitro de linfócitos T CD4 + ao estímulo com IL-2 também foi observada, sendo relacionada à falta de ativação da via Jak/STAT nessas células após tratamento com as proteínas gp120 e gp41 em conjunto (150). Indícios da interferência de peptídeos do envelope do HIV-1, como o gp160( ) e o gp160( ), na proliferação tanto de linfócitos T CD4 + quanto CD8 +, induzida por diferentes estímulos, como anticorpos anti-cd3, Concanavalina A e IL-2, foram também obtidos por outros grupos (151, 152). O conjunto de trabalhos sobre propriedades imunossupressoras do envelope do HIV-1 apresentado acima deu suporte à nossa hipótese inicial de que o fenômeno observado na co-imunização de HIVBr27 e HIVenv7 se devia a isso. Entretanto, a falta de consistência dos nossos resultados nos leva a acreditar que o ocorrido inicialmente se devia a problemas técnicos ou a outro fenômeno não relacionado à imunossupressão. De fato, a interferência na resposta imune induzida por plasmídeos foi previamente observada em estudos que avaliavam a imunogenicidade de vacinas de DNA administradas em conjunto (153, 154). Foi sugerido em um dos estudos que a competição entre plasmídeos pela maquinaria de transcrição celular pode levar à redução da expressão proteica de cada plasmídeo e, consequentemente, da resposta imune induzida pelos mesmos. Portanto, acreditamos que a co-imunização com vacinas de DNA deve ser seguida de estudos detalhados sobre as possíveis interferências entre seus componentes. Isso pode trazer informações importantes para a formulação de vacinas mais imunogênicas.

75 59 6. CONCLUSÕES Nossos resultados demonstram que o algoritmo TEPITOPE é eficiente na seleção de peptídeos que se ligam a múltiplas moléculas HLA de classe II. Além disso, acreditamos que a seleção de peptídeos conservados da sequência consenso do grupo M do HIV-1 foi eficiente para a construção de uma vacina indutora de resposta imune celular cruzada entre os diversos subtipos virais. Portanto, concluímos que a vacina HIVBr27 possui potencial de induzir uma resposta imune de grande cobertura populacional e direcionada a diferentes variantes do HIV-1 Concluímos também que a vacina HIVenv7 não possui propriedades imunossupressoras consistentes e que sua influência sobre a imunogenicidade da vacina HIVBr27 em certos experimentos se deveu a outros fatores, sendo eles técnicos ou não.

76 60 7. ANEXO 7.1. Anexo A Aprovação do Comitê de Ética em Pesquisa

77 Anexo B Aval para utilização de amostras de pacientes infectados pelo HIV-1

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87 of T Lymphocyte Responses to Conserved HIV-1 Regions Compared with Conserved- Region-Only HIV-1 Immunogens in Rhesus Monkeys. Journal of Virology. 2012;86(21): Borthwick N, Ahmed T, Ondondo B, Hayes P, Rose A, Ebrahimsa U, et al. Vaccine-elicited Human T Cells Recognizing Conserved Protein Regions Inhibit HIV- 1. Molecular Therapy. 2014;22(2): Kopycinski J, Hayes P, Ashraf A, Cheeseman H, Lala F, Czyzewska-Khan J, et al. Broad HIV Epitope Specificity and Viral Inhibition Induced by Multigenic HIV-1 Adenovirus Subtype 35 Vector Vaccine in Healthy Uninfected Adults. Plos One. 2014;9(3) Nchinda G, Kuroiwa J, Oks M, Trumpfheller C, Park CG, Huang Y, et al. The efficacy of DNA vaccination is enhanced in mice by targeting the encoded protein to dendritic cells. Journal of Clinical Investigation. 2008;118(4): Zhou J, Cheung AKL, Tan Z, Wang H, Yu W, Du Y, et al. PD1-based DNA vaccine amplifies HIV-1 GAG-specific CD8(+) T cells in mice. Journal of Clinical Investigation. 2013;123(6): Lacap PA, Huntington JD, Luo M, Nagelkerke NJD, Bielawny T, Kimani J, et al. Associations of human leukocyte antigen DRB with resistance or susceptibility to HIV-1 infection in the Pumwani Sex Worker Cohort. Aids. 2008;22(9): Ferre AL, Hunt PW, McConnell DH, Morris MM, Garcia JC, Pollard RB, et al. HIV Controllers with HLA-DRB1*13 and HLA-DQB1*06 Alleles Have Strong, Polyfunctional Mucosal CD4(+) T-Cell Responses. Journal of Virology. 2010;84(21): Bansal A, Gough E, Ritter D, Wilson C, Mulenga J, Allen S, et al. Group M- based HIV-1 Gag peptides are frequently targeted by T cells in chronically infected US and Zambian patients. Aids. 2006;20(3): Chirmule N, Kalyanaraman VS, Oyaizu N, Slade HB, Pahwa S. INHIBITION OF FUNCTIONAL-PROPERTIES OF TETANUS ANTIGEN-SPECIFIC T-CELL CLONES BY ENVELOPE GLYCOPROTEIN GP120 OF HUMAN IMMUNODEFICIENCY VIRUS. Blood. 1990;75(1): Kryworuchko M, Pasquier V, Theze J. Human immunodeficiency virus-1 envelope glycoproteins and anti-cd4 antibodies inhibit interleukin-2-induced Jak/STAT signalling in human CD4 T lymphocytes. Clinical and Experimental Immunology. 2003;131(3): Wang H, Nishanian P, Fahey JL. CHARACTERIZATION OF IMMUNE SUPPRESSION BY A SYNTHETIC HIV GP41 PEPTIDE. Cellular Immunology. 1995;161(2): Wilson SE, Habeshaw JA, Addawe MA, Hounsell EF, Oxford JS. HIV type 1 envelope glycoprotein 120 carboxy-terminal peptide-induced human T cell lines selectively suppress heterogeneous proliferative T cell responses to soluble antigens. Aids Research and Human Retroviruses. 1997;13(15): Sedegah M, Charoenvit Y, Minh L, Belmonte M, Majam VF, Abot S, et al. Reduced immunogenicity of DNA vaccine plasmids in mixtures. Gene Therapy. 2004;11(5): Wang KY, Guo YJ, Zhang YL, Lv K, Sun SH. Combined DNA vaccination against three animal viruses elicits decreased immunogenicity of a single plasmid in mice. Vaccine. 2007;25(22):

88 9. PUBLICAÇÕES 72

89 Vaccines & Vaccination Rosa et al. J Vaccines Vaccin 2011, 2:4 Research Article Open Access A Recombinant Adenovirus Encoding Multiple HIV-1 Epitopes Induces Stronger CD4 + T cell Responses than a DNA Vaccine in Mice Daniela Santoro Rosa 1,2,4, Susan Pereira Ribeiro 1,2, Rafael Ribeiro Almeida 1, Eliane Conti Mairena 2,3, Jorge Kalil 1,2,3 and Edecio Cunha- Neto 1,2,3 * 1 Laboratory of Clinical Immunology and Allergy-LIM60, Division of Clinical Immunology and Allergy, Department of Medicine, Brazil 2 Institute for Investigation in Immunology-INCT, Sao Paulo, Brazil 3 Heart Institute (InCor), University of Sao Paulo School of Medicine, Sao Paulo, Brazil 4 Division of Immunology-Federal University of Sao Paulo-UNIFESP, Sao Paulo, Brazil Abstract T-cell based vaccines against SIV/HIV may reduce both transmission and disease progression by inducing broad and functionally relevant T cell responses. Mounting evidence points toward a critical role for CD4 + T cells in the control of immunodeficiency and virus replication. We have previously shown that a DNA vaccine (HIVBr18), encoding 18 HIV CD4 epitopes capable of binding to multiple HLA class II molecules was able to elicit broad, polyfunctional, and long-lived CD4 + and CD8 + T cell responses in BALB/c and multiple HLA class II transgenic mice. By virtue of inducing broad responses against conserved CD4 + T cell epitopes that could be recognized across diverse common HLA class II alleles, this vaccine concept may cope with HIV-1 genetic variability and increase population coverage. Given the low immunogenicity of DNA vaccines in clinical trials, we tested the ability of a recombinant adenovirus serotype 5 encoding the 18 HIV epitopes (Ad5-HIVBr18) to increase specific cellular immune responses. We assessed the breadth and magnitude of HIV-specific proliferative and cytokine responses of CD4 + and CD8 + T cells induced by Ad5-HIVBr18 using different vaccination regimens/routes and compared to DNA immunization. Immunization with Ad5-HIVBr18 induced significantly higher specific CD4 + and CD8 + T cell proliferation, IFN-γ and TNF-α production than HIVBr18. The subcutaneous route of Ad5-HIVBr18 administration was associated with the highest responses. Ad5-HIVBr18 induced higher proliferative and cytokine responses than HIVBr18 up to 28 weeks post-immunization. Our results indicate that a vaccine based on an adenovirus vector encoding the HIVBr18 epitopes shows superior immunogenicity as compared to its DNA counterpart. These results support the possible testing of a vaccine encoding HIVBr18 in non-human primates and future clinical trials. Keywords: HIV vaccine; CD4 + T cells; Epitopes; Adenovirus; Polyfunctional; HIV-1; Vaccine Abbreviations: Ad5: Replication-Defective Recombinant Adenovirus Serotype 5; Ad5-βgal: Replication-Defective Recombinant Adenovirus Serotype 5 Encoding β-galactosidase; PBMC: Peripheral Blood Mononuclear Cells; CFSE: Carboxyfluorescein Succinimidyl Ester; CTL: Cytotoxic CD8 + T lymphocyte; HIV: Human Immunodeficiency Virus; SIV: Simian Immunodeficiency Virus; IM: Intramuscular; SC: Subcutaneous; SFU: Spot Forming Units; CMI: Cell Mediated Immune Responses Introduction Despite significant advances in therapy, the AIDS pandemic is responsible for devastating morbidity and mortality throughout the world ( especially in regions with limited access to antiretroviral drugs. Vaccine strategies targeting the induction of neutralizing antibodies against HIV-1 have failed to provide protection [1,3]. Alternative approaches have focused on vaccines that stimulate cell-mediated immune responses (CMI) against conserved HIV-1 proteins in an attempt to attenuate infection [4,5]. Two vaccine concepts that have recently completed clinical efficacy studies also evaluated the induction of CMI responses. The STEP trial showed negative results [5], while the RV144, which also aimed at inducing protective antibodies, presented borderline efficacy [6]. Immunological analysis from the RV 144 study showed that the vaccine-induced immune response was essentially composed of CD4 + T cells and binding antibodies [6], suggesting that CD4 + T cells could play an important role in HIV vaccine-induced immunity. Furthermore, vaccination strategies that induced SIV-specific CD4 + - along with CD8 + - T cell responses were able to lower viral load after heterologous challenge [7,8]. Searching for new HIV vaccine concept, our group has designed a DNA vaccine (HIVBr18) encoding a previously described set of eighteen conserved and multiple HLA-DR-binding HIV-1 CD4 + T cell epitopes (HIVBr18). Peptides encoding such epitopes were recognized by PBMC from over 90% of HIV-1 infected individuals [9]. Immunization of different strains of mice, including BALB/c and mice transgenic to common HLA class II alleles, with the multiepitope DNA vaccine induced responses to 17 out of the 18 epitopes encoded by the vaccine [10,11]. Moreover, the induced CD4 + and CD8 + T cells were polyfunctional and long-lived with central and memory phenotype [11]. By virtue of inducing broad responses against conserved CD4 + T cell epitopes that could be recognized across widely diverse common *Corresponding author: Edecio Cunha-Neto, Associate Professor, Division of Clinical Immunology and Allergy/LIM60, University of Sao Paulo School of Medicine (FM-USP), Sao Paulo, SP Brazil, Avenida Dr. Arnaldo, 455, sala 3209, Cerqueira Cesar, Sao Paulo, SP, Brazil, , Tel: ; Fax: ; edecunha@usp.br Received September 01, 2011; Accepted November 25, 2011; Published December 02, 2011 Citation: Rosa DS, Ribeiro SP, Almeida RR, Mairena EC, Kalil J, et al. (2011) A Recombinant Adenovirus Encoding Multiple HIV-1 Epitopes Induces Stronger CD4 + T cell Responses than a DNA Vaccine in Mice. J Vaccines Vaccin 2:124. doi: / Copyright: 2011 Saridi M, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. J Vaccines Vaccin ISSN: JVV an open access journal Volume 2 Issue

90 Citation: Rosa DS, Ribeiro SP, Almeida RR, Mairena EC, Kalil J, et al. (2011) A Recombinant Adenovirus Encoding Multiple HIV-1 Epitopes Induces Stronger CD4 + T cell Responses than a DNA Vaccine in Mice. J Vaccines Vaccin 2:124. doi: / Page 2 of 8 HLA class II alleles, this vaccine concept may cope both with HIV genetic variability and increase population coverage. A polyfunctional CD4 + T cell response is a hallmark of HIV-1 patients who control viremia, and shows an inverse correlation with viral load [12,13]. It has been demonstrated that the efficacious smallpox and yellow fever vaccines induce durable, specific polyfunctional CD4 + or CD8 + T cells [14,16]. Furthermore, vaccine-induced polyfunctional (IFNγ + IL-2 + TNFα + ) CD4 + T cell populations were shown to provide protection against Leishmania major [17] and M. tuberculosis [18] challenge. Although DNA vaccines are highly immunogenic in small animal models, one of the most significant hurdles in developing such vaccines has been transferring the success of inducing protective immunity in those models [19] to larger animal models. Recombinant viral vectors have been used as an alternative to circumvent this problem. Indeed, vaccines based on adenovirus or cytomegalovirus vectors encoding SIV proteins were able to elicit broad, polyfunctional and durable CD4 + and CD8 + T cell responses and reduced viral load after challenge in nonhuman primates [7,8]. Several clinical trials have shown that vaccines based on replication-defective adenovirus serotype 5 (Ad5) are safe and highly immunogenic [20,23], even though the Ad5-vectored trivalent HIV vaccine tested in the STEP trial showed no protection [24]. Here we sought to test whether an Ad5 vector encoding the HIVBr18 epitopes (Ad5-HIVBr18) could increase the immunogenicity detected with DNA immunization. We observed that homologous prime-boost immunization using Ad5-HIVBr18 increased IFN-γ/ TNF-α production as well as proliferation of CD4 + and CD8 + T cells against vaccine-encoded peptides when compared to DNA vaccine immunization alone. Materials and Methods Construction of a DNA vaccine and replication-defective recombinant adenovirus serotype 5 encoding multiple HIV-1 epitopes A DNA vaccine containing the codon optimized nucleotide sequence of the eighteen HIV-1 CD4 + epitopes described in Fonseca et al. [9]: p17 (73-89), p24 (33-45), p24 ( ), p6 (32-46), pol (63-77), pol ( ), pol ( ), gp41( ), gp160 (19-31), gp160 ( ), gp160 ( ), gp160 ( ), rev (11-27), vpr (58-72), vpr (65-82), vif ( ), vpu (6-20) and nef ( ) was constructed and produced as described in Ribeiro et al. and Rosa et al. [10,11]. The numbers in brackets are amino acid positions in reference HIV-1 strain HXB2. A replication-defective E1-deleted recombinant adenovirus serotype 5 (Ad5) encoding the HIVBr18 sequence (Ad5- HIVBr18) was commercially generated (ViraQuest, Inc.). Briefly, the HIVBr18 insert was subcloned into pvqad5cmvk-npa shuttle plasmid (pvqadcmv-hivbr18). HEK293 cells were co- transfected with pvqadcmv-hivbr18 and the RAPAd backbone and harvested after 7 days. The primary lysate was amplified and purified over two rounds of centrifugation on a CsCl gradient. The particles were isolated and dialyzed against A195 buffer and stored at -80 o C. Construction of radlacz, a recombinant adenovirus carrying the Escherichia coli galactosidase-coding sequence, described previously [25] was used as control and was kindly provided by Dr Maurício Rodrigues (Federal University of Sao Paulo- Brazil). Mice and Immunizations Six to eight weeks-old female BALB/c mice were used in this study. Mice were maintained and manipulated under specific pathogen-free conditions at the animal care facilities of the Institute of Tropical Medicine, University of Sao Paulo (IMT/FMUSP). Experiments were performed in accordance to the guidelines of the Internal Review Board of University of Sao Paulo School of Medicine (CAPPesq- HCFMUSP) and approved under protocol number For DNA immunization, six mice per group were injected with 7μg of cardiotoxin (Sigma) five days before vaccination and then received 3 doses of 100µg of HIVBr18 or empty vector pvax1 intramuscularly (IM) at days 0, 15 and 30. Each quadriceps was injected with 50µL of DNA at a concentration of 1µg/µL in saline. For adenovirus immunization, mice were inoculated subcutaneously (SC) or intramuscularly (IM) with viral suspension (Ad5-HIVBr18 or Ad5-βgal) containing 2x 10 8 PFU at days 0 and 15. For heterologous prime-boost regimens, animals received cardiotoxin (Sigma) five days before vaccination with HIVBr18 (IM) and fifteen days later received 2x 10 8 PFU of Ad5-HIVBr18 by IM or SC route. Peptides The eighteen multiple HLA-DR binding, frequently recognized peptides, derived from the conserved regions of HIV-1 B-subtype consensus and selected from the whole proteome [9] were synthesized in house by solid phase technology using the 9-fluorenylmethoxycarbonyl (Fmoc) strategy, with the C-terminal carboxyl group in amide form [26]. Peptide purity and quality were assessed by reverse-phase high performance liquid chromatography and mass spectrometry and was routinely above 90%. Spleen cell isolation Two or 28 weeks after the last immunization, mice were euthanized and spleens were removed aseptically. After obtaining single cell suspensions, cells were washed in RPMI Cells were then ressuspended in R-10 (RPMI supplemented with 10% of fetal bovine serum (FBS-GIBCO), 2 mm L glutamine (Sigma), 10 mm Hepes (Sigma), 1mM sodium pyruvate, 1% vol/vol non-essential aminoacids solution, 40µg/mL of Gentamicin, 20 µg/ml of Peflacin and 5x10-5 M 2- mercaptoethanol (SIGMA). Cell viability was evaluated using 0.2% Trypan Blue exclusion dye to discriminate between live and dead cells. IFN-γ ELISPOT assay Splenocytes from immunized mice were assayed for their ability to secrete IFN-γ after in vitro stimulation with 5µM of individual or pooled HIV-1 peptides. The ELISPOT assay was performed using Becton Dickinson murine IFN-γ ELISPOT kit, as previously described [10,11]. Spots were counted using AID ELISPOT Reader System (Autoimmun Diagnostika GmbH, Germany). The number of antigen specific T cells, expressed as spot-forming units (SFU)/10 6 splenocytes, was calculated after subtracting negative control values (medium only). The cutoff was calculated as the mean SFU/10 6 splenocytes + 3 SD from pvax1 and/or Ad5-βgal immunized mice, stimulated with all peptides (cutoff=15 SFU/10 6 splenocytes). CFSE-based proliferation assay To monitor the expansion and proliferation of HIV-specific T cells, splenocytes from immunized mice were labeled with carboxyfluorescein succinimidyl ester (CFSE) (27). Briefly, freshly isolated splenocytes were resuspended (50x10 6 /ml) in PBS and labeled with 1.25 µm of CFSE (Molecular Probes) at 37ºC for 10 minutes. The reaction was quenched with RPMI 1640 supplemented with 10% FBS and cells were washed before resuspending in RPMI 1640 at a density of 1.5x10 6 /ml. Cells were cultured in 96-well-round-bottomed plates (3x10 5 /well in J Vaccines Vaccin ISSN: JVV an open access journal Volume 2 Issue

