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In the Funding section, the following statement is missing: The MACS cohort study was supported by the NIH, National Institute of Allergy and Infectious Diseases grant U01-AI35041.

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.

During early HIV Infection, immunodominant T cell responses to highly variable epitopes lead to the selection and expansion of immune escape variants. As a potential therapeutic strategy, we assessed a specialized type 1-polarized monocyte-derived DC dendritic cell (MDC1)-based approach to selectively elicit functional CD8+ cytotoxic T lymphocyte (CTL) responses against highly conserved and topologically important HIV epitopes. Cells were obtained from 10 HIV-infected individuals in the Thailand RV254/SEARH010 cohort who initiated suppressive anti-retroviral therapy (ART) during Fiebig stages I to IV of early infection. Autologous MDC1 were generated for use as peptide antigen presenting cells to induce ex vivo CTL responses against HIV Gag, Pol, Env and Nef. Ultra-conserved (Epigraph) or topologically important (Network) antigens were respectively identified using the Epigraph tool and a structure-based network analysis approach, and each compared to overlapping peptides spanning the entire Gag proteome. MDC1 loaded with either overlapping Gag, Epigraph, or Network 14-21mer peptide pools were consistently capable of activating and expanding HIV-specific T cells to epitopes identified at the 9-13mer peptide level. Some CTL responses occurred outside of known or expected HLA associations, providing evidence of new HLA-associated CTL epitopes. Comparative analyses of peptide pools demonstrated more sequence conservation among the Epigraph antigens, but statistically higher magnitude of CTL responses to Network and Gag peptide groups. Importantly, when select Gag antigens used to initiate the cultures were part of the Network peptide pool, CTL responses directed against these topologically important epitopes were enhanced as compared to when they were included within the complete pool of overlapping Gag peptides. Our study supports that MDC1 can be used to effectively focus CTL responses toward potentially fitness-constrained regions of HIV as a therapeutic strategy to prevent HIV immune escape and control viral replication.Author summaryA major hurdle in the development of a successful HIV immunotherapy is the capacity of the virus to evade the immune response by efficiently establishing epitope variants in response to selective pressure. While effective at suppressing viremia, current regimens of antiretroviral therapy (ART) are not curative. Therefore, achieving immune control of HIV upon cessation of ART as a functional cure, similar to that observed in ‘elite controllers’ (EC), has been a major therapeutic goal. Such immune control is realized through the actions of antigen-specific cytotoxic T cell lymphocytes (CTL) capable of specifically targeting sequence-conserved epitopes in HIV. In this study, a specialized, antigen presenting, dendritic cell (DC)-based vaccine strategy was used to elicit HIV specific CTL responses in vitro against carefully selected, ultra-conserved and topologically important epitopes. This DC-based approach yielded broad responses against peptide epitopes of both known and unknown HLA-associations, the latter of which implies the uncovering of potentially novel epitopes. Importantly, we demonstrate that CTL responses can be re-directed or focused toward potentially more fitness-constrained regions of the virus, thus highlighting the potential for DC-based therapies to induce immune responses that circumvent the issue of viral escape.

Reducing the risk of human immunodeficiency virus type 1 (HIV-1) transmission is still a public health priority. The development of effective control strategies relies on the quantification of the effects of prophylactic and therapeutic measures in disease incidence. Although several assays can be used to estimate HIV incidence, these estimates are limited by the poor performance of these assays in distinguishing recent from long-standing infections. To address such limitation, we have developed an assay to titrate p24-specific IgG3 antibodies as a marker of recent infection. The assay is based on a recombinant p24 protein capable to detect total IgG antibodies in sera using a liquid micro array and enzyme-linked immunosorbent assay. Subsequently, the assay was optimised to detect and titrate anti-p24 IgG3 responses in a panel of sequential specimens from seroconverters over 24 months. The kinetics of p24-specific IgG3 titres revealed a transient peak in the 4 to 5-month period after seroconversion. It was followed by a sharp decline, allowing infections with less than 6 months to be distinguished from older ones. The developed assay exhibited a mean duration of recent infection of 144 days and a false-recent rate of ca. 14%. Our findings show that HIV-1 p24-specific IgG3 titres can be used as a tool to evaluate HIV incidence in serosurveys and to monitor the efficacy of vaccines and other transmission control strategies.

Background: During early HIV 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 epitopes, using HIV-infected individuals from the Thailand RV254/SEARH010 cohort who initiated antiretroviral therapy (ART) during early infection (Fiebig stages I-IV). Methods: Autologous MDC1 were used as peptide antigen presenting cells to induce ex vivo CTL responses against HIV 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-specific T cells specific 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 higher magnitude of CTL responses to Network and Gag peptide groups. Importantly, CTL responses against topologically important 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 potentially fitness-constrained regions of HIV as a means to prevent immune escape and control HIV-1 infection. Funding Statement: This study was supported by the NIH, National Institute of Allergy and Infectious Diseases grants R21-AI131763, R21-AI138716, UM1-AI126603, and U01-AI35041. Declaration of Interests: The authors have nothing to disclose. Ethics Approval Statement: The Chulalongkorn University Institutional Review Board and the Walter Reed Army Institute of Research, USA, approved this study.

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-bet(hi)) 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-bet(low)/Eomes(+) to a T-bet(hi)/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.