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.

Human Immunodeficiency Virus C.1086 Envelope gp140 Protein Boosts following DNA/Modified Vaccinia Virus Ankara Vaccination Fail To Enhance Heterologous Anti-V1V2 Antibody Response and Protection against Clade C Simian-Human Immunodeficiency Virus Challenge.

The RV144 human immunodeficiency virus type 1 (HIV-1) vaccine trial showed a strong association between anti-gp70 V1V2 scaffold (V1V2) and anti-V2 hot spot peptide (V2 HS) antibody responses and reduced risk of HIV infection. Accordingly, a primary goal for HIV vaccines is to enhance the magnitude and breadth of V1V2 and V2 HS antibody responses in addition to neutralizing antibodies. Here, we tested the immunogenicity and efficacy of HIV-1 C.1086 gp140 boosts administered sequentially after priming with CD40L-adjuvanted DNA/simian-human immunodeficiency virus (SHIV) and boosting with modified vaccinia virus Ankara (MVA)-SHIV vaccines in rhesus macaques. The DNA/MVA vaccination induced robust vaccine-specific CD4 and CD8 T cell responses with a polyfunctional profile. Two gp140 booster immunizations induced very high levels (∼2 mg/ml) of gp140 binding antibodies in serum, with strong reactivity directed against the homologous (C.1086) V1V2, V2 HS, V3, and gp41 immunodominant (ID) proteins. However, the vaccine-induced antibody showed 10-fold (peak) and 32-fold (prechallenge) weaker binding to the challenge virus (SHIV1157ipd3N4) V1V2 and failed to bind to the challenge virus V2 HS due to a single amino acid change. Point mutations in the immunogen V2 HS to match the V2 HS in the challenge virus significantly diminished the binding of vaccine-elicited antibodies to membrane-anchored gp160. Both vaccines failed to protect from infection following repeated SHIV1157ipd3N4 intrarectal challenges. However, only the protein-boosted animals showed enhanced viral control. These results demonstrate that C.1086 gp140 protein immunizations administered following DNA/MVA vaccination do not significantly boost heterologous V1V2 and V2 HS responses and fail to enhance protection against heterologous SHIV challenge.IMPORTANCE HIV, the virus that causes AIDS, is responsible for millions of infections and deaths annually. Despite intense research for the past 25 years, there remains no safe and effective vaccine available. The significance of this work is in identifying the pros and cons of adding a protein boost to an already well-established DNA/MVA HIV vaccine that is currently being tested in the clinic. Characterizing the effects of the protein boost can allow researchers going forward to design vaccines that generate responses that will be more effective against HIV. Our results in rhesus macaques show that boosting with a specific HIV envelope protein does not significantly boost antibody responses that were identified as immune correlates of protection in a moderately successful RV144 HIV vaccine trial in humans and highlight the need for the development of improved HIV envelope immunogens.

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.

Persistent Low-Level Replication of SIVΔnef Drives Maturation of Antibody and CD8 T Cell Responses to Induce Protective Immunity against Vaginal SIV Infection.

Defining the correlates of immune protection conferred by SIVΔnef, the most effective vaccine against SIV challenge, could enable the design of a protective vaccine against HIV infection. Here we provide a comprehensive assessment of immune responses that protect against SIV infection through detailed analyses of cellular and humoral immune responses in the blood and tissues of rhesus macaques vaccinated with SIVΔnef and then vaginally challenged with wild-type SIV. Despite the presence of robust cellular immune responses, animals at 5 weeks after vaccination displayed only transient viral suppression of challenge virus, whereas all macaques challenged at weeks 20 and 40 post-SIVΔnef vaccination were protected, as defined by either apparent sterile protection or significant suppression of viremia in infected animals. Multiple parameters of CD8 T cell function temporally correlated with maturation of protection, including polyfunctionality, phenotypic differentiation, and redistribution to gut and lymphoid tissues. Importantly, we also demonstrate the induction of a tissue-resident memory population of SIV-specific CD8 T cells in the vaginal mucosa, which was dependent on ongoing low-level antigenic stimulation. Moreover, we show that vaginal and serum antibody titers inversely correlated with post-challenge peak viral load, and we correlate the accumulation and affinity maturation of the antibody response to the duration of the vaccination period as well as to the SIVΔnef antigenic load. In conclusion, maturation of SIVΔnef-induced CD8 T cell and antibody responses, both propelled by viral persistence in the gut mucosa and secondary lymphoid tissues, results in protective immune responses that are able to interrupt viral transmission at mucosal portals of entry as well as potential sites of viral dissemination.

