Long-primed germinal centres with enduring affinity maturation and clonal migration.

Germinal centres are the engines of antibody evolution. Here, using human immunodeficiency virus (HIV) Env protein immunogen priming in rhesus monkeys followed by a long period without further immunization, we demonstrate germinal centre B (BGC) cells that last for at least 6 months. A 186-fold increase in BGC cells was present by week 10 compared with conventional immunization. Single-cell transcriptional profiling showed that both light- and dark-zone germinal centre states were sustained. Antibody somatic hypermutation of BGC cells continued to accumulate throughout the 29-week priming period, with evidence of selective pressure. Env-binding BGC cells were still 49-fold above baseline at 29 weeks, which suggests that they could remain active for even longer periods of time. High titres of HIV-neutralizing antibodies were generated after a single booster immunization. Fully glycosylated HIV trimer protein is a complex antigen, posing considerable immunodominance challenges for B cells1,2. Memory B cells generated under these long priming conditions had higher levels of antibody somatic hypermutation, and both memory B cells and antibodies were more likely to recognize non-immunodominant epitopes. Numerous BGC cell lineage phylogenies spanning more than the 6-month germinal centre period were identified, demonstrating continuous germinal centre activity and selection for at least 191 days with no further antigen exposure. A long-prime, slow-delivery (12 days) immunization approach holds promise for difficult vaccine targets and suggests that patience can have great value for tuning of germinal centres to maximize antibody responses.

Pathogenic simian immunodeficiency virus infection is associated with expansion of the enteric virome.

Pathogenic simian immunodeficiency virus (SIV) infection is associated with enteropathy, which likely contributes to AIDS progression. To identify candidate etiologies for AIDS enteropathy, we used next-generation sequencing to define the enteric virome during SIV infection in nonhuman primates. Pathogenic, but not nonpathogenic, SIV infection was associated with significant expansion of the enteric virome. We identified at least 32 previously undescribed enteric viruses during pathogenic SIV infection and confirmed their presence by using viral culture and PCR testing. We detected unsuspected mucosal adenovirus infection associated with enteritis as well as parvovirus viremia in animals with advanced AIDS, indicating the pathogenic potential of SIV-associated expansion of the enteric virome. No association between pathogenic SIV infection and the family-level taxonomy of enteric bacteria was detected. Thus, enteric viral infections may contribute to AIDS enteropathy and disease progression. These findings underline the importance of metagenomic analysis of the virome for understanding AIDS pathogenesis.

Adaptation of a transmitted/founder simian-human immunodeficiency virus for enhanced replication in rhesus macaques.

Transmitted/founder (TF) simian-human immunodeficiency viruses (SHIVs) express HIV-1 envelopes modified at position 375 to efficiently infect rhesus macaques while preserving authentic HIV-1 Env biology. SHIV.C.CH505 is an extensively characterized virus encoding the TF HIV-1 Env CH505 mutated at position 375 shown to recapitulate key features of HIV-1 immunobiology, including CCR5-tropism, a tier 2 neutralization profile, reproducible early viral kinetics, and authentic immune responses. SHIV.C.CH505 is used frequently in nonhuman primate studies of HIV, but viral loads after months of infection are variable and typically lower than those in people living with HIV. We hypothesized that additional mutations besides Δ375 might further enhance virus fitness without compromising essential components of CH505 Env biology. From sequence analysis of SHIV.C.CH505-infected macaques across multiple experiments, we identified a signature of envelope mutations associated with higher viremia. We then used short-term in vivo mutational selection and competition to identify a minimally adapted SHIV.C.CH505 with just five amino acid changes that substantially improve virus replication fitness in macaques. Next, we validated the performance of the adapted SHIV in vitro and in vivo and identified the mechanistic contributions of selected mutations. In vitro, the adapted SHIV shows improved virus entry, enhanced replication on primary rhesus cells, and preserved neutralization profiles. In vivo, the minimally adapted virus rapidly outcompetes the parental SHIV with an estimated growth advantage of 0.14 days-1 and persists through suppressive antiretroviral therapy to rebound at treatment interruption. Here, we report the successful generation of a well-characterized, minimally adapted virus, termed SHIV.C.CH505.v2, with enhanced replication fitness and preserved native Env properties that can serve as a new reagent for NHP studies of HIV-1 transmission, pathogenesis, and cure.

