Defining the risk of SARS-CoV-2 variants on immune protection.

The global emergence of many severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants jeopardizes the protective antiviral immunity induced after infection or vaccination. To address the public health threat caused by the increasing SARS-CoV-2 genomic diversity, the National Institute of Allergy and Infectious Diseases within the National Institutes of Health established the SARS-CoV-2 Assessment of Viral Evolution (SAVE) programme. This effort was designed to provide a real-time risk assessment of SARS-CoV-2 variants that could potentially affect the transmission, virulence, and resistance to infection- and vaccine-induced immunity. The SAVE programme is a critical data-generating component of the US Government SARS-CoV-2 Interagency Group to assess implications of SARS-CoV-2 variants on diagnostics, vaccines and therapeutics, and for communicating public health risk. Here we describe the coordinated approach used to identify and curate data about emerging variants, their impact on immunity and effects on vaccine protection using animal models. We report the development of reagents, methodologies, models and notable findings facilitated by this collaborative approach and identify future challenges. This programme is a template for the response to rapidly evolving pathogens with pandemic potential by monitoring viral evolution in the human population to identify variants that could reduce the effectiveness of countermeasures.

HIV, asymptomatic STI, and the rectal mucosal immune environment among young men who have sex with men.

Young men who have sex with men (YMSM) are disproportionately affected by HIV and bacterial sexually transmitted infections (STI) including gonorrhea, chlamydia, and syphilis; yet research into the immunologic effects of these infections is typically pursued in siloes. Here, we employed a syndemic approach to understand potential interactions of these infections on the rectal mucosal immune environment among YMSM. We enrolled YMSM aged 18-29 years with and without HIV and/or asymptomatic bacterial STI and collected blood, rectal secretions, and rectal tissue biopsies. YMSM with HIV were on suppressive antiretroviral therapy (ART) with preserved blood CD4 cell counts. We defined 7 innate and 19 adaptive immune cell subsets by flow cytometry, the rectal mucosal transcriptome by RNAseq, and the rectal mucosal microbiome by 16S rRNA sequencing and examined the effects of HIV and STI and their interactions. We measured tissue HIV RNA viral loads among YMSM with HIV and HIV replication in rectal explant challenge experiments among YMSM without HIV. HIV, but not asymptomatic STI, was associated with profound alterations in the cellular composition of the rectal mucosa. We did not detect a difference in the microbiome composition associated with HIV, but asymptomatic bacterial STI was associated with a higher probability of presence of potentially pathogenic taxa. When examining the rectal mucosal transcriptome, there was evidence of statistical interaction; asymptomatic bacterial STI was associated with upregulation of numerous inflammatory genes and enrichment for immune response pathways among YMSM with HIV, but not YMSM without HIV. Asymptomatic bacterial STI was not associated with differences in tissue HIV RNA viral loads or in HIV replication in explant challenge experiments. Our results suggest that asymptomatic bacterial STI may contribute to inflammation particularly among YMSM with HIV, and that future research should examine potential harms and interventions to reduce the health impact of these syndemic infections.

A neutralizing antibody target in early HIV-1 infection was recapitulated in rhesus macaques immunized with the transmitted/founder envelope sequence.

Transmitted/founder (T/F) HIV-1 envelope proteins (Envs) from infected individuals that developed neutralization breadth are likely to possess inherent features desirable for vaccine immunogen design. To explore this premise, we conducted an immunization study in rhesus macaques (RM) using T/F Env sequences from two human subjects, one of whom developed potent and broad neutralizing antibodies (Z1800M) while the other developed little to no neutralizing antibody responses (R66M) during HIV-1 infection. Using a DNA/MVA/protein immunization protocol, 10 RM were immunized with each T/F Env. Within each T/F Env group, the protein boosts were administered as either monomeric gp120 or stabilized trimeric gp140 protein. All vaccination regimens elicited high titers of antigen-specific IgG, and two animals that received monomeric Z1800M Env gp120 developed autologous neutralizing activity. Using early Env escape variants isolated from subject Z1800M as guides, the serum neutralizing activity of the two immunized RM was found to be dependent on the gp120 V5 region. Interestingly, the exact same residues of V5 were also targeted by a neutralizing monoclonal antibody (nmAb) isolated from the subject Z1800M early in infection. Glycan profiling and computational modeling of the Z1800M Env gp120 immunogen provided further evidence that the V5 loop is exposed in this T/F Env and was a dominant feature that drove neutralizing antibody targeting during infection and immunization. An expanded B cell clonotype was isolated from one of the neutralization-positive RM and nmAbs corresponding to this group demonstrated V5-dependent neutralization similar to both the RM serum and the human Z1800M nmAb. The results demonstrate that neutralizing antibody responses elicited by the Z1800M T/F Env in RM converged with those in the HIV-1 infected human subject, illustrating the potential of using immunogens based on this or other T/F Envs with well-defined immunogenicity as a starting point to drive breadth.

