Although scarce after annual influenza vaccination, B cells producing antibodies capable of neutralizing multiple influenza strains are abundant in humans infected with pandemic 2009 H1N1 influenza.

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. NPadjuvanted 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 Envspecific 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).

Mucosa-associated invariant T (MAIT) cells are recently characterized as a novel subset of innate-like T cells that recognize microbial metabolites as presented by the MHC-1b-related protein MR1. The significance of MAIT cells in anti-bacterial defense is well-understood but not clear in viral infections such as SIV/HIV infection. Here we studied the phenotype, distribution, and function of MAIT cells and their association with plasma viral levels during chronic SHIV infection in rhesus macaques (RM). Two groups of healthy and chronic SHIV-infected macaques were characterized for MAIT cells in blood and mucosal tissues. Similar to human, we found a significant fraction of macaque T cells co-expressing MAIT cell markers CD161 and TCRVα-7.2 that correlated directly with macaque MR1 tetramer. These cells displayed memory phenotype and expressed high levels of IL-18R, CCR6, CD28, and CD95. During chronic infection, the frequency of MAIT cells are enriched in the blood but unaltered in the rectum; both blood and rectal MAIT cells displayed higher proliferative and cytotoxic phenotype post-SHIV infection. The frequency of MAIT cells in blood and rectum correlated inversely with plasma viral RNA levels and correlated directly with total CD4 T cells. MAIT cells respond to microbial products during chronic SHIV infection and correlated positively with serum immunoreactivity to flagellin levels. Tissue distribution analysis of MAIT cells during chronic infection showed significant enrichment in the non-lymphoid tissues (lung, rectum, and liver) compared to lymphoid tissues (spleen and LN), with higher levels of tissue-resident markers CD69 and CD103. Exogenous in vitro cytokine treatments during chronic SHIV infection revealed that IL-7 is important for the proliferation of MAIT cells, but IL-12 and IL-18 are important for their cytolytic function. Overall our results demonstrated that MAIT cells are enriched in blood but unaltered in the rectum during chronic SHIV infection, which displayed proliferative and functional phenotype that inversely correlated with SHIV plasma viral RNA levels. Treatment such as combined cytokine treatments could be beneficial for enhancing functional MAIT cells during chronic HIV infection in vivo.

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-β 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.

A major obstacle to development of an effective AIDS vaccine is that along with intended beneficial responses, immunization regimen may activate CD4+ T cells that can facilitate acquisition of HIV by serving as target cells for the virus. Lu et al. reported that intra-gastric administration of chemically inactivated SIVmac239 (iSIV) and Lactobacillus plantarum (LP) (iSIV+LP) protected 15/16 Chinese-origin rhesus macaques (RMs) from high-dose intra-rectal SIVmac239 challenge at three months post-immunization. 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". Andrieu et al subsequently reported protection from infection in 23/24 RM immunized intragastrically or intravaginally with iSIV and BCG, LP or Lactobacillus rhamnosus, which they ascribed to the same tolerogenic mechanism. Using vaccine materials obtained from our co-authors, we conducted an immunization and challenge experiment in 54 Indian RMs, and included control groups receiving iSIV only or LP only, as well as unvaccinated animals. Intra-rectal challenge with SIVmac239 resulted in rapid infection in all groups of vaccinated RMs as well as unvaccinated controls. iSIV+LP vaccinated animals that became SIV infected showed viral loads similar to those observed in animals receiving iSIV only, LP only, and unvaccinated controls. The protection from SIV transmission conferred by intra-gastric iSIV+LP 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 increased understanding in 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 could act as target cells for acquisition of HIV. An alternative strategy could involve induction of a tolerizing 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 in Chinese-origin rhesus macaques by using a "tolerogenic" vaccine consisting of intra-gastric administration of Lactobacillus plantarum and AT-2-inactivated SIV (iSIV+LP) which showed highly significant protection from virus transmission. Here, we administered iSIV+LP immunizations to Indian-origin rhesus macaques, and failed to observe any protective effect from virus acquisition in this experimental setting. This work is important as it contributes to the overall assessment of the clinical potential of a new candidate AIDS vaccine platform based on the iSIV+LP.

Heterologous prime-boosting has emerged as a powerful vaccination approach against tuberculosis. However, optimal timing to boost BCG-immunity using subunit vaccines remains unclear in clinical trials. Here, we followed the adhesin Apa-specific T-cell responses in BCG-primed mice and investigated its BCG-booster potential. The Apa-specific T-cell response peaked 32-52 weeks after parenteral or mucosal BCG-priming but waned significantly by 78 weeks. A subunit-Apa-boost during the contraction-phase of BCG-response had a greater effect on the magnitude and functional quality of specific cellular and humoral responses compared to a boost at the peak of BCG-response. The cellular response increased following mucosal BCG-prime-Apa-subunit-boost strategy compared to Apa-subunit-prime-BCG-boost approach. However, parenteral BCG-prime-Apa-subunit-boost by a homologous route was the most effective strategy in-terms of enhancing specific T-cell responses during waning in the lung and spleen. Two Apa-boosters markedly improved waning BCG-immunity and significantly reduced Mycobacterium tuberculosis burdens post-challenge. Our results highlight the challenges of optimization of prime-boost regimens in mice where BCG drives persistent immune-activation and suggest that boosting with a heterologous vaccine may be ideal once the specific persisting effector responses are contracted. Our results have important implications for design of prime-boost regimens against tuberculosis in humans.