91 Citation: Rosa DS, Ribeiro SP, Almeida RR, Mairena EC, Kalil J, et al. (2011) A Recombinant Adenovirus Encoding Multiple HIV-1 Epitopes Induces Stronger CD4 + T cell Responses than a DNA Vaccine in Mice. J Vaccines Vaccin 2:124. doi: / Page 3 of 8 triplicate) for 5 days at 37 o C and 5%CO 2 with medium only or 5µM of HIV peptides. Positive control included cells stimulated with 2.5µg/mL of Concanavalin A (Sigma). Cells were then harvested, washed with 100 µl of FACS buffer (PBS with 0.5% BSA and 2mM EDTA) and stained with anti-mouse CD3 phycoerythrin (PE), anti-mouse CD4 peridinin chlorophyll protein (PerCP) and anti-mouse CD8 allophycocyanin (APC) monoclonal antibodies (BD Pharmingen, San Jose, CA) for 45 minutes at 4 0 C. Cells were then washed twice with FACS buffer, fixed with 4% paraformaldehyde, and resuspended in FACS buffer. Samples were acquired on a FACSCanto flow cytometer (BD Biosciences) and then analyzed using FlowJo software (version 9.0.2, Tree Star, San Carlo, CA). Fifty thousand events were acquired in a live lymphocyte gate. The percent of proliferating CD4 + and CD8 + CFSE low cells was determined in the CD3 + cell population and the criteria for scoring as positive included % CFSE low cells > cutoff. The cutoff of unspecific proliferative response was determined based on the percentage of proliferating cells (% of CD3 + CD4 + or CD3 + CD8 + CFSE low cells) on splenocytes from pvax1 and/ or Ad5-βgal immunized groups after stimulating with individual peptides +3 standard deviation (SD). Analysis of polyfunctional HIV-specific T-cell responses Splenocytes from immunized mice were labeled with CFSE as described above. CFSE-labeled cells were incubated at a density of 2.5x10 6 cells/ml and cultured in 96-well-round-bottomed plates (5x10 5 / well in triplicate) for 4 days at 37 o C and 5%CO 2 with medium only or pooled HIV peptides (5µM). After 4 days of incubation, cells were restimulated in the presence of 2µg/mL anti-cd28 (BD Pharmingen), 5µM of pooled HIV peptides and Brefeldin A- GolgiPlug TM (BD Pharmingen) for the last 12 hours. After the incubation period, cells were washed with FACS buffer and surface stained using monoclonal antibodies to CD8-Alexa700 and CD4- PerCP for 30 minutes at 4 0 C. Cells were fixed and permeabilized using the Cytofix/Cytoperm TM kit (BD Pharmingen). Permeabilized cells were washed with Perm/Wash buffer (BD Biosciences) and stained with monoclonal antibodies to CD3-APCCy7, IL2-PE, TNFα-PECY7 and IFNγ- APC for 30 minutes at 4 0 C. Following staining, cells were washed twice and resuspended in FACS buffer. All antibodies were from BD Pharmingen. Samples were acquired on a FACSCanto flow cytometer (BD Biosciences) and then analyzed using FlowJo software (version 9.0.2, Tree Star, San Carlo, CA). Five hundred thousand events were acquired in a live lymphocyte gate. The percent of cytokine producing and proliferating CD4 + and CD8 + cells was determined in the CD3 + cell population. In addition, we used Boolean gate (FlowJo software (version 9.0.2, Tree Star, San Carlo, CA)) platform to create several combinations of the cytokine gates among the proliferating T cells resulting in seven distinct patterns. The percentages of cytokine- producing cells were calculated by subtracting background values. For each flow cytometry experiment performed in this paper, unstained and all single-color controls were processed to allow proper compensation. Data Analysis Statistical significance (p-values) was calculated by using One-way ANOVA and Tukey s honestly significantly different (HSD) or Twoway ANOVA and Bonferroni Post Test. Results To analyze whether the Ad5-HIVBr18 vaccine could be more immunogenic than the DNA counterpart, HIVBr18, BALB/c mice were immunized with either the previously established DNA immunization protocol (3 doses of HIVBr18 [13,14]); or with 2 doses of Ad5-HIVBr18 by different routes; or with a heterologous DNA prime (HIVBr18) followed by Ad5-HIVBr18-boost by different routes. Fifteen days after the last immunization, pooled splenocytes were evaluated for specific CD4 + and CD8 + T cell proliferation against pooled HIV-1 peptides by the CFSE-based proliferation assay. CD4 + and CD8 + T cells from animals that received two doses of recombinant adenovirus subcutaneously (Ad5-HIVBr18, SC) presented significantly higher specific proliferation than the group immunized with 3 doses of the HIVBr18 DNA vaccine (Figure 1). HIV-specific proliferation was above 20% for CD4 + and above 10% for CD8 + T cells in this group, being significantly higher for the CD4 + T cell compartment than the CD8 + T cell compartment (p<0.05). Neither of the heterologous prime-boost regimens was able to increase the magnitude of CD4 + and CD8 + T cell proliferative responses as compared to the HIVBr18 DNA immunization protocol. Actually, the heterologous prime-boost protocols were associated with a decrease in the magnitude of the proliferative responses. We subsequently characterized the cytokine production profile of the specific T cells. Using the IFN-γ ELISPOT assay, we observed that splenocytes from animals that received two doses of Ad5-HIVBr18 (SC) presented significantly higher IFN-γ secretion when compared with those immunized with HIVBr18 DNA alone (Figure 2A). Both heterologous prime-boost regimens reduced the magnitude of the responses. In order to assess the functional profile of the induced CD4 + and CD8 + T cells, we performed a multiparameter flow cytometry analysis. This strategy allowed us to characterize the antigen-specific T cells based on their ability to proliferate and produce the effector cytokines IFN-g, TNF-α and IL-2 at a single cell level. Taking into account cells that proliferated and produced any of the 3 cytokines tested, we observed that subcutaneous immunization with Ad5-HIVBr18 induced significantly higher CD4 + T cells with such profile. On the other hand, heterologous prime-boost protocols induced significantly lower Figure 1: Immunization with an adenovirus encoding the multiepitope gene (Ad5-HIVBr18) elicits higher proliferative responses when compared to other regimens. BALB/c mice (6 per group) were immunized intramuscularly (IM) with the DNA vaccine HIVBr18 (3 doses); or received 2 doses of 2x10 8 PFU of recombinant adenovirus (Ad5) by intramuscular (IM) or subcutaneous (SC) route; or received a heterologous prime-boost regimen based on a DNA prime (IM), Ad5 boost (IM or SC). Control groups received pvax1 plasmid and/ or Ad5-βgal.Two weeks after the last immunization; splenocytes from each immunized group were pooled, labeled with CFSE (1.25μM) and cultured for 5 days, in the presence of 5μM of pooled HIV-1 peptides. Cells were analyzed by flow cytometry and CFSE dilution on gated CD3+CD4+ or CD3+CD8+ cells was used as readout for antigen-specific proliferation. Cutoff values were 1.7% for CD4+ and 1.5% for CD8+ T cells. Only significant differences with the DNA immunized group are depicted (*p<0.05 and ***p<0.001). DNA= HIVBr18; Ad5= adenovirus 5 encoding HIVBr18. J Vaccines Vaccin ISSN: JVV an open access journal Volume 2 Issue

92 Citation: Rosa DS, Ribeiro SP, Almeida RR, Mairena EC, Kalil J, et al. (2011) A Recombinant Adenovirus Encoding Multiple HIV-1 Epitopes Induces Stronger CD4 + T cell Responses than a DNA Vaccine in Mice. J Vaccines Vaccin 2:124. doi: / Page 4 of 8 Figure 2: Immunization with Ad5-HIVBr18 induces higher number of polyfunctional T cells when compared to other regimens. BALB/c mice (6 per group) were immunized as described in Figure 1 and Methods section. Two weeks after the last immunization, splenocytes from each immunized group were pooled and specific immune responses were evaluated in vitro against pooled HIV-1 peptides. (A) Frequencies of IFN γ secreting cells measured by ELISPOT. SFU, spot-forming units. Cutoff: 15 SFU/10 6. (B) Splenocytes were labeled with CFSE (1.25 µm) and cultured for 4 days in the presence of pooled HIV-1 peptides or medium only. On day 4, cells were pulsed for 12 hours with pooled peptides in the presence of costimulatory antibody and Brefeldin A. Cells were then surface stained with antibodies to CD4 and CD8, permeabilized and stained for intracellular cytokines (IFN-γ, TNF-α and IL-2) and CD3. Total frequencies of proliferating (CFSElow) and cytokine-producing CD4+ and CD8+ T cells are shown. (C) and (D) After gating on proliferating (CFSElow) and cytokine-producing cells, Boolean combinations were created using FlowJo software to determine the frequency of each response based on all possible combinations of cytokine expression among proliferating T cells. Splenocytes from control groups (pvax1 and/or Ad5- βgal) presented negligible numbers of IFN-γ secreting cells and proliferating-cytokine producing cells. Only significant differences with the DNA immunized group are depicted (*p<0.05, **p<0.01 and ***p<0.001). DNA= HIVBr18; Ad5= adenovirus 5 encoding HIVBr18. responses as compared to immunization with HIVBr18 DNA alone. The induced HIV-1-specific CD4 + T cell responses were significantly higher than CD8 + T cell responses in all immunization groups (p<0.01), except for the subcutaneous heterologous prime-boost regimen (Figure 2B). There was no significant difference in the magnitude of the CD8 + T cell induced immune responses among different groups. Boolean combinations allowed us to evaluate the polyfunctional profile of the induced T cells based on the ability of these cells to perform any of the above measured functions (proliferation and cytokine production). Boolean analysis of proliferating (CFSE low ) and cytokinepositive populations indicated that all immunized groups presented a similar CD4 + T cell polyfunctional profile (Figure 2C). However, Ad5-HIVBr18 immunization induced a significantly higher number of peptide-specific CD4 + T cells that were able to proliferate and produce IFN-g and TNF-α, alone or in combination, than immunization with HIVBr18 (Figure 2C). On the other hand, heterologous prime-boost regimens induced significantly lower numbers of proliferating CD4 + T cells producing TNF-α alone or in combination with IFN-γ, as compared to HIVBr18. We also found that for the CD8 + T cell subset, J Vaccines Vaccin ISSN: JVV an open access journal Volume 2 Issue

93 Citation: Rosa DS, Ribeiro SP, Almeida RR, Mairena EC, Kalil J, et al. (2011) A Recombinant Adenovirus Encoding Multiple HIV-1 Epitopes Induces Stronger CD4 + T cell Responses than a DNA Vaccine in Mice. J Vaccines Vaccin 2:124. doi: / Page 5 of 8 Figure 3: Breadth of immune responses. BALB/c mice were immunized as described in Figure 1 and Methods section. Two weeks after the last immunization, splenocytes from immunized mice (6 per group) were pooled and the specific IFN-γ responses were evaluated in vitro against 18 individual HIV-1 peptides encoded by the vaccines (5µM). The pie charts show the frequencies of HIV-1 peptide-specific IFN-γ secreting cells by displaying each number of SFU/10 6 cells for each positive peptide as a proportion of the sum of SFU/10 6 cells for all positive peptides. The sum of all positive responses (SFU/10 6 cells) is shown above each pie chart. Splenocytes from control groups (pvax1 and/or Ad5-βgal) presented negligible numbers of IFN-γ secreting cells after stimulation with each of the 18 encoded peptides. SFU, spot-forming units. Cutoff: 15 SFU/10 6. there was a higher prevalence of proliferating cells that produced IFN-γ and TNF-α, alone or simultaneously, in all immunized groups (Figure 2D). CD8 + T cells from mice subcutaneously immunized with Ad5- HIVBr18 presented higher percentages of CFSE low and IFN-γ/TNF-α producing cells than HIVBr18. Control groups (pvax1 or Ad5-βgal immunized group) presented negligible levels of cytokine producingproliferating T cells. These results showed that immunization protocols based on Ad5 vector, especially by the subcutaneous route, can generate proliferating and cytokine-producing specific T cells with higher magnitude than DNA immunization. On the other hand, the heterologous prime-boost regimens frequently reduced the responses as compared to immunization with HIVBr18. In order to verify if immunization protocols based on Ad5 vector would be able to induce a broader specific immune response, we performed IFN-γ ELISPOT assays stimulated with each of the 18 HIV-1 peptides encoded by the vaccines. While immunization with HIVBr18 induced responses to 7 peptides, subcutaneous immunization with Ad5-HIVBr18 induced responses to 2 additional peptides: (p6(32-46) and gp160( )) (Figure 3). These results show that the selected viral vector was able to increase the magnitude and, to a lesser extent, also had an impact in the breath of the immune responses against the vaccine encoded epitopes. Given the importance of long-term vaccine induced immune responses, we evaluated the presence of specific T cells 28 weeks after the last immunization. As shown in Figure 4, measurable proliferative responses were observed at this time point, and proliferative CD4 + T cell responses were significantly higher in the group immunized subcutaneously with Ad5-HIVBr18 than HIVBr18. The heterologous prime-boost regimens induced the lowest proliferative responses at the 28 week time-point, as previously observed for the 2 week time-point. The ability of T cells from immunized mice to secrete cytokines over time was also evaluated by IFN-γ ELISPOT (Figure 5A) and multiparametric flow cytometry assays (Figure 5 B-C). IFN-γ secretion was detectable in all immunized groups at the 28 week-time point and was significantly higher in the group subcutaneously immunized with Ad5-HIVBr18 than in the HIVBr18 immunized group (Figure 5A). Boolean analysis showed that different immunization protocols induced 4 to 9% of CD4 + T cells that proliferated and produced at least one cytokine in response to pooled HIV-1 peptides (Figure 5B). Proliferating/cytokine producing CD4 + T cells were significantly higher among the Ad5-HIVBr18 immunized groups. Boolean analysis at this time point (28 weeks) showed that Ad5-HIVBr18 immunization induced higher numbers of peptide-specific CD4 + T cells that proliferate (CFSE low ) and produce IFN-γ/TNF-α simultaneously (Figure 5C) and that proliferate and produce IFN-γ alone than HIVBr18 immunized group. These results showed that immunization protocols based on adenovirus 5, mainly by the subcutaneous route, can generate a more durable specific immune response than DNA immunization. On the other hand, heterologous prime boost regimens reduced the magnitude of specific immune responses. Finally, we evaluated the breadth of the induced immune responses at the 28 week-time point for all immunized groups by IFN-γ ELISPOT assay. The number of recognized peptides varies from 2 to 9 (Figure 6). The broadest response was observed in the group immunized with HIVBr18. Comparison of the 2- and 28-week time points showed different epitope targets. Responses to peptide gp160( ) found 2 weeks after last immunization with Ad5-HIVBr18 (Figure 3) were not detected anymore at the 28 week-time point, while responses to p24(33-45) and pol( ) became borderline positive in the HIVBr18-immunized group. With the exception of p17(73-89), all responses were higher in mice immunized subcutaneously with Ad5- HIVBr18 than HIVBr18. These results showed that subcutaneous administration of Ad5-HIVBr18 had an impact at the magnitude of the induced immune responses over time. Discussion In this paper, we have shown that immunization with Ad5- HIVBr18 induced stronger T cell responses than HIVBr18, the previously described DNA vaccine [10,11], without impairing the breadth of the immune responses. Ad5-HIVBr18-immunized mice displayed significantly higher proliferative, as well as polyfunctional CD4 + and CD8 + T cell responses when compared to HIVBr18. Most of these responses were highest among animals immunized with Ad5-HIVBr18 by the subcutaneous route. The same immunization protocol induced the highest T cell immune responses even at 28 weeks after the last immunization, indicating that it was also able to improve the longevity of the vaccine-induced immune responses, as compared to DNA immunization. The Ad5-HIVBr18 immunization J Vaccines Vaccin ISSN: JVV an open access journal Volume 2 Issue