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.

High Doses of GM-CSF Inhibit Antibody Responses in Rectal Secretions and Diminish Modified Vaccinia Ankara/Simian Immunodeficiency Virus Vaccine Protection in TRIM5α-Restrictive Macaques.

We tested, in rhesus macaques, the effects of a 500-fold range of an admixed recombinant modified vaccinia Ankara (MVA) expressing rhesus GM-CSF (MVA/GM-CSF) on the immunogenicity and protection elicited by an MVA/SIV macaque 239 vaccine. High doses of MVA/GM-CSF did not affect the levels of systemic envelope (Env)-specific Ab, but it did decrease the expression of the gut-homing receptor α4ß7 on plasmacytoid dendritic cells (p < 0.01) and the magnitudes of Env-specific IgA (p = 0.01) and IgG (p < 0.05) in rectal secretions. The protective effect of the vaccine was evaluated using 12 weekly rectal challenges in rhesus macaques subgrouped by tripartite motif-containing protein 5α (TRIM5α) genotypes that are restrictive or permissive for infection by the challenge virus SIVsmE660. Eight of nine TRIM5α-restrictive animals receiving no or the lowest dose (1 × 105 PFU) of MVA/GM-CSF resisted all 12 challenges. In the comparable TRIM5α-permissive group, only 1 of 12 animals resisted all 12 challenges. In the TRIM5α-restrictive animals, but not in the TRIM5α-permissive animals, the number of challenges to infection directly correlated with the magnitudes of Env-specific rectal IgG (r = +0.6) and IgA (r = +0.6), the avidity of Env-specific serum IgG (r = +0.5), and Ab dependent cell-mediated virus inhibition (r = +0.6). Titers of neutralizing Ab did not correlate with protection. We conclude that 1) protection elicited by MVA/SIVmac239 is strongly dependent on the presence of TRIM5α restriction, 2) nonneutralizing Ab responses contribute to protection against SIVsmE660 in TRIM5α-restrictive animals, and 3) high doses of codelivered MVA/GM-CSF inhibit mucosal Ab responses and the protection elicited by MVA expressing noninfectious SIV macaque 239 virus-like particles.

Virus-Like Particles Displaying Trimeric Simian Immunodeficiency Virus (SIV) Envelope gp160 Enhance the Breadth of DNA/Modified Vaccinia Virus Ankara SIV Vaccine-Induced Antibody Responses in Rhesus Macaques.

UNLABELLED: 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 with DNA (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 second MVA 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 seven DNA/MVA and 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. IMPORTANCE: The development of an effective HIV vaccine remains a global necessity for preventing HIV infection and reducing the burden of AIDS. While this goal represents a formidable challenge, the modest efficacy of the RV144 trial indicates that multicomponent vaccination regimens that elicit both cellular and humoral immune responses can prevent HIV infection in humans. However, whether protein immunizations synergize with DNA prime-viral vector boosts to enhance cellular and humoral immune responses remains poorly understood. We addressed this question in a nonhuman primate model, and our findings show benefit for sequential protein immunization combined with a potent adjuvant in boosting antibody titers induced by a preceding DNA/MVA immunization. This promising strategy can be further developed to enhance neutralizing antibody responses and boost CD8 T cells to provide robust protection and viral control.

Vaccine-Elicited Mucosal and Systemic Antibody Responses Are Associated with Reduced Simian Immunodeficiency Viremia in Infant Rhesus Macaques.

