BACKGROUND AND AIMS:The hepatitis C virus (HCV) is a major cause of liver cirrhosis and cancer in humans, and an effective vaccine is urgently needed. Here, we evaluated the immunogenicity of a DNA prime and modified vaccinia Ankara (MVA) boost vaccine expressing H77 genotype 1a HCV immunogens to induce both T- and B-cell responses. APPROACH AND RESULTS:The non-structural (NS) immunogens expressed NS3, NS4, and NS5. The structural plus p7 immunogen (S-p7) expressed Core, E1, E2, and p7. We tested the immunogenicity of these vaccines in mice and rhesus macaques (RMs) with 2 or 3 doses of DNA followed by 1 or 2 doses of MVA. The NS immunogens induced CD4 and CD8 T-cell responses against multiple NS proteins, with a dominant CD4 T-cell response to NS3 and NS5, and a dominant CD8 T-cell response to NS3. The S-p7 immunogen induced E2-binding IgG titer with neutralizing activity against autologous and heterologous HCV pseudovirus particles. In addition, the S-p7 immunogen induced a CD4 and CD8 T-cell response directed against E1 (0.08% and 0.09%, respectively) and E2 (0.13% and 0.18%, respectively) in RMs. Co-delivery of S-p7 and NS vaccines elicited a T-cell response against the majority of HCV proteins, which was comparable to the T-cell response observed in humans with HCV resolution. CONCLUSIONS:These findings show that the DNA/MVA prime/boost vaccination induces a strong and broad CD4 and CD8 T-cell response to multiple HCV proteins and a neutralizing antibody response. These results aid in the development of vaccines for HCV.

The development of an effective HIV-1 vaccine is of paramount importance to global health. Here, we compared the influence of two adjuvants, Escherichia coli double-mutant heat-labile toxin (dmLT) and alum, on the protective immunity induced by a cyclically permuted trimeric HIV-1 envelope gp120 protein (CycP-gp120) boost. Two groups of rhesus macaques received two modified vaccinia Ankara (MVA)/SHIV C.1086 primes followed by a CycP-gp120 protein boost adjuvanted with either dmLT (n = 9) or alum (n = 10). A group of unvaccinated macaques (n = 8) served as controls. All animals were intrarectally challenged with heterologous SHIV.CH505.375H.dCT weekly for 7 weeks. Following the challenge, dmLT-adjuvanted animals showed significant protection with a vaccine efficacy of 60.8% per exposure (p = 0.0246). Alum-adjuvanted animals did not show significant protection (p = 0.1575). Both adjuvants induced comparable envelope-specific binding antibody in serum and rectal secretions with broad V1V2 scaffold-binding specificity. IL-6 plasma concentration correlated positively with V1V2 scaffold-binding and increased after vaccination with both adjuvants. With respect to CD4 T cells, dmLT induced higher frequencies of proliferating central memory (TCM) and ICOS+ cells in blood compared to alum. However, these proliferating CD4 TCM cells showed a decrease in the proportion of gut-homing receptor α4β7-expressing cells in the dmLT group compared to the alum group at week 2 post-protein boost. The V1V2 scaffold-specific IgG, proliferating TCM and ICOS+ CD4 T-cell frequencies, and plasma IL-6 concentration associated positively with protection. These data demonstrate that the vaccine adjuvants dmLT and alum differentially modulate protective helper T-cell responses induced by the CycP-gp120 protein, highlighting the importance of an appropriate adjuvant for eliciting a protective immune response against HIV-1.

Broadly neutralizing antibodies (bNAbs) exhibit protective efficacy against HIV-1 infection making them an ideal archetype for HIV-1 vaccine design. Presently, no vaccine candidate has induced antibody responses capable of meaningful protection against the swathe of circulating, difficult to neutralize tier 2 HIV-1 viruses. However, the development of stabilized, native-like envelope (Env) trimers such as BG505.SOSIP.664.T332N (BG505 SOSIP) has marked a significant advancement in vaccine design, due to their ability to elicit NAbs that neutralize tier 2 viruses in rhesus macaques (RM). NAb development following envelope trimer immunization in RM remains poorly understood, with hypothesized contributions from genetic variation at the IG loci, naive B cell repertoire, and differential gene expression in B cell lineages. To address these knowledge gaps, we have developed a set of BG505 SOSIP probes capable of recovering paired clonotype identity, antigen specificity, and gene expression of B cells in a high throughput fashion. These probes were constructed by conjugating biotinylated BG505 SOSIP to streptavidin covalently linked to both sc-RNA-Seq compatible DNA oligonucleotides and flow cytometry compatible fluorophores. Using these reagents, we isolated and sequenced BG505 SOSIP specific memory B cells from the PBMCs of an RM that developed high titers of neutralizing antibodies. To benchmark the accuracy of our technology, we compared our recovered heavy and light chain sequences to those identified from the same animal using conventional methodology and recovered 100% of previously identified NAbs. We then applied this technology to recover BG505 SOSIP specific memory B cells from five additional vaccinated RMs, cloned 34 antibodies for functional characterization, and identified ten antibodies with autologous neutralizing activity.