94 Citation: Rosa DS, Ribeiro SP, Almeida RR, Mairena EC, Kalil J, et al. (2011) A Recombinant Adenovirus Encoding Multiple HIV-1 Epitopes Induces Stronger CD4 + T cell Responses than a DNA Vaccine in Mice. J Vaccines Vaccin 2:124. doi: / Page 6 of 8 Figure 4: Ad5-HIVBr18 immunization induces higher proliferative responses up to 28 weeks after the last immunization. BALB/c mice were immunized as described earlier. Twenty-eight weeks after the last immunization, splenocytes from immunized mice (6 per group) were pooled, labeled with CFSE (1.25μM) and cultured for 5 days, in the presence of 5μM of pooled HIV-1 peptides. Cells were analyzed by flow cytometry and CFSE dilution on gated CD3+CD4+ or CD3+CD8+ cells was used as readout for antigen-specific proliferation. Experimental groups presented proliferation above cutoff values in all time points. The average of proliferation on splenocytes from pvax1 and/or Ad5-βgal immunized group was subtracted from the respective experimental groups. DNA= HIVBr18; Ad5= adenovirus 5 encoding HIVBr18. DNA vs Ad5 (2x, SC), p< strategy maintained the bias toward CD4 + T cell responses, as previously observed for HIVBr18 [10,11]. An additional advantage of immunization with Ad5-HIVBr18 as compared to HIVBr18 was the reduced number of required doses (2 vs. 3 doses, respectively). We also tested heterologous prime-boost protocols, which were invariably less efficient than homologous immunization regimens. As mentioned above, we observed that homologous adenovirusbased immunization protocols increased the magnitude of specific proliferation and type 1 cytokine production, in both CD4 + and CD8 + T cell subsets. This is of great interest, since adenovirus-based vaccines that induced highly immunogenic responses in mice [28] and nonhuman primate models [8] were able to control pathogen specific challenge. In Addition, it did not impair the breadth of these responses. Interestingly, we observed that the CD4 + T cell response was significantly higher than the CD8 + response among adenovirus-based vaccine immunized groups. This is in contrast with previous reports that showed that adenovirus vector-based immunization induces a stronger CD8 + than CD4 + T cell response [29]. We believe that the CD4-biased immune responses may be due to the intrinsic design of the CD4 T cell epitope-rich insert, as previously observed by our group with HIVBr18 immunization [10,11]. Although Ad5-HIVBr18 immunization induced a CD4-biased T cell response, it also improved CD8 + specific T cell responses, when compared to HIVBr18 immunization. In the present work, all immunization strategies were able to induce polyfunctional T cells. However, we found a significantly increased number of polyfunctional CD4 + and CD8 + T cells in Ad5-HIVBr18- immunized group when compared to HIVBr18. A polyfunctional CD4 + T cell response is a hallmark of HIV-1 patients who control viremia, and shows an inverse correlation with viral load [12,13]. In mice, vaccine-induced polyfunctional CD4 + T cells have been shown to correlate with protection against Leishmania major [17] and M. tuberculosis [18]. We found that subcutaneous immunization with Ad5-HIVBr18 increased the number of recognized epitopes when compared to HIVBr18 vaccine. Indeed, the breadth of the immune responses has Figure 5: Ad5-HIVBr18 immunization induces higher numbers of polyfunctional T cells up to 28 weeks after the last immunization. BALB/c mice were immunized as described earlier. Twenty eight weeks after the last immunization, splenocytes from BALB/c mice (6 per group) were pooled and specific immune responses were evaluated in vitro against pooled HIV- 1 peptides. (A) Frequencies of IFN-γ secreting cells measured by ELISPOT. (B) Spleen cells were labeled with CFSE (1.25 μm) and cultured for 4 days in the presence of pooled HIV-1 peptides or medium only. On day 4, cells were pulsed for 12 hours with pooled peptides in the presence of costimulatory antibody and Brefeldin A. Cells were then surface stained with antibodies to CD4 and CD8, permeabilized and stained for intracellular cytokines and CD3. Total frequencies of proliferating (CFSElow) and cytokine-producing CD4+ T cells are shown. (C). Values from control groups (pvax1 and/or Ad5-βgal) were subtracted from experimental groups and are not depicted. DNA= HIVBr18; Ad5= adenovirus 5 encoding HIVBr18. DNA vs DNA (1x) + Ad5 (1x, IM), p<0.001; DNA vs DNA (1x) + Ad5 (1x, SC), p<0.001; DNA vs Ad5 (2x, SC), p< been suggested as a correlate of protection in HIV-1/SIV infection [30-33,8]. By virtue of inducing broad responses against conserved CD4 + T cell epitopes, this vaccine concept may cope with HIV genetic variability and viral escape. Beyond the magnitude and breadth of the induced immune responses, long-term immunity is critical and has an important impact on vaccine-induced protection [16]. We observed a persistent specific immune response up to 28 weeks after the last dose for all immunized J Vaccines Vaccin ISSN: JVV an open access journal Volume 2 Issue

95 Citation: Rosa DS, Ribeiro SP, Almeida RR, Mairena EC, Kalil J, et al. (2011) A Recombinant Adenovirus Encoding Multiple HIV-1 Epitopes Induces Stronger CD4 + T cell Responses than a DNA Vaccine in Mice. J Vaccines Vaccin 2:124. doi: / Page 7 of 8 Figure 6: Breadth of immune responses 28 weeks after last dose. BALB/c mice were immunized as described earlier. Twenty eight weeks after the last immunization, splenocytes from immunized mice (6 per group) were pooled and the specific IFN-γ responses were evaluated in vitro against 18 individual HIV-1 peptides encoded by the vaccines (5µM). (A) The pie charts represent the frequencies of HIV-1 peptide-specific IFN-γ secreting cells by displaying each number of SFU/10 6 cells for each positive peptide as a proportion of the sum of SFU/10 6 cells for all positive peptides. The sum of all positive responses (SFU/10 6 cells) is shown above each pie chart. Splenocytes from control groups (pvax1 and/or Ad5-βgal) presented negligible numbers of IFN γ secreting cells after stimulation with each of the 18 encoded peptides. SFU, spot-forming units. Cutoff: 15 SFU/10 6. groups, which was highest in the subcutaneously Ad5-HIVBr18 immunized group. Heterologous prime-boost regimens have been proposed as an alternative to avoid boosting vector immunity and consequent impairment of the targeted immune responses. In our study, none of the heterologous DNA prime-ad5 boost regimens were able to increase HIV- specific induced cellular immune responses when compared to HIVBr18 or Ad5-HIVBr18 alone. In fact, we have tested other heterologous DNA-prime adenovirus-boost combinations involving different number of doses of each immunogen, with similar or weaker responses (data not shown). This is in contrast with many reports that have shown that DNA prime and viral vector boost could be an effective strategy for eliciting strong T-cell responses against pathogenic viruses such as Ebola virus, HIV, and HCV [34-36]. In spite of the high immunogenicity of Ad5-HIVBr18, there is a major concern in vaccination based on Ad5 viral vector. Most humans have high titers of neutralizing antibodies against this adenovirus serotype, due to the fact that natural Ad5 infection is commonly acquired in childhood. In addition, the use of adenovirus as a vector for HIV vaccines has been questioned after the results from the STEP trial. The trend toward increased acquisition of HIV-1 infection in baseline Ad5-seropositive vaccines was concerning but not statistically significant [24]. In addition, a recent report failed to find an association between Ad5 neutralizing antibody seropositivity and incidence of HIV acquisition among populations at elevated risk of HIV infection [37]. One way to overcome the pre-existing immunity problem is to use adenovirus serotypes with low prevalence of neutralizing antibodies [38]. To achieve this goal the development of vectors based on rare human serotypes such as human Ad35 [39], AdHu28 [40] or modification of the human Ad5 capsid [41,42] has been performed. Altogether, our results showed that a recombinant adenovirus 5 vaccine expressing the CD4 epitope-rich HIVBr18 insert was highly immunogenic, especially when administered by the subcutaneous route. These results encourage the testing of an adenoviral vector-based HIVBr18 vaccine in non-human primates and future clinical trials. Acknowledgments We thank Dr. Claudio Puschel and Mr. Washington Robert da Silva for peptide synthesis; Mr. Luis Roberto Mundel for assistance at the animal facility. 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97 Broad and Cross-Clade CD4 + T-Cell Responses Elicited by a DNA Vaccine Encoding Highly Conserved and Promiscuous HIV-1 M-Group Consensus Peptides Rafael Ribeiro Almeida 1, Daniela Santoro Rosa 1,3,4, Susan Pereira Ribeiro 1,3, Vinicius Canato Santana 1, Esper Georges Kallás 1, John Sidney 5, Alessandro Sette 5, Jorge Kalil 1,2,3, Edecio Cunha-Neto 1,2,3 * 1 Laboratory of Clinical Immunology and Allergy-LIM60, Division of Clinical Immunology and Allergy, Department of Medicine, University of São Paulo School of Medicine, São Paulo, Brazil, 2 Heart Institute (InCor), University of São Paulo School of Medicine, São Paulo, Brazil, 3 Institute for Investigation in Immunology-INCT, São Paulo, Brazil, 4 Division of Immunology-Federal University of São Paulo-UNIFESP, São Paulo, Brazil, 5 Center for Infectious Disease, Allergy and Asthma Research, La Jolla Institute for Allergy and Immunology, La Jolla, California, United States of America Abstract T-cell based vaccine approaches have emerged to counteract HIV-1/AIDS. Broad, polyfunctional and cytotoxic CD4 + T-cell responses have been associated with control of HIV-1 replication, which supports the inclusion of CD4 + T-cell epitopes in vaccines. A successful HIV-1 vaccine should also be designed to overcome viral genetic diversity and be able to confer immunity in a high proportion of immunized individuals from a diverse HLA-bearing population. In this study, we rationally designed a multiepitopic DNA vaccine in order to elicit broad and cross-clade CD4 + T-cell responses against highly conserved and promiscuous peptides from the HIV-1 M-group consensus sequence. We identified 27 conserved, multiple HLA-DR-binding peptides in the HIV-1 M-group consensus sequences of Gag, Pol, Nef, Vif, Vpr, Rev and Vpu using the TEPITOPE algorithm. The peptides bound in vitro to an average of 12 out of the 17 tested HLA-DR molecules and also to several molecules such as HLA-DP, -DQ and murine IA b and IA d. Sixteen out of the 27 peptides were recognized by PBMC from patients infected with different HIV-1 variants and 72% of such patients recognized at least 1 peptide. Immunization with a DNA vaccine (HIVBr27) encoding the identified peptides elicited IFN-c secretion against 11 out of the 27 peptides in BALB/c mice; CD4 + and CD8 + T-cell proliferation was observed against 8 and 6 peptides, respectively. HIVBr27 immunization elicited cross-clade T-cell responses against several HIV-1 peptide variants. Polyfunctional CD4 + and CD8 + T cells, able to simultaneously proliferate and produce IFN-c and TNF-a, were also observed. This vaccine concept may cope with HIV-1 genetic diversity as well as provide increased population coverage, which are desirable features for an efficacious strategy against HIV-1/AIDS. Citation: Almeida RR, Rosa DS, Ribeiro SP, Santana VC, Kallás EG, et al. (2012) Broad and Cross-Clade CD4 + T-Cell Responses Elicited by a DNA Vaccine Encoding Highly Conserved and Promiscuous HIV-1 M-Group Consensus Peptides. PLoS ONE 7(9): e doi: /journal.pone Editor: Kim J. Hasenkrug, National Institute of Allergy and Infectious Diseases, United States of America Received June 18, 2012; Accepted August 15, 2012; Published September 18, 2012 Copyright: ß 2012 Almeida et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by the Brazilian National Research Council (CNPq), grant /2005-0; São Paulo State Research Funding Agency (FAPESP), grants 2004/ , 2006/ and 2008/ ; Brazilian Program for STD and AIDS, Ministry of Health (914/BRA/3014-UNESCO/Kallas); and the São Paulo City Health Department ( /Kallas). RRA, SPR and VCS are recipients of São Paulo State Research Funding Agency (FAPESP) fellowships. EGK, JK and ECN are recipients of Brazilian Council for Scientific and Technological Development - CNPq productivity awards. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * edecunha@gmail.com Introduction The development of an efficacious vaccine against human immunodeficiency virus 1 (HIV-1) still remains as the best longterm approach to control the acquired immunodeficiency syndrome (AIDS) pandemic since resource-poor endemic regions are not able to afford sustained antiretroviral therapy (ART). Clinically tested HIV-1 vaccines have shown no or modest efficacy so far [1,2]. No vaccine strategy was able to induce broadly neutralizing antibodies and T-cell based vaccines have thus emerged as an alternative to counteract AIDS by limiting both viral transmission and disease progression [3]. Indeed, a recent study using non-human primates (NHP) has demonstrated that vaccine-induced virus-specific effector memory T-cell (TEM) responses can exert a profound early control on highly pathogenic simian immunodeficiency virus (SIV) infection after mucosal challenge, which has given more hope for the development of new T-cell based vaccines against HIV-1 [4]. The breadth of T-cell responses induced against HIV-1 has become a central goal in AIDS vaccine development after the STEP trial failure [1,5]. In fact, different groups have shown that protection against SIV challenge is strongly associated with induction of either CD4 + or CD8 + T cells against multiple targets [6 9]. Thus, it is important to design novel vaccine platforms in order to broaden T-cell responses against HIV-1. T-cell based vaccines against HIV-1 are frequently focused on the induction of CD8 + T-cell responses, which are known to be responsible for killing virus-infected targets [6,10 12]. However, mounting evidence suggests that CD4 + T-cell responses may be important for controlling HIV-1 replication [13]. Although HIVspecific CD4 + T cells are preferentially targeted by the virus, the vast majority of these cells remains virus-free at any time in vivo PLOS ONE 1 September 2012 Volume 7 Issue 9 e45267