Transmitted/founder SHIV.D replicates in the brain, causes neuropathogenesis, and persists on combination antiretroviral therapy in rhesus macaques.

A biologically relevant non-human primate (NHP) model of HIV persistence in the central nervous system (CNS) is necessary. Most current NHP/SIV models of HIV infection fail to recapitulate viral persistence in the CNS without encephalitis or fail to employ viruses that authentically represent the ongoing HIV-1 pandemic. Here, we demonstrate viral replication in the brain and neuropathogenesis after combination antiretroviral therapy (ART) in rhesus macaques (RMs) using novel macrophage-tropic transmitted/founder (TF) simian-human immunodeficiency virus SHIV.D.191,859 (SHIV.D). Quantitative immunohistochemistry (IHC) and DNA/RNAscope in situ hybridization (ISH) were performed on three brain regions from six SHIV.D-infected RMs; two necropsied while viremic, two during analytical treatment interruptions, and two on suppressive ART. We demonstrated myeloid-mediated neuroinflammation, viral replication, and proviral DNA in the brain in all animals. These results demonstrate that TF SHIV.D models native HIV-1 CNS replication, pathogenesis, and persistence on ART in rhesus macaques.

Anatomic Distribution of Intravenously Injected IgG Takes Approximately 1 Week to Achieve Stratum Corneum Saturation in Vaginal Tissues.

i.v. injected Abs have demonstrated protection against simian HIV infection in rhesus macaques, paving the way for the Antibody Mediated Prevention trial in which at-risk individuals for HIV received an i.v. infusion of the HIV broadly neutralizing Ab VRC01. However, the time needed for these Abs to fully distribute and elicit protection at mucosal sites is still unknown. In this study, we interrogate how long it takes for Abs to achieve peak anatomical levels at the vaginal surface following i.v. injection. Fluorescently labeled VRC01 and/or Gamunex-C were i.v. injected into 24 female rhesus macaques (Macaca mulatta) with vaginal tissues and plasma acquired up to 2 wk postinjection. We found that Ab delivery to the vaginal mucosa occurs in two phases. The first phase involves delivery to the submucosa, occurring within 24 h and persisting beyond 1 wk. The second phase is the delivery through the stratified squamous epithelium, needing ∼1 wk to saturate the stratum corneum. This study has important implications for the efficacy of immunoprophylaxis targeting pathogens at the mucosa.

Increased Proviral DNA in Circulating Cells Correlates with Plasma Viral Rebound in Simian Immunodeficiency Virus-Infected Rhesus Macaques after Antiretroviral Therapy Interruption.

The human immunodeficiency virus (HIV) reservoir is responsible for persistent viral infection, and a small number of mosaic latent cellular reservoirs promote viral rebound upon antiretroviral therapy interruption, which is the major obstacle to a cure. However, markers that determine effective therapy and viral rebound posttreatment interruption remain unclear. In this study, we comprehensively and longitudinally tracked dynamic decay of cell-associated viral RNA/DNA in systemic and lymphoid tissues in simian immunodeficiency virus (SIV)-infected rhesus macaques on prolonged combined antiretroviral therapy (cART) and evaluated predictors of viral rebound after treatment cessation. The results showed that suppressive ART substantially reduced plasma SIV RNA, cell-associated unspliced, and multiply spliced SIV RNA to undetectable levels, yet viral DNA remained detectable in systemic tissues and lymphoid compartments throughout cART. Intriguingly, a rapid increase of integrated proviral DNA in peripheral mononuclear cells was detected once treatment was withdrawn, accompanied by the emergence of detectable plasma viral load. Notably, the increase of peripheral proviral DNA after treatment interruption correlated with the emergence and degree of viral rebound. These findings suggest that measuring total viral DNA in SIV infection may be a relatively simple surrogate marker of reservoir size and may predict viral rebound after treatment interruption and inform treatment strategies.IMPORTANCE Viral reservoirs are involved in persistent HIV infection, and a small number of mosaic latent cellular reservoirs promote viral rebound upon analytical treatment interruption, which is the major obstacle to a cure. However, early indicators that can predict resurgence of viremia after treatment interruption may aid treatment decisions in people living with HIV. Utilizing the rhesus macaque model, we demonstrated that increased proviral DNA in peripheral cells after treatment interruption, rather than levels of proviral DNA, was a useful marker to predict the emergence and degree of viral rebound after treatment interruption, providing a rapid approach for monitoring HIV rebound and informing decisions.