Inhibiting the mammalian target of rapamycin (mTOR) during acute viral infection generates highly functional memory CD8+ T cells. We investigated the effects of inhibiting mTOR by using rapamycin during the effector and contraction phases of the immune response to a DNA prime and Modified Vaccinia Ankara (MVA) boost SIV vaccination in rhesus macaques. Rapamycin administered either during MVA boosts alone (DMR) or during both primes and boosts (DRMR) reduced the contraction of effector CD8+ T cells, resulting in higher frequencies of SIV-specific memory CD8+ T cells with enhanced quality, as indicated by expression of Bcl2 and CD127. Additionally, rapamycin reduced the frequency of proliferating CCR5+CD4+ T cells in the blood following the MVA boost. After SIVmac251 infection, rapamycin-treated macaques demonstrated marked expansion of SIV-specific CD8+ T cells (reaching up to 50% in blood and 25% in gut). The heightened expansion of SIV-specific CD8+ T cells in the DMR group was associated with markedly lower (2-logs compared with unvaccinated and 1-log compared with DM) peak viral load in the gut and set-point viremia, along with improved survival after infection. Thus, inhibiting the mTOR pathway during MVA boosts of a DNA/MVA vaccine enhances vaccine efficacy by improving memory CD4+ and CD8+ T cell function.

Antigen-experienced lymphocytes can be equilibrating (continuously migratory between blood and peripheral tissues) or resident (stably surveilling within tissues). Because migration is difficult to measure outside of mice, it is common to extrapolate phenotypic proxies of residence derived from mouse studies to other species. We wished to more rigorously assess the differentiation state and function of equilibrating vs. resident memory CD8+ T cells (TRM) in nonhuman primates (NHP). To this end, we delivered a heterologous prime-boost-boost (HPBB) vaccine to both mice and Indian rhesus macaques that resulted in preternaturally abundant memory CD8 T cells that were distributed in over 30 anatomical sites. We then conducted a constellation of assays to infer migration properties of primate T cells: through single cell genomic analyses of paired mouse vaccinees that underwent parabiotic migration tests, through staged intravascular staining in NHP, and through comparisons of systemic and local routes of immunization. These data informed a cross-species signature that correlated with T cell migration properties. Functional assays were performed in NHP by reactivating memory CD8 T cells in situ and ex vivo, revealing that Trm are uniquely poised to communicate reactivation events to neighboring immune and stromal cells. Bill and Melinda Gates Foundation (OPP1116224, D.M.) NIH grants AI090732 and 5R01AI084913-14 (D.M.) U19AI096187, UM1AI124436 and UM1AI169662 (E.H and R.R.A) NCRR/NIH base grant P51 OD011132 to Emory National Primate Research Center. Lymphocyte Differentiation and Peripheral Maintenance (LYM)