98 HIV-1 M-Group Epitope-Based Vaccine [14], which may allow for their antiviral function. In fact, strong virus-specific CD4 + T-cell responses have been associated with natural control of HIV-1 infection [15,16] and cytotoxic CD4 + T cells were shown to suppress viral replication in both SIV and HIV-1-infected cells [17,18]. While the clinical associations of CD4 + T-cell responses with HIV-1 control must be carefully interpreted, due to a possible cause-effect issue, the finding that CD4 + T-cell depletion reduced vaccine-mediated protection [19] supports a direct role of such cells in HIV-1 immunity. Moreover, some groups have observed the association of vaccine-induced virus-specific CD4 + T-cell responses with protection against SIV challenge [7,20], which further supports a protective role of CD4 + T cells. Therefore, it is important to explore the anti-viral immunity exerted by CD4 + T cells in order to develop novel vaccines against HIV-1/AIDS. It is possible that the induction of CD4 + T cells will be beneficent both due to the help provided to B cells and CD8 + T cells as well as due to direct effects on HIV-1- infected targets. An important concern regarding AIDS vaccine development is how to elicit cellular immune responses to cover multiple HIV-1 circulating variants, which can differ by up to 20% within a subtype and show up to 35% of amino acid divergences between subtypes [21]. Artificially designed M-group consensus sequences display average distances to HIV-1 variants similar to those found intra-subtype and have been considered a potential alternative to circumvent the barrier posed by viral genetic diversity [22]. Indeed, studies have demonstrated that immunogens based on HIV-1 M-group consensus Env were able to provide broad crossclade T-cell responses in both mice and macaques [23,24], which suggests an important role for this strategy in HIV-1 vaccines. The high polymorphism of human leukocyte antigens (HLA), which are responsible for determining the onset of T-cell responses, is also a challenge for vaccine development. It is expected that different HLA-bearing populations respond differently to the same immunogen and this may be decisive for the vaccine success. Vaccines encoding promiscuous peptides, each binding to multiple HLA molecules, may be a solution to this problem by allowing that multiple HLA molecules spread among the population contribute to the induction of broad T-cell responses in most of the immunized individuals. This would confer broader population coverage and enhance vaccine efficacy [25 27]. Thus, novel AIDS vaccines should be rationally designed to address both viral and host genetic diversity in order to confer immunity against multiple HIV-1 circulating variants in a population with diverse HLA alleles. The inclusion of appropriate proteins in HIV-1 vaccines may be crucial for eliciting protective responses. While broad Gag- and Vif-specific responses have been correlated to vaccine-induced protection in SIV-challenged macaques [28], induction of Envspecific CD4 + T-cell responses contributed to enhanced SIV replication and accelerated progression to AIDS [29]. Env-specific CD8 + T-cell responses were also shown to be a strong predictor for disease progression in HIV-1-infected patients [30]. Furthermore, CD4 + T-cell responses targeting Gag and Env-specific epitopes were associated with spontaneous control of viral replication and progression to AIDS, respectively [31]. Recently, our group has designed a DNA vaccine encoding 18 conserved, multiple HLA-DR-binding epitopes from HIV-1 subtype B consensus sequence. This vaccine elicited broad, polyfunctional and long-lasting CD4 + T-cell responses in BALB/ c and HLA class II transgenic mice [32,33]. In this work we sought to develop a DNA vaccine that would be able to provide broad CD4 + T-cell immunity in a diverse HLA-bearing population, now targeting multiple HIV-1 M-group consensus peptides, potentially cross-reactive to a high proportion of circulating HIV-1 variants. In addition, we excluded Env peptides from our novel vaccine based on the evidence that Env-specific T-cell responses are frequently related to disease progression. To accomplish our goals, we used the TEPITOPE algorithm [34], which has been successfully applied for in silico identification of promiscuous T-cell epitopes in the context of oncology, allergy, autoimmunity and infectious diseases [35 40], to scan the HIV-1 M-group consensus sequence. We identified 27 peptides from 7 different HIV-1 proteins (Gag, Pol, Nef, Vif, Vpr, Rev and Vpu), predicted to bind to multiple HLA-DR molecules and conserved among all M-group subtypes. The identified peptides bound in vitro to several HLA-DR, -DP and -DQ molecules and also to murine IA b and IA d molecules. The peptides were antigenic in natural infection, being recognized by peripheral blood mononuclear cells (PBMC) from HIV-1-infected patients. Finally, we designed a DNA vaccine (HIVBr27) encoding the 27 peptides in tandem and immunized BALB/c mice. HIVBr27 immunization elicited broad, cross-clade and polyfunctional CD4 + and CD8 + T-cell responses. Materials and Methods Ethics Statement The research involving human participants reported in this study was approved by the institutional review board of the University of São Paulo under protocol number 0458/08. Written informed consent was obtained from all subjects. Mice were housed and manipulated under SPF conditions in the animal care facilities of the Institute of Tropical Medicine, University of São Paulo (IMT/FMUSP). Experiments were performed in accordance to the guidelines of the Ethics committee of University of São Paulo (CAPPesq- HCFMUSP). This study was approved by CAPPesq- HCFMUSP under protocol number 0653/09. Identification of HIV-1 M-group Consensus Peptides We scanned the HIV-1 M-group proteome consensus sequence available at NEWALIGN/align.html with the TEPITOPE algorithm to identify multiple HLA-DR-binding peptides [34]. The TEPITOPE algorithm predicts binding of peptides to 25 distinct HLA-DR molecules based on results from in vitro HLA-peptide binding assays. We selected the peptides predicted to bind to at least 18 out of the 25 HLA-DR molecules in the TEPITOPE matrix, using a threshold of 5%. The selected peptides were further analyzed regarding the conservation status when compared to consensus sequences of the HIV-1 subtypes A, B, C, D, F1, F2, G and H. We ended up with 27 peptides (14 24 aa in length) that had each amino acid conserved among at least 50% of the consensus sequences from the HIV-1 subtypes. Peptide Synthesis Peptides were synthesized by solid phase technology using 9- fluorenylmethoxycarbonyl (Fmoc) strategy, with the C terminal carboxyl group in amide form (GL Biochem). Peptide purity and quality were assessed by reverse-phase high performance liquid chromatography and mass spectrometry, and was routinely above 90%. HLA Class II and Murine MHC Class II Peptide-binding Assays Peptide binding assays were performed by incubating purified HLA class II molecules (HLA-DR, -DP and -DQ) or murine IA b and IA d molecules (5 500 nm) with different concentrations of PLOS ONE 2 September 2012 Volume 7 Issue 9 e45267

99 HIV-1 M-Group Epitope-Based Vaccine unlabeled peptide inhibitors and 1 10 nm 125 I-radiolabeled probe peptides for 48 h as previously described [41]. Construction of a DNA Plasmid Encoding Multiple HIV-1 Peptides We designed a multiepitopic construct encoding the 27 HIV-1 peptides: protease (53 75), protease (79 95), RT ( ), RT ( ), RT ( ), RT ( ), RT ( ), RT ( ), integrase (28 43), integrase (69 85), integrase (96 113), integrase ( ), integrase ( ), p17 (72 90), p17( )/p24(1 18), p24 (33 48), p24 ( ), p24 ( ), p24 ( ), vif (1 15), vif ( ), rev (9 27), vpr (29 42), vpr (58 80), vpu (13 26), nef (67 87) and nef ( ). Peptide sequences were codon optimized for mammalian expression and assembled in tandem in the above mentioned order with glycineproline (GPGPG) spacers at C and N termini to avoid the creation of junctional peptides, which can interfere on processing and presentation [42]. The artificial gene (EZBiolab) was subcloned into pvax1 vector (Invitrogen) using EcoRI/XhoI sites to generate the HIVBr27 plasmid, which was purified using the Endofree Plasmid Giga Kit (Qiagen) according to manufacturer s instructions. Selection of Variants from HIV-1 M-group Consensus Peptides The peptides p24( ), RT( ), RT( ) and int( ) were aligned with all HIV-1 circulating variants available at QUICK_ALIGN/QuickAlign.html. Sequences from the most frequent variants of each peptide were synthesized to perform immunological analysis. Subjects Cryopreserved peripheral-blood mononuclear cells (PBMC) were obtained from a cohort of HIV-1-infected individuals (n = 25) [43,44], as well as from healthy volunteers (n = 5), and were used for IFN-c ELISPOT assay. Infecting viral strains were characterized as previously described [45,46]. Clinical characteristics of enrolled patients are summarized in the table S1. Mice and Immunizations Six to eight week-old female BALB/c mice were used in this study. Six mice per group were injected intramuscularly, at weeks 0, 2 and 4, with HIVBr27 plasmid or empty pvax1. Each quadriceps was injected with 50 ml of DNA at a concentration of 1 mg/ml in saline such that each animal received a total of 100 mg of plasmid DNA per immunization. Two weeks after the last DNA immunization, mice were euthanized with CO 2. Spleen Cell Isolation for Immune Assays Two weeks after the last immunization, mice were euthanized and spleens were removed aseptically. After obtaining single cell suspensions, cells were washed in 10 ml of RPMI Cells were then resuspended in R-10 (RPMI supplemented with 10% of fetal bovine serum (GIBCO), 2 mm L-glutamine (Life Technologies), 1 mm sodium pyruvate (Life Technologies), 1% vol/vol non-essential amino acids solution (Life Technologies), 1% vol/vol vitamin solution (Life Technologies), 40 mg/ml of Gentamicin, 20 mg/ml of Peflacin and M2b-mercaptoetanol (Life Technologies). The viability of cells was evaluated using 0.2% Trypan Blue exclusion dye to discriminate between live and dead cells. Cell concentration was estimated with the aid of a Neubauer chamber and adjusted in cell culture medium. Detection of IFN-c Producing Human and Murine Cells by ELISPOT Assay PBMC ( cells/well) from HIV-1-infected patients and splenocytes ( cells/well) from HIVBr27 or pvax1 immunized mice were tested for their ability to secrete IFN-c after in vitro stimulation with 5 mm of individual or pooled HIV-1 peptides using ELISPOT assay. The ELISPOT assay was performed using human or murine IFN-c Becton Dickinson kit according to manufacturer s instructions. Spots were counted using an AID ELISPOT reader (Autoimmun Diagnostika GmbH). The number of antigen-specific T cells, expressed as spot-forming units (SFU)/ 10 6 PMBC or SFU/10 6 splenocytes, was calculated after subtracting negative control values (medium only). Responses in human ELISPOT assay for each patient were considered positive when $50 SFU/10 6 PBMC [47]. Responses in murine ELISPOT were considered positive when.15 SFU/10 6 splenocytes, which was calculated as the mean response +3 standard deviations (SD) of splenocytes from pvax1 immunized mice, stimulated with each peptide. CFSE-based Proliferation Assay To monitor the expansion and proliferation of HIV-1-specific T cells, splenocytes from HIVBr27 or pvax1 immunized mice were labeled with carboxyfluorescein succinimidyl ester (CFSE) [48]. Briefly, freshly isolated splenocytes were resuspended ( / ml) in PBS and labeled with 1.25 mm of CFSE (Molecular Probes) at 37uC for 10 minutes. The reaction was quenched with RPMI 1640 supplemented with 10% FBS and cells were washed before resuspending in RPMI 1640 at a density of /ml. Cells were cultured in 96 well round-bottomed plates ( /well in triplicate) for 5 days at 37uC and 5% CO 2 with medium only or 5 mm of HIV peptides. Positive controls were stimulated with 2.5 mg/ml of Concanavalin A (Sigma). Cells were then harvested, washed with 100 ml of FACS buffer (PBS with 0.5% BSA and 2 mm EDTA) and stained with anti-mouse CD3 phycoerythrin (PE), anti-mouse CD4 peridinin chlorophyll protein (PerCP) and anti-mouse CD8 allophycocyanin (APC) monoclonal antibodies (BD Pharmingen) for 45 minutes at 4uC. Samples were acquired on a FACSCanto flow cytometer (BD Biosciences) and then analyzed using FlowJo software (version 9.0.2, Tree Star). Fifty thousand events were acquired in a live lymphocyte gate. The percent of proliferating CD4 + and CD8 + CFSE low cells was determined in the CD3 + T-cell population. Positive proliferation of T cells was determined as CFSE low T cells. cutoff, which was calculated as median +3 SD of unspecific proliferative responses obtained with splenocytes from pvax1 immunized mice stimulated with HIV-1 peptides. Analysis of Polyfunctional HIV-1-specific T-cell Responses Splenocytes from immunized mice were labeled with CFSE as described above. CFSE-labeled cells were cultured in 96 well round-bottomed plates ( /well in triplicate) for 4 days at 37uC and 5% CO 2 in the presence of medium only or pooled HIV-1 peptides (5 mm). After incubation, cells were restimulated with 2 mg/ml anti-cd28 (BD Pharmingen), 5 mm of pooled HIV-1 peptides and Brefeldin A- GolgiPlugTM (BD Pharmingen) for the last 12 hours. After this period, cells were washed with FACS buffer and surface stained using the monoclonal antibodies anti-cd8-alexa700 and anti-cd4-percp for 30 minutes at 4uC. Cells were then fixed and permeabilized using the Cytofix/ CytopermTM kit (BD Pharmingen). Permeabilized cells were washed with Perm/Wash buffer (BD Biosciences) and stained with the monoclonal antibodies anti-cd3-apccy7, anti-il-2-pe, anti- PLOS ONE 3 September 2012 Volume 7 Issue 9 e45267

100 HIV-1 M-Group Epitope-Based Vaccine Figure 1. HLA-DR binding assay for M-group consensus peptides. Peptide binding assays were performed by incubating purified HLA-DR molecules (5 500 nm) with different concentrations of unlabeled peptide inhibitors and 1 10 nm 125 I-radiolabeled probe peptides for 48 h. Significant affinity threshold,1000 nm are shown in gray. A dash represents 50% inhibitory concentration (IC50) nm. doi: /journal.pone g001 TNFa-PECY7 and anti-ifnc-apc for 30 minutes at 4uC. Following staining, cells were washed twice and resuspended in FACS buffer. All antibodies were from BD Pharmingen. Samples were acquired on a FACSCanto flow cytometer (BD Biosciences) and then analyzed using FlowJo software (version 9.0.2, Tree Star, San Carlo, CA). Cells were gated on forward scatter (FSC)/side scatter (SSC) gate (500,000 events) followed by CD3 + gate and subsequent gates on CD4 + or CD8 + populations. After identification of CD4 + and CD8 + populations, we designed a gate in each positive population for IFN-c, TNF-a and IL-2 expression within or not the CFSE low gate. In addition, we used the Boolean gate (FlowJo software version 9.0.2, Tree Star) platform to create several combinations of the three cytokines (IFN-c, TNF-a and IL- 2) within CFSE low population resulting in seven distinct patterns. The percentages of cytokine producing cells within CFSE low population or not were calculated by subtracting background values. For each flow cytometry experiment performed in this paper, unstained and all single-color controls were processed to allow proper compensation. Statistical Analysis Statistical significance (p-values) was calculated by using Student s T test or Two-way ANOVA followed by Bonferroni post test. Statistical analysis and graphical representation of data was performed using GraphPad Prism version 5.0 software. Results Identification of Conserved, Multiple HLA-DR-binding Peptides in the HIV-1 M-Group Consensus Sequence The HIV-1 M-group consensus sequence was scanned with the TEPITOPE algorithm in order to identify multiple HLA- DR-binding (promiscuous) peptides. The identified peptides were aligned with consensus sequences from HIV-1 subtypes A1, B, C, D, F1, F2, G and H. Twenty-seven peptides, from 7 different HIV-1 proteins, showing each amino acid conserved among at least 50% of the aligned subtypes consensus sequences were selected (table 1). No Tat peptides were identified with the employed threshold. Env peptides were identified but excluded from further analysis due to the potential prejudice of T-cell responses targeting such protein. We also performed an alignment in order to evaluate whether the 27 peptides from M-group consensus sequence were highly conserved among HIV-1 circulating variants. According to the alignment, most of our peptides had 0 1 amino acid substitutions when compared to all sequences from HIV-1 circulating variants available at the Los Alamos HIV Database (table S2). We performed in vitro HLA-peptide binding assays to confirm the TEPITOPE accuracy and observed that each of the 27 peptides bound to an average of 12 out of the 17 tested HLA-DR molecules and that each HLA-DR molecule bound to an average of 19 out of the 27 peptides (figure 1). The peptides also bound to several HLA-DP and DQ molecules (table S3 and S4). We also performed murine MHC class II-peptide binding assays and observed that 21 and 6 peptides bound to IA d and IA b molecules, respectively (table 2). Thus, the scanning of HIV-1 M-group consensus sequence with TEPITOPE allowed the identification of highly conserved and promiscuous peptides. HIV-1 M-group Consensus Peptides are Antigenic in Natural HIV-1 Infection To evaluate whether the 27 predicted promiscuous peptides would be antigenic during natural infection we performed an IFNc ELISPOT assay using PBMC from twenty-five patients infected with different HIV-1 variants (Table S1). We observed that 7 out of the 13 Pol peptides were recognized at least once (figure 2A). Four out of the 6 Gag peptides were also recognized at least once (figure 2B). Among Vif, Rev, Vpr, Vpu and Nef, we observed recognition of 5 out of the 8 peptides (figure 2C). Overall, we found that 72% of the patients recognized at least 1 peptide and that each patient recognized an average of 2 peptides. In addition, our data shows that 16 out of the 27 peptides (60%) were recognized by HIV-1-infected patients, while no responses were observed in PBMC from healthy individuals (data not shown). Therefore, most of the conserved and promiscuous HIV-1 M- group consensus peptides identified with the TEPITOPE algorithm were showed to be antigenic in natural HIV-1 infection. PLOS ONE 4 September 2012 Volume 7 Issue 9 e45267