Th17 T Cells and Immature Dendritic Cells Are the Preferential Initial Targets after Rectal Challenge with a Simian Immunodeficiency Virus-Based Replication-Defective Dual-Reporter Vector.

Understanding the earliest events of human immunodeficiency virus (HIV) sexual transmission is critical to developing and optimizing HIV prevention strategies. To gain insights into the earliest steps of HIV rectal transmission, including cellular targets, rhesus macaques were intrarectally challenged with a single-round simian immunodeficiency virus (SIV)-based dual reporter that expresses luciferase and near-infrared fluorescent protein 670 (iRFP670) upon productive transduction. The vector was pseudotyped with the HIV-1 envelope JRFL. Regions of tissue containing foci of luminescent transduced cells were identified macroscopically using an in vivo imaging system, and individual transduced cells expressing fluorescent protein were identified and phenotyped microscopically. This system revealed that anal and rectal tissues are both susceptible to transduction 48 h after the rectal challenge. Detailed phenotypic analysis revealed that, on average, 62% of transduced cells are CCR6-positive (CCR6+) T cells-the vast majority of which express RORγT, a Th17 lineage-specific transcription factor. The second most common target cells were immature dendritic cells at 20%. These two cell types were transduced at rates that are four to five times higher than their relative abundances indicate. Our work demonstrates that Th17 T and immature dendritic cells are preferential initial targets of HIV/SIV rectal transmission. IMPORTANCE Men and women who participate in unprotected receptive anal intercourse are at high risk of acquiring HIV. While in vitro data have developed a framework for understanding HIV cell tropism, the initial target cells in the rectal mucosa have not been identified. In this study, we identify these early host cells by using an innovative rhesus macaque rectal challenge model and methodology, which we previously developed. Thus, by shedding light on these early HIV/SIV transmission events, this study provides a specific cellular target for future prevention strategies.

Immune Responses and Viral Persistence in Simian/Human Immunodeficiency Virus SHIV.C.CH848-Infected Rhesus Macaques.

Chimeric simian/human immunodeficiency viruses (SHIVs) are widely used in nonhuman primate models to recapitulate human immunodeficiency virus (HIV) infection in humans, yet most SHIVs fail to establish persistent viral infection. We investigated immunological and virological events in rhesus macaques infected with the newly developed SHIV.C.CH848 (SHIVC) and treated with combined antiretroviral therapy (cART). Similar to HIV/simian immunodeficiency virus (SIV) infection, SHIV.C.CH848 infection established viral reservoirs in CD4+ T cells and myeloid cells, accompanied by productive infection and depletion of CD4+ T cells in systemic and lymphoid tissues throughout SHIV infection. Despite 6 months of cART-suppressed viral replication, integrated proviral DNA levels remained stable, especially in CD4+ T cells, and the viral rebound was also observed after ART interruption. Autologous neutralizing antibodies to the parental HIV-1 strain CH848 were detected, with limited viral evolution at 5 months postinfection. In comparison, heterogenous neutralizing antibodies in SHIV.C.CH848-infected macaques were not detected except for 1 (1 of 10) animal at 2 years postinfection. These findings suggest that SHIV.C.CH848, a novel class of transmitted/founder SHIVs, can establish sustained viremia and viral reservoirs in rhesus macaques with clinical immunodeficiency consequences, providing a valuable SHIV model for HIV research.IMPORTANCE SHIVs have been extensively used in a nonhuman primate (NHP) model for HIV research. In this study, we investigated viral reservoirs in tissues and immune responses in an NHP model inoculated with newly generated transmitted/founder HIV-1 clade C-based SHIV.C.CH848. The data show that transmitted founder (T/F) SHIVC infection of macaques more closely recapitulates the virological and clinical features of HIV infection, including persistent viremia and viral rebound once antiretroviral therapy is discontinued. These results suggest this CCR5-tropic, SHIVC strain is valuable for testing responses to HIV vaccines and therapeutics.

