Dendritic cells focus CTL responses toward highly conserved and topologically important HIV-1 epitopes.

BACKGROUND: During early HIV-1 infection, immunodominant T cell responses to highly variable epitopes lead to the establishment of immune escape virus variants. Here we assessed a type 1-polarized monocyte-derived dendritic cell (MDC1)-based approach to selectively elicit cytotoxic T lymphocyte (CTL) responses against highly conserved and topologically important HIV-1 epitopes in HIV-1-infected individuals from the Thailand RV254/SEARCH 010 cohort who initiated antiretroviral therapy (ART) during early infection (Fiebig stages I-IV). METHODS: Autologous MDC1 were used as antigen presenting cells to induce in vitro CTL responses against HIV-1 Gag, Pol, Env, and Nef as determined by flow cytometry and ELISpot assay. Ultra-conserved or topologically important antigens were respectively identified using the Epigraph tool and a structure-based network analysis approach and compared to overlapping peptides spanning the Gag proteome. FINDINGS: MDC1 presenting either the overlapping Gag, Epigraph, or Network 14-21mer peptide pools consistently activated and expanded HIV-1-specific T cells to epitopes identified at the 9-13mer peptide level. Interestingly, some CTL responses occurred outside known or expected HLA associations, providing evidence of new HLA-associated CTL epitopes. Comparative analyses demonstrated more sequence conservation among Epigraph antigens but a higher magnitude of CTL responses to Network and Gag peptide groups. Importantly, CTL responses against topologically constrained Gag epitopes contained in both the Network and Gag peptide pools were selectively enhanced in the Network pool-initiated cultures. INTERPRETATION: Our study supports the use of MDC1 as a therapeutic strategy to induce and focus CTL responses toward putative fitness-constrained regions of HIV-1 to prevent immune escape and control HIV-1 infection. FUNDING: A full list of the funding sources is detailed in the Acknowledgment section of the manuscript.

Contrasting Roles of the PD-1 Signaling Pathway in Dendritic Cell-Mediated Induction and Regulation of HIV-1-Specific Effector T Cell Functions.

Eliciting highly functional CD8+ cytotoxic T lymphocyte (CTL) responses against a broad range of epitopes will likely be required for immunotherapeutic control of HIV-1 infection. However, the combination of CTL exhaustion and the ability of HIV-1 to rapidly establish CTL escape variants presents major hurdles toward this goal. Our previous work highlighted the use of monocyte-derived, mature, high-interleukin-12 (IL-12)-producing type 1 polarized dendritic cells (MDC1) to selectively induce more potent effector CTLs derived from naive, rather than memory, CD8+ T cell precursors isolated from HIV-1-positive participants in the Multicenter AIDS Cohort Study. In this study, we report that these highly stimulatory antigen-presenting cells also express enhanced levels of the coinhibitory molecule programmed cell death ligand 1 (PD-L1), the ligand for PD-1, which is further upregulated upon subsequent stimulation with the CD4+ T helper cell-derived factor CD40L. Interestingly, blocking the PD-1 signaling pathway during MDC1 induction of HIV-1-specific CTL responses inhibited the priming, activation, and differentiation of naive CD8+ T cells into effector T cells expressing high levels of T-box transcription factor (T-bethi) and eomesodermin (Eomes+). In contrast, PD-1 blockade enhanced the overall magnitude of memory HIV-specific CTL responses and reversed the exhausted memory phenotype from a T-betlow/Eomes+ to a T-bethi/Eomes+ phenotype. These results indicate that the PD-L1/PD-1 signaling pathway has a previously unappreciated dual role in the induction and regulation of HIV-1-specific CTL immunity, which is greatly determined by the context and differentiation stage of the responsive CD8+ T cells.IMPORTANCE Targeting the PD-1/PD-L1 immune checkpoint axis with signaling inhibitors has proven to be a powerful immunotherapeutic strategy to enhance the functional quality and survival of existing antigen-specific effector T cells. However, our study demonstrates that the context and timing of PD-1 signaling in T cells greatly impact the outcome of the effector response. In particular, we show that PD-1 activation plays a positive role during the DC-mediated initiation stage of the primary T cell response, while it serves as an inhibitory mechanism during the effector phase of the response. Therefore, caution should be taken in the design of therapies that include targeting of the PD-1/PD-L1 signaling pathway in order to avoid potential negative impacts on the induction of de novo T cell responses.