UNLABELLED: Despite significant progress in reducing peripartum mother-to-child transmission (MTCT) of human immunodeficiency virus (HIV) with antiretroviral therapy (ART), continued access to ART throughout the breastfeeding period is still a limiting factor, and breast milk exposure to HIV accounts for up to 44% of MTCT. As abstinence from breastfeeding is not recommended, alternative means are needed to prevent MTCT of HIV. We have previously shown that oral vaccination at birth with live attenuated Mycobacterium tuberculosis strains expressing simian immunodeficiency virus (SIV) genes safely induces persistent SIV-specific cellular and humoral immune responses both systemically and at the oral and intestinal mucosa. Here, we tested the ability of oral M. tuberculosis vaccine strains expressing SIV Env and Gag proteins, followed by systemic heterologous (MVA-SIV Env/Gag/Pol) boosting, to protect neonatal macaques against oral SIV challenge. While vaccination did not protect infant macaques against oral SIV acquisition, a subset of immunized animals had significantly lower peak viremia which inversely correlated with prechallenge SIV Env-specific salivary and intestinal IgA responses and higher-avidity SIV Env-specific IgG in plasma. These controller animals also maintained CD4(+) T cell populations better and showed reduced tissue pathology compared to noncontroller animals. We show that infants vaccinated at birth can develop vaccine-induced SIV-specific IgA and IgG antibodies and cellular immune responses within weeks of life. Our data further suggest that affinity maturation of vaccine-induced plasma antibodies and induction of mucosal IgA responses at potential SIV entry sites are associated with better control of viral replication, thereby likely reducing SIV morbidity. IMPORTANCE: Despite significant progress in reducing peripartum MTCT of HIV with ART, continued access to ART throughout the breastfeeding period is still a limiting factor. Breast milk exposure to HIV accounts for up to 44% of MTCT. Alternative measures, in addition to ART, are needed to achieve the goal of an AIDS-free generation. Pediatric HIV vaccines constitute a core component of such efforts. The results of our pediatric vaccine study highlight the potential importance of vaccine-elicited mucosal Env-specific IgA responses in combination with high-avidity systemic Env-specific IgG in protection against oral SIV transmission and control of viral replication in infant macaques. The induction of potent mucosal IgA antibodies by our vaccine is remarkable considering the age-dependent development of mucosal IgA responses postbirth. A deeper understanding of postnatal immune development may inform the design of improved vaccine strategies to enhance systemic and mucosal SIV/HIV antibody responses.

Mucosal vaccines: the promise and the challenge.

Most infectious agents enter the body at mucosal surfaces and therefore mucosal immune responses function as a first line of defence. Protective mucosal immune responses are most effectively induced by mucosal immunization through oral, nasal, rectal or vaginal routes, but the vast majority of vaccines in use today are administered by injection. As discussed in this Review, current research is providing new insights into the function of mucosal tissues and the interplay of innate and adaptive immune responses that results in immune protection at mucosal surfaces. These advances promise to accelerate the development and testing of new mucosal vaccines against many human diseases including HIV/AIDS.

CD40L-adjuvanted DNA/modified vaccinia virus Ankara simian immunodeficiency virus (SIV) vaccine enhances protection against neutralization-resistant mucosal SIV infection.

Here, we show that a CD40L-adjuvanted DNA/modified vaccinia virus Ankara (MVA) simian immunodeficiency virus (SIV) vaccine enhances protection against a pathogenic neutralization-resistant mucosal SIV infection, improves long-term viral control, and prevents AIDS. Analyses of serum IgG antibodies to linear peptides of SIV Env revealed a strong response to V2, with targeting of fewer epitopes in the immunodominant region of gp41 (gp41-ID) and the V1 region as a correlate for enhanced protection. Greater expansion of antiviral CD8 T cells in the gut correlated with long-term viral control.

Local control of repeated-dose rectal challenges in DNA/MVA-vaccinated macaques protected against a first series of simian immunodeficiency virus challenges.

Here, we report the results of a late boost and three additional series of simian immunodeficiency virus (SIV) challenges in seven DNA/modified vaccinia virus Ankara (MVA)-vaccinated rhesus macaques who resisted a first series of rectal challenges. During 29 additional challenges delivered over 2.3 years, all animals became infected. However, 13 blips of virus in six macaques and anamnestic Env-specific rectal IgA responses in three of the six suggested that local control of infections was occurring during the serial challenge.

Resistance to infection, early and persistent suppression of simian immunodeficiency virus SIVmac251 viremia, and significant reduction of tissue viral burden after mucosal vaccination in female rhesus macaques.