The C3/465 glycan hole cluster in BG505 HIV-1 envelope is the major neutralizing target involved in preventing mucosal SHIV infection.

Stabilized HIV-1 envelope (Env) trimers elicit tier 2 autologous neutralizing antibody (nAb) responses in immunized animals. We previously demonstrated that BG505 SOSIP.664.T332N gp140 (BG505 SOSIP) immunization of rhesus macaques (RM) provided robust protection against autologous intra-vaginal simian-human immunodeficiency virus (SHIV) challenge that was predicted by high serum nAb titers. Here, we show that nAb in these protected RM targeted a glycan hole proximal to residue 465 in gp120 in all cases. nAb also targeted another glycan hole at residues 241/289 and an epitope in V1 at varying frequencies. Non-neutralizing antibodies directed at N611-shielded epitopes in gp41 were also present but were more prevalent in RM with low nAb titers. Longitudinal analysis demonstrated that nAb broadened in some RM during sequential immunization but remained focused in others, the latter being associated with increases in nAb titer. Thirty-eight monoclonal antibodies (mAbs) isolated from a protected RM with an exceptionally high serum neutralization titer bound to the trimer in ELISA, and four of the mAbs potently neutralized the BG505 Env pseudovirus (PV) and SHIV. The four neutralizing mAbs were clonally related and targeted the 465 glycan hole to varying degrees, mimicking the serum. The data demonstrate that the C3/465 glycan hole cluster was the dominant neutralization target in high titer protected RM, despite other co-circulating neutralizing and non-neutralizing specificities. The isolation of a neutralizing mAb family argues that clonotype expansion occurred during BG505 SOSIP immunization, leading to high titer, protective nAb and setting a desirable benchmark for HIV vaccines.

Systematic Assessment of Antiviral Potency, Breadth, and Synergy of Triple Broadly Neutralizing Antibody Combinations against Simian-Human Immunodeficiency Viruses.

Daily burden and clinical toxicities associated with antiretroviral therapy (ART) emphasize the need for alternative strategies to induce long-term human immunodeficiency virus (HIV) remission upon ART cessation. Broadly neutralizing antibodies (bNAbs) can both neutralize free virions and mediate effector functions against infected cells and therefore represent a leading immunotherapeutic approach. To increase potency and breadth, as well as to limit the development of resistant virus strains, it is likely that bNAbs will need to be administered in combination. It is therefore critical to identify bNAb combinations that can achieve robust polyfunctional antiviral activity against a high number of HIV strains. In this study, we systematically assessed the abilities of single bNAbs and triple bNAb combinations to mediate robust polyfunctional antiviral activity against a large panel of cross-clade simian-human immunodeficiency viruses (SHIVs), which are commonly used as tools for validation of therapeutic strategies targeting the HIV envelope in nonhuman primate models. We demonstrate that most bNAbs are capable of mediating both neutralizing and nonneutralizing effector functions against cross-clade SHIVs, although the susceptibility to V3 glycan-specific bNAbs is highly strain dependent. Moreover, we observe a strong correlation between the neutralization potencies and nonneutralizing effector functions of bNAbs against the transmitted/founder SHIV CH505. Finally, we identify several triple bNAb combinations comprising of CD4 binding site-, V2-glycan-, and gp120-gp41 interface-targeting bNAbs that are capable of mediating synergistic polyfunctional antiviral activities against multiple clade A, B, C, and D SHIVs.IMPORTANCE Optimal bNAb immunotherapeutics will need to mediate multiple antiviral functions against a broad range of HIV strains. Our systematic assessment of triple bNAb combinations against SHIVs will identify bNAbs with synergistic, polyfunctional antiviral activity that will inform the selection of candidate bNAbs for optimal combination designs. The identified combinations can be validated in vivo in future passive immunization studies using the SHIV challenge model.

Impact of Th1 CD4 Follicular Helper T Cell Skewing on Antibody Responses to an HIV-1 Vaccine in Rhesus Macaques.