101 HIV-1 M-Group Epitope-Based Vaccine Figure 2. M-group consensus peptides are recognized by PBMC from HIV-1-infected patients. IFN-c ELISPOT assay was performed to evaluate T-cell responses against the 27 M-group consensus peptides by stimulating PBMC from patients infected with different variants of HIV-1, which are represented by colored symbols. A) IFN-c secretion against Pol peptides. B) IFN-c secretion against Gag peptides. C) IFN-c secretion against Vif, Rev, Vpr, Vpu and Nef peptides. B subtype-infected patients (n = 14), BC recombinant-infected patient (n = 1), BF recombinant-infected patients (n = 9), F subtype-infected patient (n = 1). Dotted lines represent IFN-c ELISPOT cutoff, which is $50 spots/10 6 cells. Only positive responses are shown in the graphs. doi: /journal.pone g002 PLOS ONE 5 September 2012 Volume 7 Issue 9 e45267

102 HIV-1 M-Group Epitope-Based Vaccine Figure 3. Immunization with HIVBr27 elicits T-cell responses in BALB/c mice. Two weeks after the last immunization with HIVBr27 or empty pvax1, pooled spleen cells from 6 BALB/c mice were cultured in the presence of individual or pooled HIV-1 peptides (5 mm). Frequency of IFN-c secreting cells (A) and proliferating CD4 + and CD8 + T-cells (B) against pooled HIV-1 peptides. C) Frequency of IFN-c secreting cells against individual HIV-1 peptides. Dotted line represents ELISPOT cutoff values. Data are shown as mean of three independent experiments for ELISPOT assays. Data from proliferation assay are representative of three independent experiments. doi: /journal.pone g003 A DNA Vaccine Encoding 27 Conserved and Promiscuous HIV-1 M-group Consensus Peptides Elicits Broad T-cell Responses in BALB/c Mice Given the fact that multiple peptides were able to bind to IA d molecules, we chose the BALB/c strain to undergo immunization. To evaluate the immunogenicity of the 27 conserved and promiscuous HIV-1 M-group consensus peptides we designed a DNA vaccine (HIVBr27) encoding the peptides in tandem. We assessed the magnitude and breadth of vaccine-elicited HIV-1 specific immune response by IFN-c ELISPOT and CFSE-based proliferation assays 2 weeks after the last immunization with HIVBr27 or empty pvax1. HIVBr27 immunization elicited a significantly higher number of IFN-c secreting cells when compared to empty pvax1 (figure 3A) and elicited 10% of both CD4 + and CD8 + proliferating T cells against the pool of 27 HIV-1 peptides (figure 3B). To evaluate the breadth of T-cell responses elicited by HIVBr27, we used individual HIV-1 peptides to stimulate splenocytes in vitro. Immunization of BALB/c mice resulted in IFN-c secretion against 11 peptides (figure 3C) and proliferation of CD4 + and CD8 + T-cells against 8 and 6 peptides, respectively (figure 4A and 4B). All peptides that stimulated T-cell proliferation also induced IFN-c secretion. In contrast, pvax1 immunized mice presented negligible numbers of IFN-c secreting and proliferating CD4 + and CD8 + T cells after incubation with the same HIV-1 peptides in all performed experiments. Thus, a DNA vaccine encoding the 27 identified peptides was immunogenic in BALB/c mice, eliciting broad T-cell responses. HIVBr27 Immunization Provides Cross-clade T-cell Immunity To evaluate whether HIVBr27 would provide cross-clade immunity, we synthesized the most frequent variant sequences of the 4 most immunogenic HIVBr27 encoded peptides, which are RT( ), RT( ), int( ) and p24( ) (table S5), and used them to stimulate splenocytes from immunized BALB/c mice. HIVBr27 immunization elicited comparable frequencies of IFN-c secreting cells against M-group peptides RT( ), int( ) and p24( ) when compared to most of their variants, while only IFN-c secretion against RT( ) B and D variants was reduced (figure 5A). The same pattern was observed for CD4 + and CD8 + T-cell proliferation (figure 5B and 5C). We evaluated the avidity of T-cell responses against the 4 HIVBr27 encoded peptides and their variants in IFN-c ELISPOT assay by serial peptide dilution and observed similar responses among all peptides in a concentration range from 5 mm to5pm (data not shown). Besides, most of the variant sequences kept the promiscuous profile observed in their parent peptides, according to the TEPITOPE algorithm (table S5). Taken together, these results suggest that HIVBr27 immunization elicits cross-clade T- cell responses of similar avidity, towards the most frequent variants of the M-group consensus peptides. HIVBr27 Immunization Elicits Polyfunctional CD4 + and CD8 + T-cell Responses To assess the functional profile of both CD4 + and CD8 + T-cell vaccine-induced responses we used multiparameter flow cytometry. The gating strategy is outlined in figure 6A. HIVBr27 PLOS ONE 6 September 2012 Volume 7 Issue 9 e45267

103 HIV-1 M-Group Epitope-Based Vaccine Figure 4. Immunization with HIVBr27 elicits CD4 + and CD8 + T-cell proliferation against multiple HIV-1 peptides. Two weeks after the last immunization with HIVBr27 or empty pvax1, pooled spleen cells from 6 BALB/c mice were cultured in the presence of HIV-1 peptides (5 mm) or medium only. Splenocytes were labeled with CFSE (1.25 mm) and cultured for 5 days. After staining with fluorochrome-labeled anti-cd3, -CD4 and - PLOS ONE 7 September 2012 Volume 7 Issue 9 e45267

104 HIV-1 M-Group Epitope-Based Vaccine CD8 monoclonal antibodies, cells were analyzed by flow cytometry. CFSE dilution on gated CD3 + CD4 + (A) or CD3 + CD8 + (B) cells was used as readout for antigen-specific proliferation. Data are representative of three independent experiments. doi: /journal.pone g004 immunization elicited higher frequencies of IFN-c and TNF-a producing CFSE low CD4 + T cells (4% and 3.4%, respectively) than CD8 + T cells (2% and 1.8%, respectively) against pooled HIV-1 peptides (figure 6B). It is of note that there were lower frequencies of IL-2 producing CFSE low CD4 + and CD8 + T-cells (0.34% and 0.36%, respectively) compared to other cytokine producing cells (figure 6B). To analyze whether HIVBr27 elicits polyfunctional T cells, Boolean combinations of cytokine-producing CFSE low CD4 + and CD8 + T cells were generated. HIVBr27 immunization elicited about 0.2% of IFN-c + IL-2 + TNF-a + CFSElow CD4 + T cells and less than 0.1% of IFN-c + IL-2 + TNF-a + CFSE low CD8 + T cells (figure 6C). We observed higher frequencies of both CFSE low CD4 + and CD8 + T cells able to produce the combination of IFN-c and TNF-a (2.5% and 1.12%, respectively) compared to triple cytokine producing CFSE low T cells (figure 6C). Overall, these data showed that HIVBr27 immunization can elicit polyfunctional CD4 + and CD8 + T cells that simultaneously proliferate and produce effector cytokines. Discussion In the present study, we designed a DNA vaccine encoding 27 highly conserved and promiscuous peptides from the HIV-1 M- group consensus sequence. This vaccine (HIVBr27) elicited broad, cross-clade and polyfunctional CD4 + and CD8 + T-cell responses in BALB/c mice. The 27 encoded peptides bound in vitro to multiple HLA-DR, -DP, -DQ and to murine IA b and IA d molecules. Furthermore, the peptides were shown to be antigenic in natural infection, being recognized by PBMC from patients infected with different HIV-1 variants. The 27 peptides identified with the TEPITOPE algorithm [34] bound in vitro to an average of 12 out of the 17 tested HLA-DR molecules and to several HLA-DP and -DQ molecules, showing that our peptides are highly promiscuous and that TEPITOPE is accurate in predicting promiscuous HLA-peptide binding. Moreover, each tested HLA-DR molecule bound on average to 19 out of the 27 peptides, which indicates that a vaccine encoding such peptides would have potential to induce T-cell responses against multiple targets in a wide proportion of the population. Notably, all peptides bound to at least one HLA class II molecule associated Figure 5. HIVBr27 immunization provides cross-clade immunity. Two weeks after the last immunization with HIVBr27 or empty pvax1, pooled spleen cells from 6 BALB/c mice were cultured in the presence of HIV-1 M-group consensus peptides (5 mm) (white bars) or their variants (colored bars), from diverse HIV-1 subtypes. Frequency of IFN-c secreting cells was assessed by ELISPOT assay (A) and proliferative CD4 + (B) and CD8 + (C) T-cell responses were assessed by CFSE dilution assay. Dotted lines represent ELISPOT or proliferation cutoff, which were calculated as median +3 SD of unspecific responses obtained with splenocytes from pvax1 immunized mice stimulated with HIV-1 peptides. Data are representative of two independent experiments. doi: /journal.pone g005 PLOS ONE 8 September 2012 Volume 7 Issue 9 e45267

105 HIV-1 M-Group Epitope-Based Vaccine PLOS ONE 9 September 2012 Volume 7 Issue 9 e45267

106 HIV-1 M-Group Epitope-Based Vaccine Figure 6. HIVBr27 immunization elicits polyfunctional CD4 + and CD8 + T cells. Two weeks after the last immunization with HIVBr27 or empty pvax1, pooled spleen cells from 6 BALB/c mice were collected, labeled with CFSE (1.25 mm) and cultured for 4 days in the presence of pooled HIV-1 peptides (5 mm) or medium only. On day 4, cells were pulsed for 12 hours with pooled peptides in the presence of Brefeldin A and costimulatory antibody (anti-cd28). A) Multiparameter flow cytometry strategy used to determine the frequency of IFN-c, IL-2 or TNF-a producing CFSE low CD4 + and CD8 + T cells. B) Frequency of IFN-c, IL-2 or TNF-a producing CFSE low CD4 + (left) and CD8 + (right) T-cells. C) Boolean combinations of IFN-c, IL-2 and TNF-a producing CFSE low CD4 + and CD8 + T cells from HIVBr27 immunized mice. Background responses detected in negative control tubes (cells stimulated with medium and cells from pvax1 immunized mice stimulated with pooled peptides) were subtracted from those detected in stimulated samples. Data are representative of three independent experiments. doi: /journal.pone g006 with AIDS protection, such as HLA-DRB1*0101, -DRB3*0202, - DRB1*13 and -DQB1*06 [49,50], which suggests that those peptides may be involved in protective immunity against HIV-1. The 27 peptides were also shown to be highly conserved when compared to sequences from all HIV-1 circulating variants available at the Los Alamos HIV Database, indicating that the identified peptides, or sequences highly homologous to them, are broadly represented across several HIV-1 subtypes. The peptides identified in this study showed to be antigenic during natural infection since we observed that 72% of the HIV-1- infected patients recognized at least one peptide and that 16 out of the 27 peptides were recognized. The responses were similar to those previously found by our group among clinically matched HIV-1-infected patients, towards HIV-1 B subtype consensus peptides [51]. It is of note that T-cell responses against M-group consensus Gag peptides were previously observed in HIV-1- infected patients [52]. In this study, 34% of the 15-mer M-group consensus peptides were recognized by HIV-1 B subtype-infected patients, while we observed recognition of 44% of our peptides by patients infected with the same subtype. Considering only our Gag peptides, we observed 50% of recognition by HIV-1 B subtypeinfected patients, suggesting that our TEPITOPE-selected conserved and promiscuous HIV-1 M-group consensus peptides were more frequently recognized than the 15-mer peptides employed in the previous study. HIVBr27 immunization led to broad T-cell responses in BALB/c mice, with recognition of 11 peptides in IFN-c ELISPOT assay, which is a desirable result since broad T-cell responses have been associated with protective vaccines [6,7,20]. We extended our immunological analysis and also observed broad CD4 + and CD8 + T-cell proliferation, against 8 and 6 peptides, respectively. Elicitation of HIV-1-specific proliferating CD8 + T cells with this vaccine designed to elicit CD4 + T-cell responses may have occurred due to the fact that long peptides can harbor CD8 + T- cell targets [53]. In addition, 24 out of the 27 peptides were predicted to bind to MHC class I molecules from BALB/c mice, according to the Pred Balb/c algorithm [54], which could explain the potential of HIVBr27 to induce CD8 + T-cell responses. The broad T-cell response observed in BALB/c mice, despite the fact that the peptides were selected for promiscuous HLA-DR binding, is supported by results from the in vitro MHC-peptide binding assay, in which 21 out of the 27 peptides effectively bound to IA d molecules. Moreover, previous studies have already reported cross-species recognition of peptides identified with TEPITOPE, probably by the fact that such algorithm may select for promiscuous peptides that share MHC class II binding motifs similar to many other human and non-human MHC class II molecules [33,37,55]. HIVBr27 immunization elicited comparable T-cell responses against M-group consensus peptides and their respective highfrequency variants, providing evidence that HIVBr27 is able to elicit cross-clade immunity across several HIV-1 subtypes, which is thought to be essential to afford protection against the global diversity of HIV-1. Moreover, we believe that our approach has brought significant improvement on the HIV-1 M-group consensus-based vaccine strategy, since we designed a vaccine encoding highly conserved and promiscuous CD4 + T-cell epitopes from 7 different viral genes, while no other study had addressed all these features together. Previous HIV-1 M-group consensus-based vaccines only focused on inducing cross-clade immune responses against Env [23,24,56]. These studies were able to elicit broader cross-subtype neutralizing antibodies and broader cross-clade T- cell responses when compared to immunogens based on single HIV-1 subtypes. However, their immunogens failed to include other HIV-1 proteins, as was done in HIVBr27. We conceive that multiepitopic approaches as HIVBr27 may facilitate the emergence of each peptide as individually immunogenic [25,42,57], being more attractive to broaden T-cell responses against HIV-1. Indeed, it has been shown that immunization with multiple peptides increased the breadth of the T-cell responses, as compared to those induced by whole proteins [58,59]. Given the frequent association of either Env-specific CD4 + or CD8 + T-cell responses with progression to AIDS [29 31,60,61], it is possible to speculate that an Env-based T-cell immunogen may not provide protection against AIDS progression. However, results from the RV144 trial showing predominant anti-env CD4 responses [2], and the correlation between Env-specific antibodies and protection against HIV-1 infection [62] suggest that a vaccine able to elicit strong CD4 + T-cell responses against Env would be valuable in order to improve antibody-mediated protection. The higher risk of HIV-1 infection observed in vaccinated subjects from the Step study may raise questions about the association of T-cell responses specific to other viral proteins with susceptibility of infection. However, even after extended analysis, it still seems to be an event related to the prior adenovirus 5-specific immunity [63]. Thus, it is important to carefully evaluate the inclusion of Env in HIV-1 vaccine formulations. Different approaches have been suggested to overcome HIV-1 genetic diversity. Ad26-vectored mosaic vaccines comprising Gag, Pol and Env were found to be more immunogenic than a similar vaccine based on M-group consensus sequence in rhesus macaques [64]. However, it is important to note that the M-group vaccine studied by the authors was based on whole HIV-1 proteins, unlike our approach. Another strategy proposed to elicit cross-clade T- cell responses was based on a chimaeric protein composed of conserved regions of Pol, Gag, Vif and Env from HIV-1 A, B, C and D subtypes [65]. This approach induced significant CD8 + T- cell responses in immunized mice, but limited CD4 + T-cell responses compared to those induced with HIVBr27 immunization. Indeed, HIVBr27 is the first vaccine specifically aimed to elicit broad and cross-clade CD4 + T-cell responses against HIV-1. Thus, we hypothesize that HIVBr27 may confer direct CD4 + T- cell-mediated antiviral immunity, as well as provide help for CD8 + T-cell-based vaccines, since CD4 + T cells are required for both enhancement of virus-specific CD8 + T-cell effector function and mobilization of these cells to infected tissues [66 68]. HIVBr27 immunization was also able to elicit high frequencies of both polyfunctional CD4 + and CD8 + T cells, which simultaneously proliferate and produce effector cytokines such as IFN-c and TNF-a. We observed a low but detectable frequency of CD4 + PLOS ONE 10 September 2012 Volume 7 Issue 9 e45267