The efficacy of oral, intestinal, nasal, and vaginal vaccinations with DNA simian immunodeficiency virus (SIV)/interleukin-2 (IL-2)/IL-15, SIV Gag/Pol/Env recombinant modified vaccinia virus Ankara (rMVA), and AT-2 SIVmac239 inactivated particles was compared in rhesus macaques after low-dose vaginal challenge with SIVmac251. Intestinal immunization provided better protection from infection, as a significantly greater median number of challenges was necessary in this group than in the others. Oral and nasal vaccinations provided the most significant control of disease progression. Fifty percent of the orally and nasally vaccinated animals suppressed viremia to undetectable levels, while this occurred to a significantly lower degree in intestinally and vaginally vaccinated animals and in controls. Viremia remained undetectable after CD8(+) T-cell depletion in seven vaccinated animals that had suppressed viremia after infection, and tissue analysis for SIV DNA and RNA was negative, a result consistent with a significant reduction of viral activity. Regardless of the route of vaccination, mucosal vaccinations prevented loss of CD4(+) central memory and CD4(+)/α4ß7(+) T-cell populations and reduced immune activation to different degrees. None of the orally vaccinated animals and only one of the nasally vaccinated animals developed AIDS after 72 to 84 weeks of infection, when the trial was closed. The levels of anti-SIV gamma interferon-positive, CD4(+), and CD8(+) T cells at the time of first challenge inversely correlated with viremia and directly correlated with protection from infection and longer survival.

CD40L-adjuvanted DNA/modified vaccinia virus Ankara simian immunodeficiency virus SIV239 vaccine enhances SIV-specific humoral and cellular immunity and improves protection against a heterologous SIVE660 mucosal challenge.

UNLABELLED: It remains a challenge to develop a successful human immunodeficiency virus (HIV) vaccine that is capable of preventing infection. Here, we utilized the benefits of CD40L, a costimulatory molecule that can stimulate both dendritic cells (DCs) and B cells, as an adjuvant for our simian immunodeficiency virus (SIV) DNA vaccine in rhesus macaques. We coexpressed the CD40L with our DNA/SIV vaccine such that the CD40L is anchored on the membrane of SIV virus-like particle (VLP). These CD40L containing SIV VLPs showed enhanced activation of DCs in vitro. We then tested the potential of DNA/SIV-CD40L vaccine to adjuvant the DNA prime of a DNA/modified vaccinia virus Ankara (MVA) vaccine in rhesus macaques. Our results demonstrated that the CD40L adjuvant enhanced the functional quality of anti-Env antibody response and breadth of anti-SIV CD8 and CD4 T cell responses, significantly delayed the acquisition of heterologous mucosal SIV infection, and improved viral control. Notably, the CD40L adjuvant enhanced the control of viral replication in the gut at the site of challenge that was associated with lower mucosal CD8 immune activation, one of the strong predictors of disease progression. Collectively, our results highlight the benefits of CD40L adjuvant for enhancing antiviral humoral and cellular immunity, leading to enhanced protection against a pathogenic SIV. A single adjuvant that enhances both humoral and cellular immunity is rare and thus underlines the importance and practicality of CD40L as an adjuvant for vaccines against infectious diseases, including HIV-1. IMPORTANCE: Despite many advances in the field of AIDS research, an effective AIDS vaccine that can prevent infection remains elusive. CD40L is a key stimulator of dendritic cells and B cells and can therefore enhance T cell and antibody responses, but its overly potent nature can lead to adverse effects unless used in small doses. In order to modulate local expression of CD40L at relatively lower levels, we expressed CD40L in a membrane-bound form, along with SIV antigens, in a nucleic acid (DNA) vector. We tested the immunogenicity and efficacy of the CD40L-adjuvanted vaccine in macaques using a heterologous mucosal SIV infection. The CD40L-adjuvanted vaccine enhanced the functional quality of anti-Env antibody response and breadth of anti-SIV T cell responses and improved protection. These results demonstrate that VLP-membrane-bound CD40L serves as a novel adjuvant for an HIV vaccine.

Immunogenicity of a vaccine regimen composed of simian immunodeficiency virus DNA, rMVA, and viral particles administered to female rhesus macaques via four different mucosal routes.