Generating durable humoral immunity through vaccination depends upon effective interactions of follicular helper T (Tfh) cells with germinal center (GC) B cells. Th1 polarization of Tfh cells is an important process shaping the success of Tfh-GC B cell interactions by influencing costimulatory and cytokine-dependent Tfh help to B cells. However, the question remains as to whether adjuvant-dependent modulation of Tfh cells enhances HIV-1 vaccine-induced antienvelope (anti-Env) antibody responses. We investigated whether an HIV-1 vaccine platform designed to increase the number of Th1-polarized Tfh cells enhances the magnitude and quality of anti-Env antibodies. Utilizing a novel interferon-induced protein 10 (IP-10)-adjuvanted HIV-1 DNA prime followed by a monophosphoryl lipid A and QS-21 (MPLA+QS-21)-adjuvanted Env protein boost (DIP-10 PALFQ) in macaques, we observed higher anti-Env serum IgG titers with greater cross-clade reactivity, specificity for V1V2, and effector functions than in macaques primed with DNA lacking IP-10 and boosted with MPLA-plus-alum-adjuvanted Env protein (DPALFA) The DIP-10 PALFQ vaccine regimen elicited higher anti-Env IgG1 and lower IgG4 antibody levels in serum, showing for the first time that adjuvants can dramatically impact the IgG subclass profile in macaques. The DIP-10 PALFQ regimen also increased vaginal and rectal IgA antibodies to a greater extent. Within lymph nodes, we observed augmented GC B cell responses and the promotion of Th1 gene expression profiles in GC Tfh cells. The frequency of GC Tfh cells correlated with both the magnitude and avidity of anti-Env serum IgG. Together, these data suggest that adjuvant-induced stimulation of Th1-Tfh cells is an effective strategy for enhancing the magnitude and quality of anti-Env antibody responses.IMPORTANCE The results of the RV144 trial demonstrated that vaccination could prevent HIV transmission in humans and that longevity of anti-Env antibodies may be key to this protection. Efforts to improve upon the prime-boost vaccine regimen used in RV144 have indicated that booster immunizations can increase serum anti-Env antibody titers but only transiently. Poor antibody durability hampers efforts to develop an effective HIV-1 vaccine. This study was designed to identify the specific elements involved in the immunological mechanism necessary to produce robust HIV-1-specific antibodies in rhesus macaques. By clearly defining immune-mediated pathways that improve the magnitude and functionality of the anti-HIV-1 antibody response, we will have the foundation necessary for the rational development of an HIV-1 vaccine.

Improving rigor and reproducibility in nonhuman primate research.

Nonhuman primates (NHPs) are a critical component of translational/preclinical biomedical research due to the strong similarities between NHP and human physiology and disease pathology. In some cases, NHPs represent the most appropriate, or even the only, animal model for complex metabolic, neurological, and infectious diseases. The increased demand for and limited availability of these valuable research subjects requires that rigor and reproducibility be a prime consideration to ensure the maximal utility of this scarce resource. Here, we discuss a number of approaches that collectively can contribute to enhanced rigor and reproducibility in NHP research.

Novel Modified Vaccinia Virus Ankara Vector Expressing Anti-apoptotic Gene B13R Delays Apoptosis and Enhances Humoral Responses.

Modified vaccinia virus Ankara (MVA), an attenuated poxvirus, has been developed as a potential vaccine vector for use against cancer and multiple infectious diseases, including human immunodeficiency virus (HIV). MVA is highly immunogenic and elicits strong cellular and humoral responses in preclinical models and humans. However, there is potential to further enhance the immunogenicity of MVA, as MVA-infected cells undergo rapid apoptosis, leading to faster clearance of recombinant antigens and potentially blunting a greater response. Here, we generated MVA-B13R by replacing the fragmented 181R/182R genes of MVA with a functional anti-apoptotic gene, B13R, and confirmed its anti-apoptotic function against chemically induced apoptosis in vitro In addition, MVA-B13R showed a significant delay in induction of apoptosis in muscle cells derived from mice and humans, as well as in plasmacytoid dendritic cells (pDCs) and CD141+ DCs from rhesus macaques, compared to the induction of apoptosis in MVA-infected cells. MVA-B13R expressing simian immunodeficiency virus (SIV) Gag and Pol and HIV envelope (SHIV) (MVA-B13R/SHIV) produced higher levels of envelope in the supernatants than MVA/SHIV-infected DF-1 cells in vitro Immunization of BALB/c mice showed induction of higher levels of envelope-specific antibody-secreting cells and memory B cells, higher IgG antibody titers, and better persistence of antibody titers with MVA-B13R/SHIV than with MVA/SHIV. Gene set enrichment analysis of draining lymph node cells from day 1 after immunization showed negative enrichment for interferon responses in MVA-B13R/SHIV-immunized mice compared to the responses in MVA/SHIV-immunized mice. Taken together, these results demonstrate that restoring B13R functionality in MVA significantly delays MVA-induced apoptosis in muscle and antigen-presenting cells in vitro and augments vaccine-induced humoral immunity in mice.IMPORTANCE MVA is an attractive viral vector for vaccine development due to its safety and immunogenicity in multiple species and humans even under conditions of immunodeficiency. Here, to further improve the immunogenicity of MVA, we developed a novel vector, MVA-B13R, by replacing the fragmented anti-apoptotic genes 181R/182R with a functional version derived from vaccinia virus, B13R Our results show that MVA-B13R significantly delays apoptosis in antigen-presenting cells and muscle cells in vitro and augments vaccine-induced humoral immunity in mice, leading to the development of a novel vector for vaccine development against infectious diseases and cancer.