Abstract Background Mpox is an orthopoxvirus with a rodent reservoir that causes a disease similar to mild smallpox. In 2022, a sexually-transmitted global mpox outbreak infected over 90,000 individuals. Some immunocompromised patients experienced prolonged, severe mpox resulting in significant morbidity, amputations, and death. People living with HIV (PLWH) were at particularly high risk due to impaired cell-mediated immunity, as CD4 and CD8 T-cells help eradicate the virus from infected lesions. Mpox virion total binding titer in PLWH with or without history of mpox infection Methods We compared immune responses to mpox in PLWH who were either mpox-naïve (n = 10) versus mpox-survivors (n = 15). We measured total mpox antibody levels with a novel ELISA assay using lysed mpox virions. We characterized mpox and orthopoxvirus-specific cell-mediated immunity using intracellular cytokine staining with an enhanced protocol to allow detection of low-frequency cellular populations. Poxvirus-specific T-cell responses in PLWH as determined by intracellular cytokine stimulation using live vaccinia virus (MVA), conserved orthopoxvirus peptide pools (VCD4 and VCD8), and mpox-specific peptide pools (MCD4 and MCD8). Pools provided by Alba Grifoni and Alessandro Sette (La Jolla Institute). Interferon-gamma (IFNγ), tumor necrosis factor alpha (TNFα), and interleukin 2 (IL-2) were the predominant cytokines detected. Responders defined as having any CD4 or CD8 response for any of the 9 stimulations above the 90th percentile of the control group. Results Our assays differentiated well between the mpox-naïve and mpox-survivors. All mpox-survivors had binding titers of 10 to 1,000 times background with no antibodies detected in mpox-uninfected PLWH. Mpox-specific CD4 and CD8 responses of 0.01 to 0.3% were present in all infected individuals, comparable to immunocompetent mpox-survivors. The magnitude of mpox-specific T-cell responses correlated well with vaccinia-specific T-cell responses, demonstrating induction of broad anti-orthopoxvirus cell-mediated immunity. CD4 T-cell responses were TH1-type, with higher polyfunctionality in mpox-specific T-cells compared to HIV and CMV-specific T-cells. The magnitude of the IFNγ and TNFα CD4 and CD8 responses correlated with mpox-binding antibody titers, but did not predict cross-neutralization titers from vaccinia, a closely related virus that is used to vaccinate for smallpox and mpox. Host CD4 count did not correlate with the magnitude of mpox-specific cellular or humoral immunity. Conclusion These new assays effectively quantify and characterize mpox-specific immunity, with strong induction of orthopoxvirus-specific humoral and cellular immunity in immunocompromised hosts with HIV. Although PLWH are at increased risk for complications from mpox, their potent immune responses post-infection suggest that most are at reduced risk for severe complications from re-infection. Disclosures Samuel D. Stampfer, MD/PhD, Gilead: Stocks/Bonds (Public Company) Colleen F. Kelley, MD, MPH, Gilead: Grant/Research Support|Humanigen: Grant/Research Support|Moderna: Grant/Research Support|Novavax: Grant/Research Support|ViiV: Grant/Research Support

COVID-19 vaccines provide robust protection against severe disease, hospitalization, and death. Neutralizing antibodies are a strong correlate of protection and can prevent SARS-CoV-2 infection of the lungs. We used a conventional laboratory mouse model combined with high- or low-dose vaccination to understand the early immunological response following SARS-CoV-2 infection in the lungs of vaccinated mice. The lungs of high-dose vaccinated mice were completely protected against SARS-CoV-2 infection whereas low-dose vaccinated mice were partially protected. We observed a greater influx of total monocytes, macrophages, dendritic cells, neutrophils, and eosinophils in the lungs of low-dose vaccinated mice compared to naïve infected mice. The different proportions of innate immune cells in the lungs indicated that infection in low-dose vaccinated mice induces a unique inflammatory environment compared to naïve infected or uninfected mice. A prominent feature of infection of low-dose vaccinated mice was infiltration of eosinophils in the lungs, which we observed across different COVID-19 vaccines and SARS-CoV-2 variants. Single cell transcriptional profiling of lung parenchymal immune cells showed that viral RNA was predominantly associated with eosinophils. Eosinophils from low-dose vaccinated mice were transcriptionally distinct from naïve mice after challenge and showed an IFN-γ biased signature. Further, monocytes from low-dose vaccinated mice expressed eotaxin-2, suggesting a monocyte-eosinophil signaling axis. Antibody mediated depletion of eosinophils in low-dose vaccinated mice resulted in increased virus replication and dissemination in the lungs. These findings demonstrate the protective nature of eosinophils during SARS-CoV-2 infection in the context of vaccination and highlight quantitative and qualitative differences in the immune response in a model for vaccine breakthrough infection.

Thymic output has been extensively studied. While advanced ex-vivo T cell generative approaches exist for mouse and human models, such advancements for nonhuman primate model are lacking. We report the establishment of a rhesus macaque-specific artificial thymic organoid (Rh-ATO) system enabling robust ex vivo T cell generation from CD34+ hematopoietic stem and progenitor cells (HSPCs). A continuum of distinct thymopoietic stages were recorded - robust T cell specification of HSPCs resembling thymus seeding progenitors, emergence of CD4+CD3- immature single positive, CD4+CD8+ double positive thymocytes, and finally, generation of CD4+ and CD8+ single-positive T cell subsets expressing CD38, consistent with recent thymic emigrant phenotype. These events closely mirrored Bonafide thymopoietic stages observed in the thymus. T cells expressed TCRs and exhibited polyfunctional cytokine expression. Thus, we report first demonstration of an off the shelf NHP-specific 3D system recapitulating thymopoiesis, providing a translational platform for modeling T cell development, therapeutic strategies, and immunopathogenesis.