The encouraging results of the RV144 vaccine trial have spurred interest in poxvirus prime-protein boost human immunodeficiency virus (HIV) vaccine modalities as a strategy to induce protective immunity. Because vaccine-induced protective immunity is critically determined by HIV envelope (Env) conformation, significant efforts are directed toward generating soluble trimeric Env immunogens that assume native structures. Using the simian immunodeficiency virus (SIV)-macaque model, we tested the immunogenicity and efficacy of sequential immunizations withDNA(D), modified vaccinia virus Ankara (MVA) (M), and protein immunogens, all expressing virus-like particles (VLPs) displaying membrane-anchored trimeric Env. A single VLP protein boost displaying trimeric gp160 adjuvanted with nanoparticle-encapsulated Toll-like receptor 4/7/8 (TLR4/7/8) agonists, administered 44 weeks after the secondMVA immunization, induced up to a 3-fold increase in Env-specific IgG binding titers in serum and mucosa. Importantly, the VLP protein boost increased binding antibody against scaffolded V1V2, antibody-dependent phagocytic activity against VLP-coated beads, and antibody breadth and neutralizing antibody titers against homologous and heterologous tier 1 SIVs. Following 5 weekly intrarectal SIVmac251 challenges, two of sevenDNA/MVAand VLP (DM+VLP)-vaccinated animals were completely protected compared to productive infection in all seven DM-vaccinated animals. Vaccinated animals demonstrated stronger acute viral pulldown than controls, but a trend for higher acute viremia was observed in the DM+VLP group, likely due to a slower recall of Gag-specific CD8 T cells. Our findings support immunization with VLPs containing trimeric Env as a strategy to augment protective antibody but underscore the need for optimal engagement of CD8 T cells to achieve robust early viral control.

The influence of preexisting immunity to viral vectors is a major issue for the development of viral vectored vaccines. Here, we investigate the effect of preexisting vaccinia virus immunity on the immunogenicity and efficacy of a DNA/MVA SIV vaccine in rhesus macaques using a pathogenic intrarectal SIV251 challenge. Preexisting immunity decreased SIV-specific CD8 and CD4 T cell responses, but preserved the SIV-specific humoral immunity. In addition, preexisting immunity did not diminish the control of a SIV challenge mediated by the DNA/MVA vaccine. The peak and set point viremia was 150- and 17-fold lower, respectively in preimmune animals compared to control animals. The peak and set point viremia correlated directly with colorectal virus at 2 weeks post challenge suggesting that early control of virus replication at the site of viral challenge was critical for viral control. Factors that correlated with early colorectal viral control included (i) the presence of anti-SIV IgA in rectal secretions, (ii) high avidity binding antibody for the native form of Env and (iii) low magnitude of vaccine-elicited SIV-specific CD4 T cells displaying the CCR5 viral co-receptor. The frequency of SIV-specific CD8 T cells in blood and colorectal tissue at 2 weeks post challenge did not correlate with early colorectal viral control. These results suggest that preexisting vaccinia virus immunity may not limit the potential of recombinant MVA vaccines to elicit humoral immunity and highlight the importance of immunodeficiency virus vaccines achieving early control at the mucosal sites of challenge.

Background: In this study, we analyzed the protective efficacy of a simian immunodeficiency virus (SIV) macaque 239 (SIVmac239) analogue of the clinically tested GOVX-B11 deoxyribonucleic acid (DNA)/modified vaccinia Ankara (MVA) human immunodeficiency virus vaccine. Methods: The tested vaccine used a DNA immunogen mutated to mimic the human vaccine and a regimen with DNA deliveries at weeks 0 and 8 and MVA deliveries at weeks 16 and 32. Twelve weekly rectal challenges with 0.3 animal infectious doses of SIV sootey mangabey E660 (SIVsmE660) were administered starting at 6 months after the last immunization. Results: Over the first 6 rectal exposures to SIVsmE660, <10-year-old tripartite motif-containing protein 5 (TRIM5)α-permissive rhesus macaques showed an 80% reduction in per-exposure risk of infection as opposed to a 46% reduction in animals over 10 years old; and, over the 12 challenges, they showed a 72% as opposed to a 10% reduction. Analyses of elicited immune responses suggested that higher antibody responses in the younger animals had played a role in protection. Conclusions: The simian analogue of the GOVX-B11 HIV provided strong protection against repeated rectal challenges in young adult macaques.

Since the initial proof-of-concept studies examining the ability of antigen-encoded plasmid DNA to serve as an immunogen, DNA vaccines have evolved as a clinically safe and effective platform for priming HIV-specific cellular and humoral responses in heterologous “prime-boost” vaccination regimens. Direct injection of plasmid DNA into the muscle induces T- and B-cell responses against foreign antigens. However, the insufficient magnitude of this response has led to the development of approaches for enhancing the immunogenicity of DNA vaccines. The last two decades have seen significant progress in the DNA-based vaccine platform with optimized plasmid constructs, improved delivery methods, such as electroporation, the use of molecular adjuvants and novel strategies combining DNA with viral vectors and subunit proteins. These innovations are paving the way for the clinical application of DNA-based HIV vaccines. Here, we review preclinical studies on the DNA-prime/modified vaccinia Ankara (MVA)-boost vaccine modality for HIV. There is a great deal of interest in enhancing the immunogenicity of DNA by engineering DNA vaccines to co-express immune modulatory adjuvants. Some of these adjuvants have demonstrated encouraging results in preclinical and clinical studies, and these data will be examined, as well.