Dynamics of SIV-specific CXCR5+ CD8 T cells during chronic SIV infection.

A significant challenge to HIV eradication is the elimination of viral reservoirs in germinal center (GC) T follicular helper (Tfh) cells. However, GCs are considered to be immune privileged for antiviral CD8 T cells. Here, we show a population of simian immunodeficiency virus (SIV)-specific CD8 T cells express CXCR5 (C-X-C chemokine receptor type 5, a chemokine receptor required for homing to GCs) and expand in lymph nodes (LNs) following pathogenic SIV infection in a cohort of vaccinated macaques. This expansion was greater in animals that exhibited superior control of SIV. The CXCR5+ SIV-specific CD8 T cells demonstrated enhanced polyfunctionality, restricted expansion of antigen-pulsed Tfh cells in vitro, and possessed a unique gene expression pattern related to Tfh and Th2 cells. The increase in CXCR5+ CD8 T cells was associated with the presence of higher frequencies of SIV-specific CD8 T cells in the GC. Following TCR-driven stimulation in vitro, CXCR5+ but not CXCR5- CD8 T cells generated both CXCR5+ as well as CXCR5- cells. However, the addition of TGF-ß to CXCR5- CD8 T cells induced a population of CXCR5+ CD8 T cells, suggesting that this cytokine may be important in modulating these CXCR5+ CD8 T cells in vivo. Thus, CXCR5+ CD8 T cells represent a unique subset of antiviral CD8 T cells that expand in LNs during chronic SIV infection and may play a significant role in the control of pathogenic SIV infection.

Intragastric Administration of Lactobacillus plantarum and 2,2'-Dithiodipyridine-Inactivated Simian Immunodeficiency Virus (SIV) Does Not Protect Indian Rhesus Macaques from Intrarectal SIV Challenge or Reduce Virus Replication after Transmission.

A major obstacle to development of an effective AIDS vaccine is that along with the intended beneficial responses, the immunization regimen may activate CD4+ T cells that can facilitate acquisition of human immunodeficiency virus (HIV) by serving as target cells for the virus. Lu et al. (W. Lu et al., Cell Rep 2:1736-1746, 2012, https://doi.org/10.1016/j.celrep.2012.11.016) reported that intragastric administration of chemically inactivated simian immunodeficiency virus SIVmac239 and Lactobacillus plantarum (iSIV-L. plantarum) protected 15/16 Chinese-origin rhesus macaques (RMs) from high-dose intrarectal SIVmac239 challenge at 3 months postimmunization. They attributed the observed protection to induction of immune tolerance, mediated by "MHC-Ib/E-restricted CD8+ regulatory T cells that suppressed SIV-harboring CD4+ T cell activation and ex vivo SIV replication in 15/16 animals without inducing SIV-specific antibodies or cytotoxic T." J.-M. Andrieu et al. (Front Immunol 5:297, 2014, https://doi.org/10.3389/fimmu.2014.00297) subsequently reported protection from infection in 23/24 RMs immunized intragastrically or intravaginally with iSIV and Mycobacterium bovis BCG, L. plantarum, or Lactobacillus rhamnosus, which they ascribed to the same tolerogenic mechanism. Using vaccine materials obtained from our coauthors, we conducted an immunization and challenge experiment with 54 Indian RMs and included control groups receiving iSIV only or L. plantarum only as well as unvaccinated animals. Intrarectal challenge with SIVmac239 resulted in rapid infection in all groups of vaccinated RMs as well as unvaccinated controls. iSIV-L. plantarum-vaccinated animals that became SIV infected showed viral loads similar to those observed in animals receiving iSIV only or L. plantarum only or in unvaccinated controls. The protection from SIV transmission conferred by intragastric iSIV-L. plantarum administration reported previously for Chinese-origin RMs was not observed when the same experiment was conducted in a larger cohort of Indian-origin animals.IMPORTANCE Despite an increased understanding of immune responses against HIV, a safe and effective AIDS vaccine is not yet available. One obstacle is that immunization may activate CD4+ T cells that may act as target cells for acquisition of HIV. An alternative strategy may involve induction of a tolerance-inducing response that limits the availability of activated CD4+ T cells, thus limiting the ability of virus to establish infection. In this regard, exciting results were obtained for Chinese-origin rhesus macaques by using a "tolerogenic" vaccine, consisting of intragastric administration of Lactobacillus plantarum and 2,2'-dithiodipyridine-inactivated SIV, which showed highly significant protection from virus transmission. In the present study, we administered iSIV-L. plantarum to Indian-origin rhesus macaques and failed to observe any protective effect on virus acquisition in this experimental setting. This work is important because it contributes to the overall assessment of the clinical potential of a new candidate AIDS vaccine platform based on iSIV-L. plantarum.