107 HIV-1 M-Group Epitope-Based Vaccine T cells able to proliferate and produce IFN-c, IL-2 and TNF-a. These results indicate that HIVBr27 has potential to induce T-cell responses with a functional profile related to natural control of viral replication and non progression to AIDS, as previously observed in HIV-1-infected patients [50,69 71] and vaccinemediated protection in NHP models [9,72]. We hereby demonstrate that immunization with the HIVBr27 vaccine encoding multiple conserved and promiscuous HIV-1 M- group consensus peptides is able to elicit broad, cross-clade and polyfunctional T-cell responses. This vaccine concept may cope with HIV-1 genetic diversity as well as provide increased population coverage, which are desirable features for an efficacious strategy against HIV-1/AIDS. Supporting Information Table S1 Clinical characteristics of HIV-1-infected patients. 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109 REFERENCES CONTENT ALERTS T Cells Target APOBEC3 Proteins in Human Immunodeficiency Virus Type 1-Infected Humans and Simian Immunodeficiency Virus-Infected Indian Rhesus Macaques Stéphane Champiat, Keith E. Garrison, Rui André Saraiva Raposo, Benjamin J. Burwitz, Jason Reed, Ravi Tandon, Vanessa A. York, Laura P. Newman, Francesca A. Nimityongskul, Nancy A. Wilson, Rafael R. Almeida, Jeffrey N. Martin, Steven G. Deeks, Michael G. Rosenberg, Andrew A. Wiznia, Gerald E. Spotts, Christopher D. Pilcher, Fredrick M. Hecht, Mario A. Ostrowski, Jonah B. Sacha and Douglas F. Nixon J. Virol. 2013, 87(11):6073. DOI: /JVI Published Ahead of Print 27 March Updated information and services can be found at: These include: This article cites 36 articles, 18 of which can be accessed free at: Receive: RSS Feeds, etocs, free alerts (when new articles cite this article), more» Downloaded from on June 4, 2014 by USP/REITORIA/SIBI Information about commercial reprint orders: To subscribe to to another ASM Journal go to:

110 T Cells Target APOBEC3 Proteins in Human Immunodeficiency Virus Type 1-Infected Humans and Simian Immunodeficiency Virus- Infected Indian Rhesus Macaques Stéphane Champiat, a,b Keith E. Garrison, a,c Rui André Saraiva Raposo, a Benjamin J. Burwitz, d Jason Reed, d Ravi Tandon, a Vanessa A. York, a Laura P. Newman, e Francesca A. Nimityongskul, e Nancy A. Wilson, e Rafael R. Almeida, a,f,g Jeffrey N. Martin, h Steven G. Deeks, h Michael G. Rosenberg, i Andrew A. Wiznia, i Gerald E. Spotts, j Christopher D. Pilcher, j Fredrick M. Hecht, j Mario A. Ostrowski, k Jonah B. Sacha, d,l Douglas F. Nixon a Division of Experimental Medicine, Department of Medicine, University of California San Francisco, San Francisco, California, USA a ; Université Pierre et Marie Curie-Paris 6, Medical School, Pitié-Salpêtrière Hospital, Paris, France b ; Department of Biology, Saint Mary s College of California, Moraga, California, USA c ; Vaccine & Gene Therapy Institute, Oregon Health & Science University, Beaverton, Oregon, USA d ; Department of Pathology and Laboratory Medicine, University of Wisconsin Madison, Madison, Wisconsin, USA e ; Laboratory of Clinical Immunology and Allergy, LIM60, Division of Clinical Immunology and Allergy, Department of Medicine, University of São Paulo School of Medicine, São Paulo, São Paulo, Brazil f ; Institute for Investigation in Immunology (INCT), São Paulo, São Paulo, Brazil g ; Epidemiology and Prevention Interventions Center, Division of Infectious Diseases, and The Positive Health Program, San Francisco General Hospital, University of California San Francisco, San Francisco, California, USA h ; Jacobi Medical Center, Albert Einstein College of Medicine, Bronx, New York, USA i ; Positive Health Program, Department of Medicine, San Francisco General Hospital, University of California San Francisco, San Francisco, California, USA j ; Department of Immunology, University of Toronto, Toronto, Ontario, Canada k ; Division of Pathobiology & Immunology, Oregon National Primate Research Center, Oregon Health & Science University, Beaverton, Oregon, USA l APOBEC3 proteins mediate potent antiretroviral activity by hypermutating the retroviral genome during reverse transcription. To counteract APOBEC3 and gain a replicative advantage, lentiviruses such as human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) have evolved the Vif protein, which targets APOBEC3 proteins for proteasomal degradation. However, the proteasome plays a critical role in the generation of T cell peptide epitopes. Whether Vif-mediated destruction of APOBEC3 proteins leads to the generation and presentation of APOBEC3-derived T cell epitopes on the surfaces of lentivirus-infected cells remains unknown. Here, using peptides derived from multiple Vif-sensitive APOBEC3 proteins, we identified APOBEC3-specific T cell responses in both HIV-1-infected patients and SIV-infected rhesus macaques. These results raise the possibility that these T cell responses may be part of the larger antiretroviral immune response. Viral sequence diversity is the Achilles heel of traditional vaccine approaches, as exemplified by influenza vaccinology. The influenza vaccine must change yearly in anticipation of the predominant circulating viral strains, which differ from one another byonly1to2%(1, 2). In contrast, circulating human immunodeficiency virus type 1 (HIV-1) strains differ from each other by 20% in the more conserved proteins and up to 35% in the Env protein (1, 2). This enormous sequence diversity is a major stumbling block to the development of a conventional HIV-1 vaccine. Indeed, a neutralizing antibody-based vaccine has proven elusive, in part due to the extremely high sequence variation of the HIV-1 Env protein (3). While CD8 T cell-based vaccines directed against more-conserved viral proteins have shown some ability to blunt viral replication (4, 5), they are also hindered by HIV-1 sequence variation (6). Even minor variations in sequence can have a dramatic effect on CD8 T cell efficacy, as just one amino acid change can abrogate recognition and suppression of virus replication (7 9). Additionally, HIV-1 rapidly mutates to escape effective cytotoxic T lymphocyte (CTL) responses, thereby raising the possibility that any effective vaccine will only have a transient effect (6, 10, 11). On a population level, CD8 T cells are driving HIV-1 evolution toward fixation of epitope escape variants that are more likely to evade the immune responses of the population in which they circulate (12, 13). Therefore, a successful HIV-1 vaccine must overcome the formidable challenge of HIV-1 sequence diversity. In this study, we sought to determine whether invariant self-antigens overexpressed within an HIV-1-infected cell could act as potential immune targets for vaccine development. Specifically, we examined HIV-infected patients and simian immunodeficiency virus (SIV)-infected macaques for the presence of APOBEC-specific T cell responses. Mammalian host cells have developed intrinsic mechanisms to prevent lentiviral replication, as well as to maintain genomic stability by restricting the movement of retroelements. These mechanisms include postentry interference by tripartite motif-containing protein 5 (TRIM5 ), transcriptional silencing through DNA methylation, posttranscriptional silencing via RNA interference, tetherin, and mutational inactivation of elements in the course of their retrotransposition cycle by cellular cytosine deaminases (14, 15). The apolipoprotein B mrna-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) family of cytidine deaminases consists of 7 members (APOBEC3A to APOBEC3H) in humans, all of which are encoded on the same gene cluster of chromosome 22. These enzymes are expressed in the majority of human cells and have been extensively studied since the discovery that Received 5 March 2012 Accepted 12 March 2013 Published ahead of print 27 March 2013 Address correspondence to Douglas F. Nixon, douglas.nixon@ucsf.edu, or Jonah B. Sacha, sacha@ohsu.edu. S.C. and K.E.G. contributed equally to this article. Copyright 2013, American Society for Microbiology. All Rights Reserved. doi: /jvi The authors have paid a fee to allow immediate free access to this article. Downloaded from on June 4, 2014 by USP/REITORIA/SIBI June 2013 Volume 87 Number 11 Journal of Virology p jvi.asm.org 6073

111 Champiat et al. APOBEC3G acts as a viral restriction factor in HIV-1 infection (16). Since that time, multiple additional APOBEC3s have been implicated in HIV restriction (17 19). APOBEC3G is packaged into budding HIV-1 virions through an interaction with the nucleocapsid region of HIV-1 Gag, and its proclivity for binding single-stranded nucleic acids facilitates this process (20, 21). Following HIV-1 infection of a target cell, the viral RNA genome is uncoated, and reverse transcriptase generates a single strand of DNA complementary to the viral genome. During this transcription process, reverse transcriptase also degrades the viral RNA strand through its RNase H activity, leaving only the single strand of cdna (20). APOBEC3G exerts its enzymatic activity on this single-stranded DNA, mutating cytosines to uracils. These extensive mutations can cause degradation of the viral genome or inhibit viral replication due to altered reading frames. Alternatively, APOBEC3G has been shown to inhibit HIV-1 replication independent of its enzymatic function. First, it can impair reverse transcriptase activity by blocking the binding of trna Lys3 (the primer that initiates reverse transcription). Second, it can stifle the cleavage of trna Lys3 from the single-stranded DNA intermediate, leading to aberrant viral DNA ends (19). Finally, it can bind to HIV-1 integrase, a component of the viral preintegration complex, suggesting that it may obstruct nuclear homing of provirus (19). HIV-1 expresses 6 accessory proteins that are integral to its replication and persistence. One of these accessory proteins is the viral infectivity factor (Vif) protein. The function of Vif was initially elucidated from experiments showing that Vif-deficient HIV could replicate in certain cells (permissive), but not in others (nonpermissive). Infection of a hybridoma of these two cell types with Vif-deficient HIV-1 yielded noninfectious virions, implying that a host cellular restriction factor was obstructing further infections. Subsequent analysis of two highly related cell lines, one permissive and the other nonpermissive, using a cdna subtraction strategy revealed that this cellular restriction factor was APOBEC3G (22). Since this discovery, Vif has been shown to ubiquitinate APOBEC3G, leading to degradation by the proteasome. Indeed, the levels of APOBEC3G are drastically reduced in virion-producing cells infected with HIV-1, and APOBEC3G is not packaged into progeny virions in the presence of Vif (23). Vif has been shown to inhibit APOBEC3G and APOBEC3F, but not APOBEC3B (19, 24). Thus, although the physiological role of APOBEC3 proteins is not fully understood, they represent an ancient form of antiviral or antiretroelement defense in mammals (19). The proteasome is an important first step in the antigen presentation pathway for HLA class I peptide epitopes recognized by CD8 T cells. We hypothesized that HIV-1 infection would give rise to CD8 T cell responses against APOBEC3 proteins, due to increased Vif-triggered proteasomal processing and HLA class I presentation. In a cell not infected by HIV-1, there would be limited presentation of APOBEC3-derived peptide epitopes on the surface as part of the normal repertoire of self-antigens presented by major histocompatibility complex class I (MHC-I) molecules. The immense sequence diversity of HIV-1 remains a major roadblock to a prophylactic HIV-1 vaccine. Targeting CD8 T cells to HIV-1-infected cells by their higher expression of APOBEC3 antigens may be a novel way to circumvent the obstacle presented by viral sequence diversity. Here, we present the first data showing APOBEC3-specific T cell responses in both HIV-1- infected humans and SIV-infected rhesus macaques. MATERIALS AND METHODS Peptide antigens for humans. We used in silico T cell immunogenicity prediction methods to identify peptide epitopes for the HLA-B*58, -A*2, and -B*7 superfamilies within APOBEC3F and APOBEC3G. Peptides were identified within the APOBEC3F and APOBEC3G proteins with NetCTL 1.2 software ( Top scoring epitopes for the HLA-B*58, -A*2, and -B*7 supertypes were selected for peptide synthesis. We selected peptides on the basis of their NetCTL software affinity score and their capacity to be presented by several HLA supertypes and excluded one peptide similar to the activationinduced cytidine deaminase protein (AID). Priority was given to peptides shared by APOBEC3F and APOBEC3G proteins. A total of 12 peptides in total were synthesized and tested together in a peptide pool or individually in the assay. An APOBEC3A/B peptide pool consisting of the four top scoring epitopes for HLA-B*58, -A*2, and -B*7 was also used where specified. Peptide antigens for rhesus macaques. For the studies in macaques, we used the following two sets of peptides: predicted MHC-I-binding peptides for rhesus APOBEC3F and 15-mer peptides overlapping rhesus APOBEC3C, APOBEC3G, and APOBEC3H by 11 amino acids. For predicted binders, we used the MHCPathway Macaque algorithm ( to generate 36 CD8 T cell epitopes derived from the rhesus APOBEC3F protein predicted to bind two common MHC-I molecules, Mamu-A1*001:01 and Mamu-A1*002:01. We then lumped these peptides into 3 Mamu-A1*001:01 pools and 6 Mamu-A1*002:01 pools, where each pool contained between 2 and 8 peptides. All peptides were synthesized by either Pepscan or Genscript and resuspended in 100% dimethyl sulfoxide (DMSO). Study participants. In this study, we measured T cell reactivity to APOBEC3F and APOBCEC3G peptides in the following groups: (i) lowrisk healthy volunteers (n 33); (ii) vertically exposed, but uninfected children (n 7); (iii) vertically exposed, HIV-1-infected children (n 73); (iv) long-term nonprogressors (LTNP), who were defined as having asymptomatic, untreated HIV-1 infection for at least 7 years with no consistent decline in peripheral blood CD4 count and low or undetectable levels of plasma viremia ( 10,000 copies/ml, bdna) (n 7); (v) HIV-1- infected adults with primary HIV-1 infection (n 13); (vi) chronically infected adults not treated with an antiretroviral agent with viral loads below 5,000 copies RNA/ml (viral-load-controlled virus in the absence of therapy ( controllers ; n 19); (vii) non-antiretroviral-treated individuals who had higher levels of viremia ( noncontrollers ; n 21); and (viii) individuals treated with highly active antiretroviral therapy (HAART), defined by the treatment-mediated maintenance of an undetectable viral load ( HAART suppressed ; n 20). HIV-1-negative controls were obtained from purchased buffy coats from low-risk donors attending the Stanford Blood Bank Center. Blood donors are asked about risk activity and are included or excluded as a blood donor based upon criteria established by blood banks. HIV-1-positive donors are from studies of adults with primary infections (Options cohort) and chronic HIV-1 infection (SCOPE cohort) at the University of California San Francisco (UCSF) and individuals with vertical infection under the care of clinicians at the Jacobi Hospital in the Bronx in New York, New York. All participants were enrolled in institutional review board (IRB)-approved studies, conducted under the directive of the Declaration of Helsinki. Nonhuman primates. The animals in this study were Indian rhesus macaques (Macaca mulatta) from either the Wisconsin or Oregon National Primate Research Center colony. The animals were infected with SIV for other unrelated studies and were cared for according to the regulations and guidelines of the Institutional Animal Care and Use Committee of the University of Wisconsin or the Oregon Health & Science University. Downloaded from on June 4, 2014 by USP/REITORIA/SIBI 6074 jvi.asm.org Journal of Virology