Mucosal integrin α4ß7 blockade fails to reduce the seeding and size of viral reservoirs in SIV-infected rhesus macaques.

Cellular viral reservoirs are rapidly established in tissues upon HIV-1/SIV infection, which persist throughout viral infection, even under long-term antiretroviral therapy (ART). Specific integrins are involved in the homing of cells to gut-associated lymphoid tissues (GALT) and inflamed tissues, which may promote the seeding and dissemination of HIV-1/SIV to these tissue sites. In this study, we investigated the efficacy of prophylactic integrin blockade (α4ß7 antibody or α4ß7/α4ß1 dual antagonist TR-14035) on viral infection, as well as dissemination and seeding of viral reservoirs in systemic and lymphoid compartments post-SIV inoculation. The results showed that blockade of α4ß7/α4ß1 did not decrease viral infection, replication, or reduce viral reservoir size in tissues of rhesus macaques after SIV infection, as indicated by equivalent levels of plasma viremia and cell-associated SIV RNA/DNA to controls. Surprisingly, TR-14035 administration in acute SIV infection resulted in consistently higher viremia and more rapid disease progression. These findings suggest that integrin blockade alone fails to effectively control viral infection, replication, dissemination, and reservoir establishment in HIV-1/SIV infection. The use of integrin blockade for prevention or/and therapeutic strategies requires further investigation.

Immunopathogenesis in HIV-associated pediatric tuberculosis.

Tuberculosis (TB) is an increasing global emergency in human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS) patients, in which host immunity is dysregulated and compromised. However, the pathogenesis and efficacy of therapeutic strategies in HIV-associated TB in developing infants are essentially lacking. Bacillus Calmette-Guerin vaccine, an attenuated live strain of Mycobacterium bovis, is not adequately effective, which confers partial protection against Mycobacterium tuberculosis (Mtb) in infants when administered at birth. However, pediatric HIV infection is most devastating in the disease progression of TB. It remains challenging whether early antiretroviral therapy (ART) could maintain immune development and function, and restore Mtb-specific immune function in HIV-associated TB in children. A better understanding of the immunopathogenesis in HIV-associated pediatric Mtb infection is essential to provide more effective interventions, reducing the risk of morbidity and mortality in HIV-associated Mtb infection in infants. IMPACT: Children living with HIV are more likely prone to opportunistic infection, predisposing high risk of TB diseases. HIV and Mtb coinfection in infants may synergistically accelerate disease progression. Early ART may probably induce immune reconstitution inflammatory syndrome and TB pathology in HIV/Mtb coinfected infants.

Novel Transmitted/Founder Simian-Human Immunodeficiency Viruses for Human Immunodeficiency Virus Latency and Cure Research.