Background: T cell regeneration in the thymus is intrinsically linked to the T cell-biased lineage differentiation of hematopoietic stem and progenitor cells (HSPCs). Although nonhuman primates (NHPs) serve as indispensable models for studying thymic output under physiological and pathological conditions, a non-animal technology facilitating efficient TCR-selected T cell development and evaluating T cell output from NHP-derived HSPCs has been lacking. To address this gap, we established a rhesus macaque-specific artificial thymic organoid (RhATO) modeling primary thymus-tissue-free thymopoiesis. Methods: The RhATO was developed by expressing Rhesus macaque (RM) Delta-like Notch ligand 1 in mouse bone marrow stromal cell line (MS5-RhDLL1). The bone marrow-derived HSPCs were aggregated with MS5-RhDLL1 and cultured forming 3D artificial thymic organoids. These organoids were maintained under defined cytokine conditions to support complete T cell developmental ontogeny. T cell developmental progression was assessed by flow cytometry, and TCR-selected subsets were analyzed for phenotypic and functional properties. Results: RhATOs recapitulated the complete spectrum of thymopoietic events, including emergence of thymus-seeding progenitors, CD4+CD3− immature single-positive and CD4+CD8+ double-positive early thymocytes, and mature CD4+ or CD8+ single-positive subsets. These subsets expressed CD38, consistent with the recent thymic emigrant phenotype, and closely mirrored canonical T cell ontogeny described in humans. RhATO-derived T cells were TCR-selected and demonstrated cytokine expression upon stimulation. Conclusions: This study provides the first demonstration of an NHP-specific artificial thymic technology that faithfully models thymopoiesis. RhATO represents a versatile ex vivo platform for studying T cell development, immunopathogenesis, and generating TCR selected T cells.

The US National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institute of Health (NIH), convened a virtual workshop on August 8-9 th , 2023 to explore potential synergies between HIV vaccine approaches that are designed to induce cellular or humoral immune responses. The goal of this workshop was to review data on leading vaccine candidates and to discuss the best strategies for combining these approaches to optimize immunity against HIV. Here, we summarize the findings reviewed at the workshop and discuss the knowledge gaps and priorities for future studies that will help accelerate the development of a preventive HIV vaccine.

Cytokines are key mediators of immune regulation, orchestrate communication between immune cells, and play a pivotal role in shaping the immune landscape during chronic infection and cancer. The therapeutic potential of IL-15/IL-15Rα and IL-12 has been explored individually in various immunotherapeutic strategies, though not as a combination. Therefore, we investigated whether the combination of IL-15/IL-15Rα and IL-12 treatment would enhance the potency and quality of either NK cells, SIV-specific CD8 T cells, or both, compared with single cytokine treatment. Our findings reveal that in vitro IL-15/IL-15Rα and IL-15/IL-15Rα plus IL-12 treatment results in an expansion of functional CD8 T cells and NK cells from uninfected and chronically infected macaques with simian/human immunodeficiency virus. Additionally, the cytokine combination significantly reduced CCR5 expression on total CD4 T cells, limiting the number of viral targets. This study supports the potential utilization of combined IL-15/IL-15Rα plus IL-12 treatment for chronic viral infections and cancer.

Background: A goal of mucosal human immunodeficiency virus type 1 (HIV-1) vaccines is to generate mucosal plasma cells producing polymeric IgA (pIgA)-neutralizing antibodies at sites of viral entry. However, vaccine immunogens capable of eliciting IgA neutralizing antibodies (nAbs) that recognize tier 2 viral isolates have not yet been identified. Methods: To determine if stabilized native-like HIV-1 envelope (Env) trimers could generate IgA nAbs, we purified total IgA and IgG from the banked sera of six rhesus macaques that had been found in a previous study to develop serum nAbs after subcutaneous immunization with BG505.664 SOSIP and 3M-052 adjuvant, which is a TLR7/8 agonist. The neutralization of autologous tier 2 BG505 T332N pseudovirus by the IgA and IgG preparations was measured using the TZM-bl assay. Anti-SOSIP binding antibodies (bAbs) were measured by ELISA. Results: The IgG samples were found to have significantly greater levels of both nAb and bAb. However, after normalizing the nAb titer relative to the concentration of bAb, SOSIP-specific IgA purified from 2/6 animals was found to neutralize just as effectively as SOSIP-specific IgG, and in 3/6 animals, neutralization by the specific IgA was significantly greater. The more potent neutralization by IgA in these three animals was associated with a higher percentage of anti-SOSIP J chain-bound (polymeric) antibody. Conclusions: The parenteral vaccination of nonhuman primates with BG505.664 SOSIP generates HIV-1 tier 2 IgA nAbs in serum, including SOSIP-specific polymeric IgA, which appears to neutralize more efficiently than monomeric IgA or IgG. Mucosal delivery of this SOSIP or other stable Env trimers could generate locally synthesized polymeric IgA nAbs in mucosal tissues and secretions.