Membrane and envelope virus proteins co-expressed as lysosome associated membrane protein (LAMP) fused antigens: a potential tool to develop DNA vaccines against flaviviruses

Vaccination is the most practical and cost-effective strategy to prevent the majority of the flavivirus infection to which there is an available vaccine. However, vaccines based on attenuated virus can potentially promote collateral side effects and even rare fatal reactions. Given this scenario, the developent of alternative vaccination strategies such as DNA-based vaccines encoding specific flavivirus sequences are being considered. Endogenous cytoplasmic antigens, characteristically plasmid DNA-vaccine encoded, are mainly presented to the immune system through Major Histocompatibility Complex class I - MHC I molecules. The MHC I presentation via is mostly associated with a cellular cytotoxic response and often do not elicit a satisfactory humoral response. One of the main strategies to target DNA-encoded antigens to the MHC II compartment is expressing the antigen within the Lysosome-Associated Membrane Protein (LAMP). The flavivirus envelope protein is recognized as the major virus surface protein and the main target for neutralizing antibodies. Different groups have demonstrated that co-expression of flavivirus membrane and envelope proteins in mammalian cells, fused with the carboxyl-terminal of LAMP, is able to induce satisfactory levels of neutralizing antibodies. Here we reviewed the use of the envelope flavivirus protein co-expression strategy as LAMP chimeras with the aim of developing DNA vaccines for dengue, West Nile and yellow fever viruses.

A vacinação é a estratégia mais prática e o melhor custo-benefício para prevenir a maioria das infecções dos flavivirus, para os quais existe vacina disponível. Entretanto, as vacinas baseadas em vírus atenuados podem potencialmente promover efeitos colaterais e, mais raramente, reações fatais. Diante deste cenário, o desenvolvimento de estratégias alternativas de vacinação, como vacinas baseadas em DNA codificando seqüências específicas dos flavivirus, está sendo considerado. Antí-genos citoplasmáticos endógenos, caracteristicamente codificados por vacinas de DNA plasmidial, são majoritariamente apresentados ao sistema imune através de moléculas do Complexo Maior de Histocompatibilidade de classe I - MHC I. A via de apresentação MHC I é mais associada à resposta celular citotóxica e, frequentemente, não elicita uma resposta humoral satisfatória. Uma das principais estratégias para direcionar antígenos codificados pelas vacinas de DNA para o compartimento MHC II é expressar estes antígenos dentro da Proteína de Associação à Membrana Lisossomal (LAMP). A proteína do envelope dos flavivirus é reconhecidamente a principal proteína de superfície viral e o principal alvo para anticorpos neutralizantes. Diferentes grupos têm demonstrado que a co-expressão das proteínas de membrana e do envelope dos flavivirus em células de mamíferos, fusionada com a porção carboxi-terminal de LAMP, é capaz de induzir níveis satisfatórios de anticorpos neutralizantes. Neste trabalho revisamos a estratégia de co-expressão da proteína do envelope dos flavivírus, como quimeras de LAMP, com o objetivo de desenvolver vacinas de DNA contra a febre do Oeste do Nilo, dengue e febre amarela.

Membrane and envelope virus proteins co-expressed as lysosome associated membrane protein (LAMP) fused antigens: a potential tool to develop DNA vaccines against flaviviruses.

Vaccination is the most practical and cost-effective strategy to prevent the majority of the flavivirus infection to which there is an available vaccine. However, vaccines based on attenuated virus can potentially promote collateral side effects and even rare fatal reactions. Given this scenario, the development of alternative vaccination strategies such as DNA-based vaccines encoding specific flavivirus sequences are being considered. Endogenous cytoplasmic antigens, characteristically plasmid DNA-vaccine encoded, are mainly presented to the immune system through Major Histocompatibility Complex class I - MHC I molecules. The MHC I presentation via is mostly associated with a cellular cytotoxic response and often do not elicit a satisfactory humoral response. One of the main strategies to target DNA-encoded antigens to the MHC II compartment is expressing the antigen within the Lysosome-Associated Membrane Protein (LAMP). The flavivirus envelope protein is recognized as the major virus surface protein and the main target for neutralizing antibodies. Different groups have demonstrated that co-expression of flavivirus membrane and envelope proteins in mammalian cells, fused with the carboxyl-terminal of LAMP, is able to induce satisfactory levels of neutralizing antibodies. Here we reviewed the use of the envelope flavivirus protein co-expression strategy as LAMP chimeras with the aim of developing DNA vaccines for dengue, West Nile and yellow fever viruses.