Adjuvanting a Simian Immunodeficiency Virus Vaccine with Toll-Like Receptor Ligands Encapsulated in Nanoparticles Induces Persistent Antibody Responses and Enhanced Protection in TRIM5α Restrictive Macaques.

Our previous work has shown that antigens adjuvanted with ligands specific for Toll-like receptor 4 (TLR4) and TLR7/8 encapsulated in poly(lactic-co-glycolic) acid (PLGA)-based nanoparticles (NPs) induce robust and durable immune responses in mice and macaques. We investigated the efficacy of these NP adjuvants in inducing protective immunity against simian immunodeficiency virus (SIV). Rhesus macaques (RMs) were immunized with NPs containing TLR4 and TLR7/8 agonists mixed with soluble recombinant SIVmac239-derived envelope (Env) gp140 and Gag p55 (protein) or with virus-like particles (VLPs) containing SIVmac239 Env and Gag. NP-adjuvanted vaccines induced robust innate responses, antigen-specific antibody responses of a greater magnitude and persistence, and enhanced plasmablast responses compared to those achieved with alum-adjuvanted vaccines. NP-adjuvanted vaccines induced antigen-specific, long-lived plasma cells (LLPCs), which persisted in the bone marrow for several months after vaccination. NP-adjuvanted vaccines induced immune responses that were associated with enhanced protection against repeated low-dose, intravaginal challenges with heterologous SIVsmE660 in animals that carried TRIM5α restrictive alleles. The protection induced by immunization with protein-NP correlated with the prechallenge titers of Env-specific IgG antibodies in serum and vaginal secretions. However, no such correlate was apparent for immunization with VLP-NP or alum as the adjuvant. Transcriptional profiling of peripheral blood mononuclear cells isolated within the first few hours to days after primary vaccination revealed that NP-adjuvanted vaccines induced a molecular signature similar to that induced by the live attenuated yellow fever viral vaccine. This systems approach identified early blood transcriptional signatures that correlate with Env-specific antibody responses in vaginal secretions and protection against infection. These results demonstrate the adjuvanticity of the NP adjuvant in inducing persistent and protective antibody responses against SIV in RMs with implications for the design of vaccines against human immunodeficiency virus (HIV). IMPORTANCE: The results of the RV144 HIV vaccine trial, which demonstrated a rapid waning of protective immunity with time, have underscored the need to develop strategies to enhance the durability of protective immune responses. Our recent work in mice has highlighted the capacity of nanoparticle-encapsulated TLR ligands (NP) to induce potent and durable antibody responses that last a lifetime in mice. In the present study, we evaluated the ability of these NP adjuvants to promote robust and durable protective immune responses against SIV in nonhuman primates. Our results demonstrate that immunization of rhesus macaques with NP adjuvants mixed with soluble SIV Env or a virus-like particle form of Env (VLP) induces potent and durable Env-specific antibody responses in the serum and in vaginal secretions. These responses were superior to those induced by alum adjuvant, and they resulted in enhanced protection against a low-dose intravaginal challenge with a heterologous strain of SIV in animals with TRIM5a restrictive alleles. These results highlight the potential for such NP TLR L adjuvants in promoting robust and durable antibody responses against HIV in the next generation of HIV immunogens currently being developed.

A simultaneous oral and intramuscular prime/sublingual boost with a DNA/Modified Vaccinia Ankara viral vector-based vaccine induces simian immunodeficiency virus-specific systemic and mucosal immune responses in juvenile rhesus macaques.