CD4 T follicular helper cells (Tfh) are essential for establishing serological memory and have distinct helper attributes that impact both the quantity and quality of the antibody response. Insights into Tfh subsets that promote antibody persistence and functional capacity can critically inform vaccine design. Based on the Tfh profiles evoked by the live attenuated measles virus vaccine, renowned for its ability to establish durable humoral immunity, we investigated the potential of a Tfh1/17 recall response during the boost phase to enhance persistence of HIV-1 Envelope (Env) antibodies in rhesus macaques. Using a DNA-prime encoding gp160 antigen and Tfh polarizing cytokines (interferon protein-10 (IP-10) and interleukin-6 (IL-6)), followed by a gp140 protein boost formulated in a cationic liposome-based adjuvant (CAF01), we successfully generated germinal center (GC) Tfh1/17 cells. In contrast, a similar DNA-prime (including IP-10) followed by gp140 formulated with monophosphoryl lipid A (MPLA)+QS-21 adjuvant predominantly induced GC Tfh1 cells. While the generation of GC Tfh1/17 cells with CAF01 and GC Tfh1 cells with MPLA+QS-21 induced comparable peak Env antibodies, the latter group demonstrated significantly greater antibody concentrations at week 8 after final immunization which persisted up to 30 weeks (gp140 IgG ng/ml- MPLA; 5500; CAF01, 2155; p <0.05). Notably, interferon γ+ Env-specific Tfh responses were consistently higher with gp140 in MPLA+QS-21 and positively correlated with Env antibody persistence. These findings suggest that vaccine platforms maximizing GC Tfh1 induction promote persistent Env antibodies, important for protective immunity against HIV.

A key barrier to the development of vaccines that induce broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV) and other viruses of high antigenic diversity is the design of priming immunogens that induce rare bnAb-precursor B cells. The high neutralization breadth of the HIV bnAb 10E8 makes elicitation of 10E8-class bnAbs desirable; however, the recessed epitope within gp41 makes envelope trimers poor priming immunogens and requires that 10E8-class bnAbs possess a long heavy chain complementarity determining region 3 (HCDR3) with a specific binding motif. We developed germline-targeting epitope scaffolds with affinity for 10E8-class precursors and engineered nanoparticles for multivalent display. Scaffolds exhibited epitope structural mimicry and bound bnAb-precursor human naive B cells in ex vivo screens, protein nanoparticles induced bnAb-precursor responses in stringent mouse models and rhesus macaques, and mRNA-encoded nanoparticles triggered similar responses in mice. Thus, germline-targeting epitope scaffold nanoparticles can elicit rare bnAb-precursor B cells with predefined binding specificities and HCDR3 features.