A robust simian-human immunodeficiency virus (SHIV)-macaque model of latency is critical to investigate eradicative and suppressive strategies that target HIV-1 Env. To this end, we previously reported a novel strategy for constructing SHIVs that bear primary or transmitted/founder (TF) Envs with modifications at Env residue 375 that enable efficient replication in Indian rhesus macaques (RM). Such TF SHIVs, however, have not been examined for their suitability for HIV-1 latency and cure research. Here, we evaluate two promising TF SHIVs, SHIV.D.191859 and SHIV.C.CH848, which encode TF subtype D and C HIV-1 Envs, respectively, for their viral kinetics and persistence during suppressive combination antiretroviral therapy (cART) and treatment interruption in RM. Our results suggest that the viral kinetics of these SHIVs in RM during acute, early, and chronic infection, and upon cART initiation, maintenance and discontinuation, mirror those of HIV-1 infection. We demonstrate consistent early peak and set point viremia, rapid declines in viremia to undetectable plasma titers following cART initiation, infection of long-lived cellular subsets and establishment of viral latency, and viral rebound with return to pretreatment set point viremia following treatment interruption. The viral dynamics and reservoir biology of SHIV.D.191859, and to a lesser extent SHIV.C.CH848, during chronic infection, cART administration, and upon treatment interruption suggest that these TF SHIVs are promising reagents for a SHIV model of HIV-1 latency and cure.IMPORTANCE Simian-human immunodeficiency viruses (SHIVs) have been successfully used for over 2 decades to study virus-host interactions, transmission, and pathogenesis in rhesus macaques. The majority of Env trimers of most previously studied SHIVs, however, do not recapitulate key properties of transmitted/founder (TF) or primary HIV-1 isolates, such as CCR5 tropism, tier 2 neutralization resistance, and native trimer conformation. Here, we test two recently generated TF SHIVs, SHIV.D.191859 and SHIV.C.CH848, which were designed to address these issues as components of a nonhuman primate model of HIV-1 latency. We conclude that the TF SHIV-macaque model reflects several hallmarks of HIV and SIV infection and latency. Results suggest that this model has broad applications for evaluating eradicative and suppressive strategies against the HIV reservoir, including Env-specific interventions, therapeutic vaccines, and engineered T cells.

A MUC16 IgG Binding Activity Selects for a Restricted Subset of IgG Enriched for Certain Simian Immunodeficiency Virus Epitope Specificities.

We have recently shown that MUC16, a component of the glycocalyx of some mucosal barriers, has elevated binding to the G0 glycoform of the Fc portion of IgG. Therefore, IgG from patients chronically infected with human immunodeficiency virus (HIV), who typically exhibit increased amounts of G0 glycoforms, showed increased MUC16 binding compared to uninfected controls. Using the rhesus macaque simian immunodeficiency virus SIVmac251 model, we can compare plasma antibodies before and after chronic infection. We find increased binding of IgG to MUC16 after chronic SIV infection. Antibodies isolated for tight association with MUC16 (MUC16-eluted antibodies) show reduced FcγR engagement and antibody-dependent cellular cytotoxicity (ADCC) activity. The glycosylation profile of these IgGs was consistent with a decrease in FcγR engagement and subsequent ADCC effector function, as they contain a decrease in afucosylated bisecting glycoforms that preferentially bind FcγRs. Testing of the SIV antigen specificity of IgG from SIV-infected macaques revealed that the MUC16-eluted antibodies were enriched for certain specific epitopes, including regions of gp41 and gp120. This enrichment of specific antigen responses for fucosylated bisecting glycoforms and the subsequent association with MUC16 suggests that the immune response has the potential to direct specific epitope responses to localize to the glycocalyx through interaction with this specific mucin.IMPORTANCE Understanding how antibodies are distributed in the mucosal environment is valuable for developing a vaccine to block HIV infection. Here, we study an IgG binding activity in MUC16, potentially representing a new IgG effector function that would concentrate certain antibodies within the glycocalyx to trap pathogens before they can reach the underlying columnar epithelial barriers. These studies reveal that rhesus macaque IgG responses during chronic SIV infection generate increased antibodies that bind MUC16, and interestingly, these MUC16-tethered antibodies are enriched for binding to certain antigens. Therefore, it may be possible to direct HIV vaccine-generated responses to associate with MUC16 and enhance the antibody's ability to mediate immune exclusion by trapping virions within the glycocalyx and preventing the virus from reaching immune target cells within the mucosa. This concept will ultimately have to be tested in the rhesus macaque model, which is shown here to have MUC16-targeted antigen responses.