BACKGROUND: A pediatric vaccine to prevent breast milk transmission of human immunodeficiency virus (HIV) may generate greater immune responses at viral entry sites if given by an oral route. METHODS: We compared immune responses induced in juvenile macaques by prime/boosting with simian immunodeficiency virus (SIV)-expressing DNA/modified vaccinia Ankara virus (MVA) by the intramuscular route (IM), the oral (O)/tonsillar routes (T), the O/sublingual (SL) routes, and O+IM/SL routes. RESULTS: O/T or O/SL immunization generated SIV-specific T cells in mucosal tissues but failed to induce SIV-specific IgA in saliva or stool or IgG in plasma. IM/IM or O+IM/SL generated humoral and cellular responses to SIV. IM/IM generated greater frequencies of TFH in spleen, but O+IM/SL animals had higher avidity plasma IgG and more often demonstrated mucosal IgA responses. CONCLUSION: These results suggest that codelivery of HIV DNA/MVA vaccines by the oral and IM routes might be optimal for generating both systemic and mucosal antibodies.

Breakthrough of SIV strain smE660 challenge in SIV strain mac239-vaccinated rhesus macaques despite potent autologous neutralizing antibody responses.

Although the correlates of immunological protection from human immunodeficiency virus or simian immunodeficiency virus infection remain incompletely understood, it is generally believed that medium to high titers of serum neutralizing antibodies (nAbs) against the challenge virus will prevent infection. This paradigm is based on a series of studies in which passive transfer of HIV-specific nAbs protected rhesus macaques (RMs) from subsequent mucosal challenge with a chimeric human/simian immunodeficiency virus. However, it is unknown whether nAb titers define protection in the setting of active immunization. Here we determined serum nAb titers against breakthrough transmitted/founder (T/F) SIVsmE660-derived envelope glycoprotein (Env) variants from 14 RMs immunized with SIVmac239-based DNA-prime/modified vaccinia virus Ankara-boost vaccine regimens that included GM-CSF or CD40L adjuvants and conferred significant but incomplete protection against repeated low-dose intrarectal challenge. A single Env variant established infection in all RMs except one, with no identifiable genetic signature associated with vaccination breakthrough compared with T/F Envs from four unvaccinated monkeys. Breakthrough T/F Env pseudoviruses were potently neutralized in vitro by heterologous pooled serum from chronically SIVsmE660-infected monkeys at IC50 titers exceeding 1:1,000,000. Remarkably, the T/F Env pseudoviruses from 13 of 14 monkeys were also susceptible to neutralization by autologous prechallenge serum at in vitro IC50 titers ranging from 1:742-1:10,832. These titers were similar to those observed in vaccinated RMs that remained uninfected. These data suggest that the relationship between serum nAb titers and protection from mucosal SIV challenge in the setting of active immunization is more complex than previously recognized, warranting further studies into the balance between immune activation, target cell availability, and protective antibody responses.

Signatures in Simian Immunodeficiency Virus SIVsmE660 Envelope gp120 Are Associated with Mucosal Transmission but Not Vaccination Breakthrough in Rhesus Macaques.

UNLABELLED: Mucosal surfaces are vulnerable to human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) infection and thus are key sites for eliciting vaccine-mediated protection. Vaccine protocols carried out at the Yerkes Primate Research Center utilized SIVmac239-based immunization strategies with intrarectal and intravaginal SIVsmE660 challenge of rhesus macaques. We investigated whether there were genetic signatures associated with SIVsmE660 intrarectal and intravaginal transmissions in vaccinated and unvaccinated monkeys. When transmitted/founder (T/F) envelope (Env) sequences from 49 vaccinated and 15 unvaccinated macaques were compared to each other, we were unable to identify any vaccine breakthrough signatures. In contrast, when the vaccinated and control T/F Envs were combined and compared to the challenge stock, residues at gp120 positions 23, 45, 47, and 70 (Ile-Ala-Lys-Asn [I-A-K-N]) emerged as signatures of mucosal transmission. However, T/F Envs derived from intrarectal and intravaginal infections were not different. Our data suggest that the vaginal and rectal mucosal environments both imposed a strong selection bias for SIVsmE660 variants carrying I-A-K-N that was not further enhanced by immunization. These findings, combined with the strong conservation of A-K-N in most HIV-2/SIVsmm isolates and the analogous residues in HIV-1/SIVcpz isolates, suggest that these residues confer increased transmission fitness to SIVsmE660. IMPORTANCE: Most HIV-1 infections occur across a mucosal barrier, and it is therefore important to understand why these sites are vulnerable and how to protect them with a vaccine. To gain insight into these questions, we studied rhesus macaques that were vaccinated with SIVmac239 and unvaccinated controls to determine whether the SIVsmE660 viral variants that infected these two groups were different. We did not find differences between viral variants in the absence versus presence of vaccination-induced immunity, but we did find that the SIVsmE660 viral variants that infected the monkeys, regardless of vaccination, were different from the dominant population found in the viral challenge inoculum. Our data suggest that the mucosal environments of the vagina and rectum both impose a strong selection for the SIVsmE660 variants in the challenge inoculum that are most like SIV and HIVs that circulate in nature.