Abstract Background In 2022, UNAIDS estimated out of the 1.7 million children living with HIV, only 57.1% had access to antiretroviral therapy (ART). Though ART has significantly decreased HIV-associated morbidity and mortality, it cannot eliminate the latent viral reservoir. Consequently, most people living with HIV demonstrate viral rebound when ART is interrupted. There is therefore a critical need for alternative therapeutic to achieve long term ART free viral control. Broadly neutralizing antibodies (bnAb) have shown promising results in preclinical and clinical studies, but their potential efficacy is limited by the emergence of escape variants, especially with monotherapy. We previously tested the neutralizing and non-neutralizing functions of different bnAb combinations and identified a triple bnAb combination that showed robust neutralization potency and breadth, as well as non-neutralizing functions. In this project, we investigated the impact of combination bnAb therapy on viral rebound in SHIV infected infant rhesus macaques (RMs). Methods Ten infant rhesus macaques were orally challenged with SHIV.C.CH505 at 4 weeks of age. Oral challenge is a well-established method that is used to mimic breastmilk transmission of HIV, which contributes to almost 50% of pediatric HIV infections every year. A triple bnAb combination of simianized 3BNC117 (CD4 binding site), PGDM1400 (V2 glycan dependent), and PGT151 (interface) was subcutaneously administered twice at 40 mg/kg of each antibody. The initial infusion was administered at ART initiation (8 weeks post-infection) while the second infusion with administered at the time of analytical treatment interruption (ATI) (49 weeks post-infection). RMs were monitored weekly from the beginning of infection until 12 weeks post-ATI. Enzyme-linked Immunosorbent Assay (ELISA) was used to monitor plasma concentrations of each bNab and Env-specific IgG binding responses. Results Env-specific antibody binding levels against HIV envelope proteins gp120 and gp41 peaked in plasma 8 weeks after infection. The antibody levels then slightly decreased during ART and increased again after viral rebound following ATI. BnAb plasma concentrations peaked 1-2 weeks after each bnAb infusion, then slowly declined. After the initial infusion, all RMs had undetectable levels of 3BNC117 and PGT151 by 8 weeks post initial infusion, and only 2/10 RMs had detectable PGDM1400 levels. All animals experienced viral rebound following ATI.). Importantly, the average time to virus rebound (7 weeks, range 3-10 weeks) was significantly longer than a s control group of RMs without any immune-based intervention (~2.5 weeks). By the time of viral rebound, most animals had undetectable plasma levels of 3BNC117, PGDM1400 or PGT151. However, in some animals, rebound occurs when passively administered antibodies were still detectable, suggesting the emergence of bnAb resistant variants. Conclusion Passive immunization with a triple-bnAb combination of 3BNC117, PGT151 and PGDM1400 was associated with delayed viral rebound, but more investigation is needed to understand the potential role of this strategy in the HIV therapeutic portfolio. Future work will investigate the sensitivity of the rebound viruses to the bnAb combination to evaluate the contribution of bnAb escape to viral rebound.

Crosstalk between the microbiome and gut mucosa-resident immune cells plays a pivotal role in modulating immune responses to pathogens, including responses to HIV infection. However, how these interactions may differ between young men who have sex with men (YMSM) disproportionately impacted by HIV, as compared with older adult MSM (AMSM), is not well understood. A broad analysis of associations between the microbiome and rectal transcriptome revealed 10 microbial families/genera correlated with immunologic gene pathways. Specifically, the rectal transcriptome of YMSM was characterized by upregulation of T cell activation/differentiation pathways and signaling from multiple cytokine families compared with AMSM. The microbiome of YMSM was enriched with pathogenic genera, including Peptostreptococcus, shown to be positively correlated with type I IFN pathways important for antiviral immunity. These findings demonstrate that YMSM have a unique immune phenotype and rectal microenvironment and support further evaluation of biological factors that influence rectal HIV transmission.

Abstract HIV infection leads to alterations in HIV-specific CD4 T cells including increased expression of inhibitory receptors and skewing toward Tfh cell signature. Tfh cells have been shown to increase during chronic HIV infection. However, the heterogeneous nature of antigen-specific GC-Tfh cells and the relative contribution of specific GC-Tfh subset to viral persistence is not understood in HIV/SIV infection. Here, we characterized the phenotype and function of antigen-specific GC-Tfh cells based on the expression of chemokine receptors, Th1 (CXCR3), Th2 (CCR4), and Th17 (CCR6) along with activation-induced markers (AIM assay) CD25, OX40 and 41BB in the lymph nodes following chronic SHIV1157ipd3N4 infection in rhesus macaques. In SHIV naïve RM, only a small fraction of total GC-Tfh expressed CXCR3, CCR4, and CCR6. During chronic SHIV infection, there was an increase in GC-Tfh cells. Interestingly the majority of antigen-specific GC-Tfh cells expressed CXCR3 (Tfh1), while a significant proportion of them also expressed CCR4+ (Tfh2) and CCR6+ (Tfh17) cells. These cells expressed OX40, 41BB, and CD25 at higher levels than non-Tfh cells. Importantly, the expansion of GC-Tfh1 and GC-Tfh-17 cells is associated with high viremia. These data demonstrate that chronic SHIV infection promotes the expansion of antigen-specific GC-Tfh cells with heterogeneous populations, which are distinct from conventional GC-Tfh, and associated with higher viral RNA levels during chronic SHIV infection.