112 APOBEC3-Specific T Cells in HIV-1 and SIV Infection TABLE 1 T cell responses against APOBEC3 peptides HIV status and group No. of subjects Mean CD4 count (/mm 3 ) Mean viral load (no. of copies/ml) ELISPOT analysis. Peptides were tested, as previously described, in a gamma interferon (IFN- ) enzyme-linked immunosorbent spot assay (ELISPOT) using either fresh or cryopreserved peripheral blood mononuclear cells (PBMC) (25). Peptides were tested in duplicate wells at a concentration of 10 g/ml or 10 M (either individually or in pools) with 100,000 PBMC per well. The total numbers of spots for duplicate wells were averaged, and all the numbers of spots were normalized to the numbers of IFN- spot-forming cells (SFC) per PBMC. The numbers of spots from control wells (no stimulation) were subtracted to determine the responses to each peptide. Responses were considered positive if they exceeded two times the standard deviation of the background count as previously described (8). We did not consider responses of 50 SFC per PBMC as positive responses. Flow cytometry. Cryopreserved PBMC from chronically HIV-1-infected individuals or PBMC/bronchoalveolar lavage (BAL) cells were stimulated with or without 10 g/ml APOBEC3F/G or APOBEC3A/B peptide pools for 6 h with 1 g/ml costimulatory anti-cd28 and anti- CD49d antibodies (Biolegend) and in the presence of 5 g/ml brefeldin A. An exogenous cytomegalovirus (CMV) pp65 peptide pool was used as a positive control. The cells were stained with fluorophore-conjugated antibodies to CD3 (clone UCHT1; Beckman Coulter), CD4 (clone RPA-T4; BD Pharmingen), CD8 (clone 3B5; Life Technologies), and IFN- (clone B27; BD Pharmingen) to determine phenotype and function, and an amine dye was used to discriminate between live and dead cells. Data were acquired with a BD LSR-II system. At least 100,000 events were collected and analyzed with FlowJo software (TreeStar, Inc.). Generation of an APOBEC-specific cell line in vitro. CD8 T cell lines were produced as described previously (26). Briefly, PBMC were isolated using Ficoll-Paque Plus (Amersham Biosciences) density centrifugation. CD8 cells were isolated using CD8 microbeads and columns purchased from Miltenyi Biotec and used according to the manufacturer s instructions. Isolated CD8 cells were repeatedly stimulated with autologous, irradiated B lymphoblastoid cells pulsed with the peptide of interest. Cells were cultured in R (RPMI 1640 medium containing 15% fetal calf serum [FCS] and 100 U/ml interleukin 2 [IL-2]). Restricting allele assay. Transient expression of cloned MHC class I cdna was achieved by electroporation of plasmid DNA into the MHC class I-deficient human B cell line Briefly, 5 g of plasmid DNA encoding the MHC-I allele of interest was added to cells in 100 l of Nucleofector solution C and electroporated using program G-16 on a Nucleofector I device (Amaxa, Köln, Germany). Maximum cell No. of responders against APOBEC3 pool Mean APOBEC3 pool response (no. of SFU/10 6 PBMC) a Range of APOBEC3 pool responses (SFU/10 6 PBMC) a HIV infected Chronic infection LTNP b , Natural controllers HAART suppressed Viremic , Primary infection , Children with chronic infection , , HIV-exposed children but uninfected Nonexposed, healthy HIVnegative adults a SFU, spot-forming units. The background SFU have been subtracted from the values shown. b LTNP, long-term nonprogressors. %of responders surface expression occurred 4 days postelectroporation. MHC class I surface expression on stable and transient MHC class I transfectants was measured by W6/32 antibody surface staining. Staining was also performed on cells as a negative control and on immortalized macaque B cell lines as a positive control. These cells were incubated with the peptide of interest for 1.5 h at 37 C and then washed three times with warm phosphate-buffered saline (PBS) to remove excess peptide. The freshly washed peptide-pulsed cells were then incubated with CD8 T cell lines specific for the peptide of interest for intracellular staining (ICS) or ELISPOT as described above to determine the restricting MHC-I molecule. MHC-I tetramer production and staining. MHC class I tetramers were constructed with minor modifications as previously described (8, 27). Ex vivo MHC class 1 tetramer stains were performed on CD8 T cell lines or cryopreserved PBMC as previously described (8). Briefly, cells were stained with 5 l of 0.1-mg/ml tetramer stocks, 3 l ofcd3 conjugated to fluorescein isothiocyanate (FITC) (clone SP34-2; BD Biosciences), and 5 l of CD8 conjugated to peridinin chlorophyll protein (PerCP) (clone SK1; BD Biosciences) in approximately 100 l ofr10 (RPMI 1640 with 10% FCS). After the cells were fixed in 1% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA), approximately to lymphocyte-gated events were acquired on a FACSCalibur (BD Biosciences) and analyzed using FlowJo (TreeStar, Inc.). RESULTS APOBEC3-specific responses in HIV-1-infected patients. APOBEC3F and APOBEC3G are both potent inhibitors of HIV-1 replication and sensitive to HIV-1 Vif-induced proteasomal degradation (16, 19, 22). Therefore, we focused on these two proteins in our initial studies. Using the NetCTL MHC-I binding algorithm ( we generated peptide epitopes from APOBEC3F and APOBEC3G predicted to bind tightly to members of the common HLA-B2*8, -A*2, and -B*7 supertypes. We selected the top two predicted epitopes for each HLA allele derived from each APOBEC3 protein, resulting in 12 predicted APOBEC3 peptides. We then combined these peptides into a single APOBEC3 peptide pool and screened a cohort of healthy adults not infected with HIV-1, chronically HIV-1-infected adults, exposed uninfected children, and chronically HIV- Downloaded from on June 4, 2014 by USP/REITORIA/SIBI June 2013 Volume 87 Number 11 jvi.asm.org 6075

113 Champiat et al. Downloaded from on June 4, 2014 by USP/REITORIA/SIBI FIG 1 APOBEC3-specific T cell responses in HIV-1-infected children. (A) PBMC from 13 chronically HIV-1-infected children were screened against 9 of the 12 APOBEC3 peptides present in the APOBEC3 peptide pool. Results are shown as the mean numbers of spot-forming units (SFU) per million PBMC of duplicate wells in the IFN- ELISPOT, and background SFUs have been subtracted from the values. The sequences of the individual peptide epitopes are shown on the x axis. (B) Examples of wells from an IFN- ELISPOT assay. One hundred thousand PBMC from three chronically infected adults were left unstimulated (Media) or stimulated with phytohemagglutinin (PHA), CMV pp65 peptide pool, APOBEC3A/B peptide pool, or APOBEC3F/G peptide pool. The number of spotforming units/10 5 cells are indicated in the lower left corner for each well. (C) IFN- production by the CD8 lymphocyte population from PBMC isolated from three chronically HIV-1-infected patients from the SCOPE cohort. The percentage of cells within the gate is shown in each plot: unstimulated, stimulated with a pool of CMV pp65 peptides, stimulated with a pool of APOBEC3A/B peptides, and stimulated with APOBEC3F/G pool. 1-infected children using IFN- ELISPOT (Table 1). We observed little or no response in the healthy, non-hiv-1-infected adults and exposed uninfected children. In contrast, we observed weak, yet detectable responses in the chronically HIV-1-infected adults. Intriguingly, we observed the strongest APOBEC3-specific responses in the long-term nonprogressors (5/7), with a mean of 486 SFC/10 6 PBMC (Table 1). In primary HIV-1-infected subjects, 5/13 had responses, albeit at lower magnitude, with a mean of jvi.asm.org Journal of Virology

114 APOBEC3-Specific T Cells in HIV-1 and SIV Infection SFU/10 6 PBMC. HIV-1 noncontrollers had the lowest mean T cell response to the APOBEC3 pool (34 SFC/10 6 PBMC), although there was no substantial difference compared to the responses of the HAART-suppressed group (54 SFC/10 6 PBMC) or the controllers (45 SFC/10 6 PBMC). There were 13/73 responders in the group of HIV-1-infected children (88 SFC/10 6 PBMC) (Table 1). APOBEC3-specific responses in HIV-1-infected patients are CD8 T cell mediated. To further investigate these APOBEC3- specific T cell responses, we selected 13 chronically HIV-1-infected children with responses against the APOBEC3F/G peptide pool and tested them against individual APOBEC3 peptides using IFN- ELISPOT (Fig. 1A). Figure 1B illustrates typical results of a IFN- ELISPOT of three chronically infected adults. We then tested PBMC IFN- production by flow cytometry in three chronically infected adults comparing APOBEC3F/G-specific CD8 T cell responses to those generated by a known ubiquitous T cell immunogen, CMV pp65 (Fig. 1C). In addition, a pool consisting of four predicted peptides derived from Vif-insensitive APOBEC3A/B proteins was included as a negative control. IFN- -producing T cells were shown to be CD8, and there was little to no response to APOBEC3A/B peptide pools, suggesting that Vifinduced proteasomal destruction of APOBEC3F/G but not APOBEC3A/B, may occur in HIV-1-infected cells (Fig. 1C). Together with the responses observed in LTNP, these data suggest that APOBEC3-specific T cell responses may expand during HIV-1 infection. No CD4 T cell responses were identified using flow cytometry from these human studies, although we cannot rule out the possibility that some APOBEC3-specific IFN- production in the ELISPOT assay was produced by CD4 T cells. Identification of APOBEC3-specific T cell responses in SIVinfected rhesus macaques. Given that HIV-1-infected patients, LTNP in particular, mounted APOBEC3G- and APOBEC3F-specific CD8 T cell responses, we next investigated whether SIVinfected Indian rhesus macaques might also target SIVmac239 Vif-sensitive APOBEC3 protein sequences (28). To this end, we screened PBMC from a total of 52 chronically SIV-infected Indian rhesus macaques for T cell responses against rhesus APOBEC3F, APOBEC3C, APOBEC3H, and APOBEC3G by IFN- ELISPOT (Fig. 2A, B, and C). The majority of animals did not respond to any of the APOBEC3 peptides, but similar to what we had observed in HIV-1-infected humans, we detected the presence of low-magnitude responses, particularly against APOBEC3G (Fig. 2C). Because the strongest APOBEC3-specific responses in humans were observed in LTNP, we focused subsequent analysis predominantly on the small number of LTNP Indian rhesus macaques in our cohort, which have spontaneously controlled SIV replication to low levels. Interestingly, we observed the highest frequency response against APOBEC3C pool B in an animal that had previously undergone experimental depletion of CD8 cells (Fig. 3A). To confirm this high frequency APOBEC3C T cell response, we next screened PBMC from this animal against the individual 15-mer peptides in APOBEC3C pool B and identified a single 15-mer peptide eliciting a high frequency IFN- T cell response (Fig. 3A). We also confirmed a high frequency T cell response against APOBEC3G in a second LTNP macaque and identified a single 15-mer peptide triggering this response (Fig. 3B). To further characterize that APOBEC3G-specific T cell response, we performed cytokine flow cytometry. Surprisingly, we identified that this APOBEC3G-specific response was CD4 T cell mediated FIG 2 APOBEC3 responses in chronically SIV-infected rhesus macaques. (A to C) PBMC from SIV-infected rhesus macaques were screened against peptides derived from rhesus APOBEC3F and predicted to bind Mamu- A1*001:01 or Mamu-A1*002:01 (A), against overlapping 15-mer peptides spanning rhesus APOBEC3C and APOBEC3H (B), or against overlapping 15-mer peptides spanning rhesus APOBEC3G (C). The mean of each response minus the background from each animal is plotted. The broken line indicates the threshold for positivity. (Fig. 3C). Thus, we determined that both CD4 and CD8 T cells target APOBEC3 proteins. Finally, we identified a LTNP macaque with no detectable viral load that mounted a T cell response against a pool of predicted Mamu-A1*002:01-binding, APOBEC3F-derived peptides (Fig. 4A). Although this response was not robust, the magnitude of this response was similar to the magnitude of the Mamu-A1*002:01- restricted immunodominant SIVmac239 Gag GY9 response in this animal (Fig. 4A). Next, we stimulated PBMC from this animal with the predicted APOBEC3F Mamu-A1*002:01 pool to generate an APOBEC3-specific cell line in vitro. Using this cell line, we mapped the APOBEC3 pool response to a single predicted CD8 T cell epitope, APOBEC3F LY11 (Fig. 4B). Using single MHC-I-expressing transfectants, we then confirmed the restricting allele of APOBEC3F LY11-specific CD8 T cells to be Mamu-A1*002:01 as predicted (data not shown). As we had Downloaded from on June 4, 2014 by USP/REITORIA/SIBI June 2013 Volume 87 Number 11 jvi.asm.org 6077

115 Champiat et al. identified both the minimal optimal peptide epitope and its restricting allele, we generated a CD8 T cell tetramer that specifically stained our in vitro cell line (Fig. 4C). We next used a Vif WY8 tetramer and our APOBEC3 LY11 tetramer reagent to longitudinally examine the immune response in this LTNP macaque to both APOBEC3F and the SIVmac239 Vif protein. As expected, tetramers for APOBEC3F LY11 stained only cells specific for that epitope and not Vif WY8. We noted that while CD8 T cells specific for both Vif and APOBEC3F were detected at low frequencies, they expanded and contracted asynchronously with respect to each other (Fig. 4C). Furthermore, consistent with our other observations, APOBEC3-specific CD8 T cells appeared to be in the peripheral blood compartment. DISCUSSION We show here for the first time that APOBEC3-specific CD8 T cells exist in humans infected with HIV-1 and in macaques infected with SIV. It is intriguing that the long-term nonprogressors had the highest magnitude responses to the APOBEC3 peptides, suggesting that they may be involved in helping control HIV-1 replication. Within the remaining HIV-1-infected subjects, APOBEC3-specific T cell responses were detected at lower magnitude and there was a trend toward higher responses within the controller and HAART-suppressed groups. The data suggest that immune responses to APOBEC3 proteins may be triggered by lentivirus infection. Indeed, we found APOBEC3-specific T cell responses in healthy individuals at a very low frequency. Since APOBEC3-derived peptides will stabilize nascent HLA molecules and be transported to the surface of any nucleated cell at low levels as part of the repertoire of self-peptides processed within the cell, we hypothesize that APOBEC3-specific T cells are present normally at low frequency as part of the normal low-level self-peptide T cell repertoire. However, we also hypothesize that in the inflammatory context of HIV-1 or SIV infection, Vif-induced degradation of APOBEC3 will result in a marked increase in the amount of APOBEC3-derived peptides on the surfaces of virus-infected cells. Thus, while APOBEC3 peptides will be present at low levels on the surfaces of healthy cells, they should be present at much higher levels in virus-infected cells due to Vif-induced destruction of APOBEC3. Alternatively, the generalized immune deregulation in the context of HIV-1 infection might provide cytokine-driven expansion of self-antigen responses. Recognition of APOBEC3-derived peptides is theoretically independent of the sequence of the infecting viral sequence. This could be used to circumvent the obstacle of virus sequence diversity by targeting a stable target induced by the viral replication cycle. To escape APOBEC3-specific T cell immune responses, HIV-1 would need to mutate Vif such that it would no longer interact with and degrade APOBEC proteins. Such a mutation would render the virus vulnerable to APOBEC3-induced hypermutation. APOBEC3 is a self-protein that is widely expressed (22, 29). Downloaded from on June 4, 2014 by USP/REITORIA/SIBI FIG 3 Confirmation of APOBEC3-specific responses in SIV-infected rhesus macaques. (A) PBMC from LTNP rhesus macaques were screened against the APOBEC3C pool B and each individual 15-mer peptide within that pool. Results are shown as means plus standard deviations (error bars) of duplicate wells. (B) PBMC from LTNP rhesus macaques were screened against the APOBEC3G pool B and each individual 15-mer peptide within that pool. Black bars indicate positive responses. Results are shown as means plus standard deviations of duplicate wells. The broken line represents the threshold for positivity. (C) Bronchoalveolar lavage cells were stimulated with media (no stimulation) or the rhesus APOBEC3G pool B single 15-mer peptide identified in panel B. The percentages of cells positive for IFN- and tumor necrosis factor alpha (TNF- ) are shown jvi.asm.org Journal of Virology