Decreased T Follicular Regulatory Cell/T Follicular Helper Cell (TFH) in Simian Immunodeficiency Virus-Infected Rhesus Macaques May Contribute to Accumulation of TFH in Chronic Infection.

T follicular helper cells (TFH) are critical for the development and maintenance of germinal center (GC) and humoral immune responses. During chronic HIV/SIV infection, TFH accumulate, possibly as a result of Ag persistence. The HIV/SIV-associated TFH expansion may also reflect lack of regulation by suppressive follicular regulatory CD4(+) T cells (TFR). TFR are natural regulatory T cells (TREG) that migrate into the follicle and, similar to TFH, upregulate CXCR5, Bcl-6, and PD1. In this study, we identified TFR as CD4(+)CD25(+)FOXP3(+)CXCR5(+)PD1(hi)Bcl-6(+) within lymph nodes of rhesus macaques (RM) and confirmed their localization within the GC by immunohistochemistry. RNA sequencing showed that TFR exhibit a distinct transcriptional profile with shared features of both TFH and TREG, including intermediate expression of FOXP3, Bcl-6, PRDM1, IL-10, and IL-21. In healthy, SIV-uninfected RM, we observed a negative correlation between frequencies of TFR and both TFH and GC B cells, as well as levels of CD4(+) T cell proliferation. Post SIV infection, the TFR/TFH ratio was reduced with no change in the frequency of TREG or TFR within the total CD4(+) T cell pool. Finally, we examined whether higher levels of direct virus infection of TFR were responsible for their relative depletion post SIV infection. We found that TFH, TFR, and TREG sorted from SIV-infected RM harbor comparable levels of cell-associated viral DNA. Our data suggest that TFR may contribute to the regulation and proliferation of TFH and GC B cells in vivo and that a decreased TFR/TFH ratio in chronic SIV infection may lead to unchecked expansion of both TFH and GC B cells.

Characterization and Implementation of a Diverse Simian Immunodeficiency Virus SIVsm Envelope Panel in the Assessment of Neutralizing Antibody Breadth Elicited in Rhesus Macaques by Multimodal Vaccines Expressing the SIVmac239 Envelope.

UNLABELLED: Antibodies that can neutralize diverse viral strains are likely to be an important component of a protective human immunodeficiency virus type 1 (HIV-1) vaccine. To this end, preclinical simian immunodeficiency virus (SIV)-based nonhuman primate immunization regimens have been designed to evaluate and enhance antibody-mediated protection. However, these trials often rely on a limited selection of SIV strains with extreme neutralization phenotypes to assess vaccine-elicited antibody activity. To mirror the viral panels used to assess HIV-1 antibody breadth, we created and characterized a novel panel of 14 genetically and phenotypically diverse SIVsm envelope (Env) glycoproteins. To assess the utility of this panel, we characterized the neutralizing activity elicited by four SIVmac239 envelope-expressing DNA/modified vaccinia virus Ankara vector- and protein-based vaccination regimens that included the immunomodulatory adjuvants granulocyte-macrophage colony-stimulating factor, Toll-like receptor (TLR) ligands, and CD40 ligand. The SIVsm Env panel exhibited a spectrum of neutralization sensitivity to SIV-infected plasma pools and monoclonal antibodies, allowing categorization into three tiers. Pooled sera from 91 rhesus macaques immunized in the four trials consistently neutralized only the highly sensitive tier 1a SIVsm Envs, regardless of the immunization regimen. The inability of vaccine-mediated antibodies to neutralize the moderately resistant tier 1b and tier 2 SIVsm Envs defined here suggests that those antibodies were directed toward epitopes that are not accessible on most SIVsm Envs. To achieve a broader and more effective neutralization profile in preclinical vaccine studies that is relevant to known features of HIV-1 neutralization, more emphasis should be placed on optimizing the Env immunogen, as the neutralization profile achieved by the addition of adjuvants does not appear to supersede the neutralizing antibody profile determined by the immunogen. IMPORTANCE: Many in the HIV/AIDS vaccine field believe that the ability to elicit broadly neutralizing antibodies capable of blocking genetically diverse HIV-1 variants is a critical component of a protective vaccine. Various SIV-based nonhuman primate vaccine studies have investigated ways to improve antibody-mediated protection against a heterologous SIV challenge, including administering adjuvants that might stimulate a greater neutralization breadth. Using a novel SIV neutralization panel and samples from four rhesus macaque vaccine trials designed for cross comparison, we show that different regimens expressing the same SIV envelope immunogen consistently elicit antibodies that neutralize only the very sensitive tier 1a SIV variants. The results argue that the neutralizing antibody profile elicited by a vaccine is primarily determined by the envelope immunogen and is not substantially broadened by including adjuvants, resulting in the conclusion that the envelope immunogen itself should be the primary consideration in efforts to elicit antibodies with greater neutralization breadth.