Informed consent disclosure to vaccine trial subjects of risk of COVID-19 vaccines worsening clinical disease.

AIMS OF THE STUDY: Patient comprehension is a critical part of meeting medical ethics standards of informed consent in study designs. The aim of the study was to determine if sufficient literature exists to require clinicians to disclose the specific risk that COVID-19 vaccines could worsen disease upon exposure to challenge or circulating virus. METHODS USED TO CONDUCT THE STUDY: Published literature was reviewed to identify preclinical and clinical evidence that COVID-19 vaccines could worsen disease upon exposure to challenge or circulating virus. Clinical trial protocols for COVID-19 vaccines were reviewed to determine if risks were properly disclosed. RESULTS OF THE STUDY: COVID-19 vaccines designed to elicit neutralising antibodies may sensitise vaccine recipients to more severe disease than if they were not vaccinated. Vaccines for SARS, MERS and RSV have never been approved, and the data generated in the development and testing of these vaccines suggest a serious mechanistic concern: that vaccines designed empirically using the traditional approach (consisting of the unmodified or minimally modified coronavirus viral spike to elicit neutralising antibodies), be they composed of protein, viral vector, DNA or RNA and irrespective of delivery method, may worsen COVID-19 disease via antibody-dependent enhancement (ADE). This risk is sufficiently obscured in clinical trial protocols and consent forms for ongoing COVID-19 vaccine trials that adequate patient comprehension of this risk is unlikely to occur, obviating truly informed consent by subjects in these trials. CONCLUSIONS DRAWN FROM THE STUDY AND CLINICAL IMPLICATIONS: The specific and significant COVID-19 risk of ADE should have been and should be prominently and independently disclosed to research subjects currently in vaccine trials, as well as those being recruited for the trials and future patients after vaccine approval, in order to meet the medical ethics standard of patient comprehension for informed consent.

A Subcutaneous Implant of Tenofovir Alafenamide Fumarate Causes Local Inflammation and Tissue Necrosis in Rabbits and Macaques.

We describe the in vitro and in vivo evaluation of a subcutaneous reservoir implant delivering tenofovir alafenamide hemifumarate (TAF) for the prevention of HIV infection. These long-acting reservoir implants were able to deliver antiretroviral drug for over 90 days in vitro and in vivo We evaluated the implants for implantation site histopathology and pharmacokinetics in plasma and tissues for up to 12 weeks in New Zealand White rabbit and rhesus macaque models. A dose-ranging study in rabbits demonstrated dose-dependent pharmacokinetics and local inflammation up to severe necrosis around the active implants. The matched placebos showed normal wound healing and fibrous tissue encapsulation of the implant. We designed a second implant with a lower release rate and flux of TAF and achieved a median cellular level of tenofovir diphosphate of 42 fmol per 106 rhesus macaque peripheral blood mononuclear cells at a TAF dose of 10 µg/kg/day. This dose and flux of TAF also resulted in adverse local inflammation and necrosis near the implant in rhesus macaques. The level of inflammation in the primates was markedly lower in the placebo group than in the active-implant group. The histological inflammatory response to the TAF implant at 4 and 12 weeks in primates was graded as a severe reaction. Thus, while we were able to achieve a sustained target dose, we observed an unacceptable inflammatory response locally at the implant tissue interface.

Inhibition of p38 MAPK in combination with ART reduces SIV-induced immune activation and provides additional protection from immune system deterioration.