116 APOBEC3-Specific T Cells in HIV-1 and SIV Infection FIG 4 A LTNP macaque targets APOBEC3F. (A) PBMC from a LTNP (with no detectable plasma virus) were screened against APOBEC3F pools predicted to bind Mamu-A1*001:01 or Mamu-A1*002:01, and the SIVmac239 Gag GY9 epitope in an IFN- ELISPOT. Results are shown as means plus standard deviations of duplicate wells. (B) A CD8 T cell line was generated by multiple rounds of stimulation using the APOBEC3F Mamu-A1*002:01 pool C. The cell line reacts against the autologous B lymphocyte cell line (BLCL) presenting both the pool and the single epitope APOBEC3F LY11. Graphs were generated by gating on CD8 cells. (C) Tetramers specific for either APOBEC3F LY11 or SIVmac239 Vif WY8 were used to stain cryopreserved PBMC, an in vitro CD8 T cell line specific for Mamu-A1*002:01-restricted APOBEC3F LY11, and an in vitro CD8 T cell clone specific for Mamu-A1*002:01-restricted SIVmac239 Vif WY8. Dot plots were generated by gating on CD3 or CD8 cells. The percentages of tetramer-positive events are shown. rhaj11, recombinant human AJ11; WPI, weeks post-sivmac239 infection. Self-directed T cell responses raise the possibility of autoimmunity. However, as we have shown in our data, elite controllers of HIV-1 infection make anti-apobec3-specific T cell responses without apparent deleterious clinical autoimmune manifestations. The anti-apobec3 T cell responses observed do not appear to have led to any overt systemic autoimmunity in these patients or primates. The immune system exists with continuous surveillance and recognition of self-antigens, which in the absence of danger signals does not result in an immunopathogenic state (30, 31). Analogous to a cancer cell with aberrant protein expression, an HIV-1-infected cell will express APOBEC3-derived peptide epitopes at a higher frequency than uninfected cells. In the cancer field, many studies have approached cancer immunotherapy with self-antigens expressed in tumors (32, 33). In addition, the cancer therapeutic vaccination field has used self-antigens (e.g., melanomaassociated antigen [MAGE] peptides for melanoma) with good safety profiles and successful induction of immune responses (34). Further studies will be needed to investigate the potential of APOBEC3 epitopes as safe antigens in a vaccine against HIV-1. An important caveat for this proposed vaccination approach is that noninfected cells might also upregulate APO- BEC3 during infections and could also possibly be marked for destruction. On the basis of the observations presented here, we hypothesize that lentiviral infection generates APOBEC3-specific T cells, which are capable of recognizing and eliminating virally infected cells. Further, a vaccine immunogen using APOBEC3 sequences could generate APOBEC3-specific T cells, which might recognize and kill a cell infected with any variant of HIV-1. This novel approach targets infected cells based on their presentation of APOBEC3-derived peptides, not HIV-1 peptides. These studies are, therefore, the first steps toward a novel vaccine approach, which circumvents the obstacle of HIV-1 sequence diversity by targeting a surrogate marker of HIV-1 infection. ACKNOWLEDGMENTS We thank Emily Eriksson, Mark Peakman, and Mike Malim for helpful discussions. This work was supported in part by the AIDS biology program of the AIDS Research Institute, UCSF, NIH grants AI and P01 AI071713, and an Explorations grant from the Bill & Melinda Gates Foundation, grant This work was also supported in part by grant NIH OD Steven G. Deeks and Jeffrey N. Martin are supported in part by the National Institutes of Health (grants AI and AI071713) and the UCSF Center for AIDS Research (grant AI027763). Rafael R. Almeida is a recipient of São Paulo State Research Funding Agency (FAPESP) fellowships. Downloaded from on June 4, 2014 by USP/REITORIA/SIBI June 2013 Volume 87 Number 11 jvi.asm.org 6079

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118 Bicistronic DNA Vaccines Simultaneously Encoding HIV, HSV and HPV Antigens Promote CD8 + T Cell Responses and Protective Immunity Vinicius C. Santana 1, Mariana O. Diniz 1, Francisco A. M. O. Cariri 1, Armando M. Ventura 1, Edécio Cunha- Neto 2, Rafael R. Almeida 2, Marco A. Campos 3, Graciela K. Lima 3, Luís C. S. Ferreira 1 * 1 Department of Microbiology, Biomedical Sciences Institute, University of São Paulo, São Paulo, Brazil, 2 Laboratory of Clinical Immunology and Allergy-LIM60, Division of Clinical Immunology and Allergy, Department of Medicine, University of São Paulo School of Medicine, São Paulo, Brazil, 3 René Rachou Research Center, Fiocruz, Belo Horizonte, Brazil Abstract Millions of people worldwide are currently infected with human papillomavirus (HPV), herpes simplex virus (HSV) or human immunodeficiency virus (HIV). For this enormous contingent of people, the search for preventive and therapeutic immunological approaches represents a hope for the eradication of latent infection and/or virus-associated cancer. To date, attempts to develop vaccines against these viruses have been mainly based on a monovalent concept, in which one or more antigens of a virus are incorporated into a vaccine formulation. In the present report, we designed and tested an immunization strategy based on DNA vaccines that simultaneously encode antigens for HIV, HSV and HPV. With this purpose in mind, we tested two bicistronic DNA vaccines (pires I and pires II) that encode the HPV-16 oncoprotein E7 and the HIV protein p24 both genetically fused to the HSV-1 gd envelope protein. Mice i.m. immunized with the DNA vaccines mounted antigen-specific CD8 + T cell responses, including in vivo cytotoxic responses, against the three antigens. Under experimental conditions, the vaccines conferred protective immunity against challenges with a vaccinia virus expressing the HIV-derived protein Gag, an HSV-1 virus strain and implantation of tumor cells expressing the HPV-16 oncoproteins. Altogether, our results show that the concept of a trivalent HIV, HSV, and HPV vaccine capable to induce CD8 + T celldependent responses is feasible and may aid in the development of preventive and/or therapeutic approaches for the control of diseases associated with these viruses. Citation: Santana VC, Diniz MO, Cariri FAMO, Ventura AM, Cunha-Neto E, et al. (2013) Bicistronic DNA Vaccines Simultaneously Encoding HIV, HSV and HPV Antigens Promote CD8 + T Cell Responses and Protective Immunity. PLoS ONE 8(8): e doi: /journal.pone Editor: Mauricio Martins Rodrigues, Federal University of São Paulo, Brazil Received May 28, 2013; Accepted July 4, 2013; Published August 8, 2013 Copyright: ß 2013 Santana et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by CNPq (INCTV), FAPESP and USP (Vaccine Research Core) grants. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * lcsf@usp.br Introduction The diseases caused by human immunodeficiency virus (HIV), human papillomavirus (HPV) and herpes simplex virus (HSV) represent serious public health threats, as they affect millions of people irrespective of economic or social status [1]. The mortality and morbidity associated with HIV or HSV infection were significantly reduced after the discovery and dissemination of antiviral therapies that reduce viral loads and relieve symptoms in infected people. However, the currently available drugs are not able to eradicate the viruses, and infections with these viruses remain in a chronic latent state and recur after treatment interruption (HIV) or after debilitation of the immune defenses (HSV). Despite decades of intense scientific work and enormous investments, no effective anti-hiv or anti-hsv vaccine is presently available [2]. Regarding HPV, two prophylactic vaccines that are able to induce antibody responses have been shown to confer protection against virus infection and therefore reduce the long-term incidence of HPV-associated tumors [3,4]. However, the impact on the incidence of HPV-associated cancers is expected to be observed only after the widespread use of these vaccines. Nonetheless, those already infected with high-risk HPV types or afflicted with HPV-associated cancer or neoplastic lesions are not expected to benefit from preventive anti-viral vaccines. Therefore, the development of therapeutic cancer vaccines that target HPVinfected cells is a priority for several research groups [5]. The concept of therapeutic vaccines relies on the fact that the activation of immunological mechanisms leading to cytotoxic responses, particularly antigen-specific CD8 + T cell activation, permanently eradicates virus-infected or tumor cells [6]. Although theoretically sound and technologically feasible, the development of vaccines that efficiently activate antigen-specific CD8 + T cell populations to control the replication of viruses, such as HIV, remains elusive, as dramatically illustrated by the STEP program [7]. Similarly, numerous attempts to develop both prophylactic and therapeutic anti-hsv vaccines have systematically failed, and new insights regarding the immunological control of HSV-1 and HSV-2 infections are eagerly awaited [8,9]. Vaccines targeting the tumors induced by HPV, under both experimental and clinical conditions, stand as the best and most promising examples of the viability of therapeutic vaccines as immunological tools for the control of infectious and degenerative diseases [10 15]. PLOS ONE 1 August 2013 Volume 8 Issue 8 e71322

119 Trivalent HIV, HSV and Anti-Tumor HPV Vaccines DNA vaccines have been widely used as therapies against tumors and viruses because of their capability to induce antigenspecific CD8 + T cell responses as well as their rather simple manipulation [16,17]. DNA vaccines are also amenable to the development of multivalent formulations either by a mixture of plasmids encoding single antigens or by multiple antigens expressed as fused epitopes [18] or proteins derived from the same or different pathogens [19 21]. Multivalent DNA vectors can also be engineered to encode polycistronic transcripts under the control of a single promoter, leading to the simultaneous expression of multiple antigens in transfected host cells [22 26]. We have previously shown that DNA vaccines encoding the HPV-16 E7 oncoprotein genetically fused to HSV-1 glycoprotein D (gd) enhance both the induction of E7-specific CD8 + T cell responses and therapeutic/prophylactic anti-tumor effects compared to vaccines encoding the non-fused HPV oncoproteins in mice [15,27,28]. Additional evidence has indicated that these gddependent immunological effects, particularly the activation of CD8 + T cell responses to bystander antigens, involve the binding of gd to herpes virus entry mediator (HVEM) and the blockage of a co-inhibitory immune mechanism involving the B- and T- lymphocyte attenuator (BTLA) cell receptor [13,29]. In addition, further experimental evidence has indicated that the binding of gd to HVEM not only represents an important step in virus entry but also triggers an NF-kB-activation pathway and inhibits apoptosis in HVEM-expressing cells, such as antigen-presenting cells [30,31]. More recently, the fusion of gd with the influenza virus nucleoprotein further demonstrated that an adjuvant effect on CD8 + T cell responses can be achieved with different antigens and reveals the possibility of the development of multivalent anti-virus vaccine formulations that are able to induce prophylactic and/or therapeutic protective immune responses [32]. In the present study, we applied this vaccine technology to develop trivalent DNA vaccines encoding the HSV-1 protein gd genetically fused with both the HPV-16 oncoprotein E7 and the HIV-1 protein p24 by the same transfected cell. To achieve the simultaneous expression of the two hybrid proteins using a single vaccine vector, we employed a bicistronic expression system using an internal ribosome entry site (IRES) sequence [23,24]. Two vaccine vectors were constructed, pires I and pires II, which expressed the recombinant hybrid proteins under the control of a strong virus promoter (CMV) but differed in the order of the cistrons with respect to the promoter. Mammalian cells transfected with the recombinant vectors simultaneously expressed the HSV, HIV and HPV proteins at the cell surface. More importantly, C57BL/6 or BALB/c mice immunized with the tested vaccines elicited antigenspecific CD8 + T cell responses and were protected to challenges with a vaccinia virus expressing the HIV-1 protein Gag and a HSV-1 strain. In addition, vaccine administration could therapeutically eradicate the growth of tumor cells expressing the HPV- 16 oncoproteins implanted in C57BL/6 mice. Altogether, the present study demonstrates that the generation of a trivalent HIV, HSV and HPV anti-cancer vaccine is a feasible goal and should be pursued as a potential tool to prevent and/or treat infections with these viruses. Results Generation of Trivalent DNA Vaccines that Simultaneously Encode HIV, HSV and HPV Antigens The DNA vaccines tested in this study were obtained by amplifying the chimeric HPV-16 E7 or HIV-1 p24 coding sequences, both of which were fused to the HSV-1 gd sequence, and cloning them into the pires vector. This process generated two bifunctional plasmids, pires I and pires II, which only differ in the order of their cloned chimeric genes (Figure 1A). pires I carries the gdp24 gene fusion upstream of the IRES sequence and the gde7 gene fusion downstream of this regulatory sequence. In contrast, the pires II vector carries the gde7 and gdp24 sequences upstream and downstream of the IRES sequence, respectively. To confirm the expression and cellular localization of the encoded hybrid proteins, the recombinant DNA vaccines were introduced into mammalian COS-7 cells, and the encoded proteins were observed by immunofluorescence. The HSV-1 protein gd is a viral envelope glycoprotein that is expressed on the surface of infected cells via a C-terminal anchor sequence [33]. Thus, the recombinant proteins were detected in non-permeabilized cells treated with specific monoclonal (gd) or polyclonal (p24 and E7) antibodies against the target antigens. Cells transfected with pires I or pires II were simultaneously stained with antibodies against all three viral antigens (Figure 1B). Signals corresponding to the three proteins were detected mainly at the surface of the non-permeabilized cells. These results indicate that the hybrid proteins were correctly folded and properly targeted to the cell membrane compartment of the pires I- and pires II-transfected cells. pires I and pires II Vaccination Induces the Activation of Antigen-specific CD8 + T Cells and Simultaneous Protective Responses against Gag-expressing Vaccinia Virus and Tumor Cells Expressing HPV-16 Antigens Mice were immunized with pires I and pires II and monitored for the activation of E7-specific (in C57BL/6 mice) and p24-specific (in BALB/c mice) CD8 + T cells, the key immunological response required for both the control of virus-induced tumors and intracellularly replicating viruses. The CD8 + T cell mediated responses were first monitored in the vaccinated mice by detecting the level of antigen-specific IFN-c + -producing CD8 + T cells using both ICS and ELISPOT assays for total spleen cells harvested fourteen days after the last immunization dose. The cells harvested from the vaccinated mice were incubated with synthetic peptides corresponding to the immunodominant HPV-16 E7 and HIV-1 p24 MHC-I-restricted CD8 + T cell-specific epitopes for the H-2K b and H-2K d haplotypes, respectively. Cells stimulated with the H-2K d -restricted p24 peptide for 6 h resulted in the activation of p24-specific IFN-c + /CD8 + T cells at frequencies of approximately 1% and 0.6% in BALB/c mice immunized with pires I and pires II, respectively, as determined by ICS (Figure 2A). Following the same stimulation procedure with the p24-specific peptide but evaluating the results with an ELISPOT assay, only the animals vaccinated with pires I reached a statistically higher response compared with the control group immunized with the empty DNA vector (Figure 2B). The E7-specific IFN-c + /CD8 + T cell responses elicited in the vaccinated C57BL/6 mice were determined using the H-2K b -restricted E7-specific peptide. Intracellular IFN-c staining showed that the animals immunized with pires I or pires II developed higher E7-specific IFN-c + / CD8 + cell responses compared with mice immunized with the control vector, while only the animals immunized with the pires II vector generated positive responses compared with the control group when the responses were analyzed with the ELISPOT assay (Figure 2C and D). In addition, the E7-specific responses detected in the mice immunized with pires II were statistically higher than those detected in the mice immunized with pires I, as monitored by ICS (Figure 2C). Collectively, these results indicate that immunization with pires I and pires II induces PLOS ONE 2 August 2013 Volume 8 Issue 8 e71322

120 Trivalent HIV, HSV and Anti-Tumor HPV Vaccines Figure 1. Construction of bicistronic DNA vaccines encoding HPV, HIV and HSV antigens for expression in mammalian cells. (A) Schematic linear representation of the trivalent DNA vaccines. pires I and pires II contain gdp24 and gde7 chimeric gene fusions, which are inverted with regard to the CMV promoter and IRES sequence. The empty vector pires Ø was used as a control. The nucleotide numbers corresponding to the IRES sequence and the cloned chimeric genes are indicated. (B) In vitro expression of the chimeric proteins encoded by pires I (left panels) and pires II (right panels). Non-permeabilized pires I- or pires II-transfected COS-7 cells were labeled with antigen-specific antibodies for the simultaneous detection of the HSV-1 protein gd and the HIV-1 protein p24 or the HPV-16 oncoprotein E7. Green, gd; red, p24 or E7; yellow, co-localization of gd with p24 or E7; blue, DAPI nuclear staining. doi: /journal.pone g001 p24- and E7-specific CD8+ T cell responses in mice, but higher E7-specific IFN-c+/CD8+ T cell responses are present in mice immunized with the vector in which the E7/gD fusion is encoded by the first cistron (pires II). To further demonstrate that the antigen-specific activation of CD8+ T cells was functional in the vaccinated mice, we measured the in vivo antigen-specific cytotoxic responses elicited in mice immunized with pires I or pires II. Splenocytes harvested from non-vaccinated mice were surface labeled with both a fluorescent label (CFSE) and either the MHC-I restricted p24- or E7-specific peptide. The labeled cells were intravenously introduced into vaccinated mice, which were monitored 24 h later for the specific lysis of the labeled cells in their spleens. The relative reduction in PLOS ONE the level of peptide-labeled cells compared with cells labeled only with CFSE was indicative of antigen-specific CD8+ T celldependent cytolytic activity. Mice immunized with pires I mounted a p24-specific CD8+ T cell-dependent cytotoxic response, while mice immunized with pires II showed a statistically significant E7-specific CD8+ T cell-dependent cytotoxic response compared with mice immunized with the empty vector (Figure 3A and B). These results further demonstrate that effective antigen-specific CD8+ T cell-dependent cytotoxic responses are induced by the vaccines in which the target gene is expressed by the first cistron of the bicistronic transcript. As a final step to evaluate the protective immunological status of the mice immunized with the DNA vaccines encoding the HPV 3 August 2013 Volume 8 Issue 8 e71322