Vaccine-Induced Linear Epitope-Specific Antibodies to Simian Immunodeficiency Virus SIVmac239 Envelope Are Distinct from Those Induced to the Human Immunodeficiency Virus Type 1 Envelope in Nonhuman Primates.

To evaluate antibody specificities induced by simian immunodeficiency virus (SIV) versus human immunodeficiency virus type 1 (HIV-1) envelope antigens in nonhuman primate (NHP), we profiled binding antibody responses to linear epitopes in NHP studies with HIV-1 or SIV immunogens. We found that, overall, HIV-1 Env IgG responses were dominated by V3, with the notable exception of the responses to the vaccine strain A244 Env that were dominated by V2, whereas the anti-SIVmac239 Env responses were dominated by V2 regardless of the vaccine regimen.

DNA and virus particle vaccination protects against acquisition and confers control of viremia upon heterologous simian immunodeficiency virus challenge.

We have previously shown that macaques vaccinated with DNA vectors expressing SIVmac239 antigens developed potent immune responses able to reduce viremia upon high-dose SIVmac251 challenge. To further improve vaccine-induced immunity and protection, we combined the SIVmac239 DNA vaccine with protein immunization using inactivated SIVmac239 viral particles as protein source. Twenty-six weeks after the last vaccination, the animals were challenged intrarectally at weekly intervals with a titrated dose of the heterologous SIVsmE660. Two of DNA-protein coimmunized macaques did not become infected after 14 challenges, but all controls were infected by 11 challenges. Vaccinated macaques showed modest protection from SIVsmE660 acquisition compared with naïve controls (P = 0.050; stratified for TRIM5α genotype). Vaccinees had significantly lower peak (1.6 log, P = 0.0048) and chronic phase viremia (P = 0.044), with 73% of the vaccinees suppressing viral replication to levels below assay detection during the 40-wk follow-up. Vaccine-induced immune responses associated significantly with virus control: binding antibody titers and the presence of rectal IgG to SIVsmE660 Env correlated with delayed SIVsmE660 acquisition; SIV-specific cytotoxic T cells, prechallenge CD4(+) effector memory, and postchallenge CD8(+) transitional memory cells correlated with control of viremia. Thus, SIVmac239 DNA and protein-based vaccine protocols were able to achieve high, persistent, broad, and effective cellular and humoral immune responses able to delay heterologous SIVsmE660 infection and to provide long-term control of viremia. These studies support a role of DNA and protein-based vaccines for development of an efficacious HIV/AIDS vaccine.

Identification of novel markers for mouse CD4(+) T follicular helper cells.

CD4(+) T follicular helper (TFH) cells are central for generation of long-term B-cell immunity. A defining phenotypic attribute of TFH cells is the expression of the chemokine R CXCR5, and TFH cells are typically identified by co-expression of CXCR5 together with other markers such as PD-1, ICOS, and Bcl-6. Herein, we report high-level expression of the nutrient transporter folate R 4 (FR4) on TFH cells in acute viral infection. Distinct from the expression profile of conventional TFH markers, FR4 was highly expressed by naive CD4(+) T cells, was downregulated after activation and subsequently re-expressed on TFH cells. Furthermore, FR4 expression was maintained, albeit at lower levels, on memory TFH cells. Comparative gene expression profiling of FR4(hi) versus FR4(lo) Ag-specific CD4(+) effector T cells revealed a molecular signature consistent with TFH and TH1 subsets, respectively. Interestingly, genes involved in the purine metabolic pathway, including the ecto-enzyme CD73, were enriched in TFH cells compared with TH1 cells, and phenotypic analysis confirmed expression of CD73 on TFH cells. As there is now considerable interest in developing vaccines that would induce optimal TFH cell responses, the identification of two novel cell surface markers should be useful in characterization and identification of TFH cells following vaccination and infection.

Tailoring Tfh profiles enhances antibody persistence to a clade C HIV-1 vaccine in rhesus macaques.

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.