Differences in immune activation were identified as the most significant difference between AIDS-susceptible and resistant species. p38 MAPK, activated in HIV infection, is key to induction of interferon-stimulated genes and cytokine-mediated inflammation and is associated with some of the pathology produced by HIV or SIV infection in AIDS-susceptible primates. As small molecule p38 MAPK inhibitors are being tested in human trials for inflammatory diseases, we evaluated the effects of treating SIV-infected macaques with the p38 MAPK inhibitor PH-797804 in conjunction with ART. PH-797804 had no side effects, did not impact negatively the antiviral immune response and, used alone, had no significant effect on levels of immune activation and did not reduced the viremia. When administered with ART, it significantly reduced numerous immune activation markers compared to ART alone. CD38+/HLA-DR+ and Ki-67+ T-cell percentages in blood, lymph node and rectal CD4+ and CD8+ T cells, PD-1 expression in CD8+ T cells and plasma levels of IFNα, IFNγ, TNFα, IL-6, IP-10, sCD163 and C-reactive protein were all significantly reduced. Significant preservation of CD4+, CD4+ central memory, CD4+/IL-22+ and CD4+/IL-17+ T-cell percentages and improvement of Th17/Treg ratio in blood and rectal mucosa were also observed. Importantly, the addition of PH-797804 to ART initiated during chronic SIV infection reduced immune activation and restored immune system parameters to the levels observed when ART was initiated on week 1 after infection. After ART interruption, viremia rebounded in a similar fashion in all groups, regardless of when ART was initiated. We concluded that the inhibitor PH-797804 significantly reduced, even if did not normalized, the immune activation parameters evaluated during ART treatment, improved preservation of critical populations of the immune system targeted by SIV, and increased the efficacy of ART treatment initiated in chronic infection to levels similar to those observed when initiated in acute infection but did not affect positively or negatively viral reservoirs.

Chemokine receptor CCR5 correlates with functional CD8+ T cells in SIV-infected macaques and the potential effects of maraviroc on T-cell activation.

C-C chemokine receptor 5 (CCR5) plays an essential role in HIV pathogenesis as the major coreceptor on CD4+ T cells used by HIV, yet the function of CCR5 on CD8 T cells is not well understood. Furthermore, the immunologic effects of the CCR5 inhibitor maraviroc (MVC), despite approval for clinical use, have not yet been well evaluated for their potential effects on cytotoxic T-cell responses. In this study, we characterized the development and function of CCR5+CD8+ T cells in rhesus macaques with or without Simian immunodeficiency virus (SIV) infection. We also investigated the effects of the CCR5 antagonist MVC on functional CCR5+CD8+ T-cell responses in vitro. The data show that CCR5+CD8+ T cells have an effector memory phenotype and increase with age in systemic and mucosal lymphoid tissues as a heterogeneous population of polyfunctional CD8 T cells. In addition, CCR5 is highly expressed on SIV gag-specific (CM9+) CD8+ T cells in SIV-infected macaques, yet CCR5+CD8+ T cells are significantly reduced in mucosal lymphoid tissues with disease progression. Furthermore, in vitro MVC treatment reduced activation and cytokine secretion of CD8+ T cells via a CCR5-independent pathway. These findings suggest that surface CCR5 protein plays an important role in differentiation and activation of CD8+ T cells. Although MVC may be helpful in reducing chronic inflammation and activation, it may also inhibit virus-specific CD8+ T-cell responses. Thus optimal use of CCR5 antagonists either alone or in combination with other drugs should be defined by further investigation.-Wang, X., Russell-Lodrigue, K. E., Ratterree, M. S., Veazey, R. S., Xu, H. Chemokine receptor CCR5 correlates with functional CD8+ T cells in SIV-infected macaques and the potential effects of maraviroc on T-cell activation.

BCL6 BTB-specific inhibition via FX1 treatment reduces Tfh cells and reverses lymphoid follicle hyperplasia in Indian rhesus macaque (Macaca mulatta).

BACKGROUND: The BTB domain of B-cell lymphoma 6 (BCL6) protein was identified as a therapeutic target for B-cell lymphoma. This study compared the pharmacokinetics (PK) of the BCL6 BTB inhibitor (FX1) between mice and macaques, as well as evaluating its lymphoid suppressive effect in uninfected macaques with lymphoid hyperplasia. MATERIALS AND METHODS: Eight uninfected adult Indian rhesus macaques (Macaca mulatta) were used in the study, four animals carrying lymphoid tissue hyperplasia. Plasma FX1 levels were measured by HPLC-MS/MS. Lymph node biopsies were used for H&E and immunohistochemistry staining, as well as mononuclear cell isolation for flow cytometry analysis. RESULTS: Inhibition of the BCL6 BTB domain with FX1 led to a reduction in the frequency of GC, Tfh CD4+ , and Tfh precursor cells, as well as resolving lymphoid hyperplasia, in rhesus macaques. CONCLUSIONS: B-cell lymphoma 6 inhibition may represent a novel strategy to reduce hyperplastic lymphoid B-cell follicles and decrease Tfh cells.

Impaired Development and Expansion of Germinal Center Follicular Th Cells in Simian Immunodeficiency Virus-Infected Neonatal Macaques.

Germinal center (GC) CD4+ follicular Th (Tfh) cells are critical for cognate B cell help in humoral immune responses to pathogenic infections. Although Tfh cells are expanded or depleted in HIV/SIV-infected adults, the effects of pediatric HIV/SIV infection on Tfh cells remain unclear. In this study, we examined changes in lymphoid follicle formation in lymph nodes focusing on GC Tfh cells, B cell development, and differentiation in SIV-infected neonatal rhesus macaques (Macaca mulatta) compared with age-matched cohorts. Our data showed that follicles and GCs of normal infants rapidly formed in the first few weeks of age, in parallel with increasing GC Tfh cells in various lymphoid tissues. In contrast, GC development and GC Tfh cells were markedly impaired in SIV-infected infants. There was a very low frequency of GC Tfh cells throughout SIV infection in neonates and subsequent infants, accompanied by high viremia, reduction of B cell proliferation/resting memory B cells, and displayed proinflammatory unresponsiveness. These findings indicate neonatal HIV/SIV infection compromises the development of GC Tfh cells, likely contributing to ineffective Ab responses, high viremia, and eventually rapid disease progression to AIDS.

An HSV-2 Trivalent Vaccine Is Immunogenic in Rhesus Macaques and Highly Efficacious in Guinea Pigs.

A genital herpes vaccine is urgently needed to prevent pain and suffering, reduce the incidence of neonatal herpes, and decrease the risk of HIV acquisition and transmission that accompanies genital infection. We evaluated a trivalent HSV-2 subunit antigen vaccine administered with CpG and alum in rhesus macaques and guinea pigs. The vaccine contains glycoproteins C, D and E (gC2, gD2, gE2) to block virus entry by gD2 and immune evasion by gC2 and gE2. In rhesus macaques, the trivalent vaccine induced plasma and mucosa neutralizing antibodies, antibodies that block gC2 and gE2 immune evasion activities, and stimulated CD4 T cell responses. After intravaginal challenge, a self-limited vaginal infection of brief duration was detected by histopathology and immunohistochemistry in naïve, but not in trivalent immunized macaques. Vaccine efficacy was evaluated in female guinea pigs. Animals were mock immunized, or immunized with gD2, the trivalent vaccine or the trivalent vaccine followed by a booster dose of gD2 (trivalent + gD2). The trivalent and trivalent + gD2 groups were 97% and 99% efficacious, respectively in preventing genital lesions and both outperformed gD2 alone. As a marker of transmission risk, vaginal swabs were evaluated daily for HSV-2 DNA and replication competent virus between five and seven weeks after challenge. HSV-2 DNA shedding was reduced in all groups compared with mock. Shedding of replication competent virus occurred on fewer days in the trivalent than gD2 immunized animals while the trivalent + gD2 group had no shedding of replication competent virus. Overall, the trivalent group had genital lesions on < 1% days and shedding of replication competent virus on 0.2% days. The vaccine has outstanding potential for prevention of genital herpes in humans.