A comparative evaluation of the immunity stimulated with a vaccine regimen that includes simian immunodeficiency virus (SIV), interleukin 2 (IL-2), and IL-15 DNAs, recombinant modified vaccinia virus Ankara (rMVA), and inactivated SIVmac239 particles administered into the oral and nasal cavities, small intestine, and vagina was carried out in female rhesus macaques to determine the best route to induce diverse anti-SIV immunity that may be critical to protection from SIV infection and disease. All four immunizations generated mucosal SIV-specific IgA. Oral immunization was as effective as vaginal immunization in inducing SIV-specific IgA in vaginal secretions and generated greater IgA responses in rectal secretions and saliva samples compared to the other immunization routes. All four immunizations stimulated systemic T-cell responses against Gag and Env, albeit to a different extent, with oral immunization providing greater magnitude and nasal immunization providing wider functional heterogeneity. SIV-specific T cells producing gamma interferon (IFN-γ) dominated these responses. Limited levels of SIV-specific IgG antibodies were detected in plasma samples, and no SIV-specific IgG antibodies were detected in secretions. Vaccination also induced CD4(+) and CD8(+) T-cell responses in the rectal and vaginal mucosa with greater functional heterogeneity than in blood samples. Rectal T-cell responses were significantly greater in the orally vaccinated animals than in the other animals. The most balanced, diverse, and higher-magnitude vaginal T-cell responses were observed after intestinal vaccination. Significantly higher CD8(+) granzyme B-positive T-cell responses were observed systemically after intestinal vaccination and in rectal cells after oral immunization. The majority of SIV-specific T cells that produced granzyme B did not produce cytokines. Of the immunization routes tested, oral vaccination provided the most diverse and significant response to the vaccine.

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.

Long-term control of simian immunodeficiency virus mac251 viremia to undetectable levels in half of infected female rhesus macaques nasally vaccinated with simian immunodeficiency virus DNA/recombinant modified vaccinia virus Ankara.

The efficacy of two SIV DNA plus recombinant modified vaccinia virus Ankara nasal vaccine regimens, one combined with plasmids expressing IL-2 and IL-15, the other with plasmids expressing GM-CSF, IL-12, and TNF-α, which may better stimulate humoral responses, was evaluated in two female rhesus macaque groups. Vaccination stimulated significant SIV-specific mucosal and systemic cell-mediated immunity in both groups, whereas SIV-specific IgA titers were sporadic and IgG titers negative. All vaccinated animals, except one, became infected after intravaginal SIV(mac251) low-dose challenge. Half of the vaccinated, infected animals (7/13) promptly controlled virus replication to undetectable viremia for the duration of the trial (130 wk) and displayed virological and immunological phenotypes similar to those of exposed, uninfected individuals. When all vaccinated animals were considered, a 3-log viremia reduction was observed, compared with controls. The excellent viral replication containment achieved in vaccinated animals translated into significant preservation of circulating α4ß7(high+)/CD4(+) T cells and of circulating and mucosal CD4(+)/C(M) T cells and in reduced immune activation. A more significant long-term survival was also observed in these animals. Median survival was 72 wk for the control group, whereas >50% of the vaccinated animals were still disease free 130 wk postchallenge, when the trial was closed. There was a statistically significant correlation between levels of CD4(+)/IFN-γ(+) and CD8(+)/IFN-γ(+) T cell percentages on the day of challenge and the control of viremia at week 60 postchallenge or survival. Postchallenge immunological correlates of protection were systemic anti-SIV Gag + Env CD4(+)/IL-2(+), CD4(+)/IFN-γ(+), and CD8(+)/TNF-α(+) T cells and vaginal anti-SIV Gag + Env CD8(+) T cell total monofunctional responses.

Genetic immunization in the lung induces potent local and systemic immune responses.

Successful vaccination against respiratory infections requires elicitation of high levels of potent and durable humoral and cellular responses in the lower airways. To accomplish this goal, we used a fine aerosol that targets the entire lung surface through normal respiration to deliver replication-incompetent recombinant adenoviral vectors expressing gene products from several infectious pathogens. We show that this regimen induced remarkably high and stable lung T-cell responses in nonhuman primates and that it also generated systemic and respiratory tract humoral responses of both IgA and IgG isotypes. Moreover, strong immunogenicity was achieved even in animals with preexisting antiadenoviral immunity, overcoming a critical hurdle to the use of these vectors in humans, who commonly are immune to adenoviruses. The immunogenicity profile elicited with this regimen, which is distinct from either intramuscular or intranasal delivery, has highly desirable properties for protection against respiratory pathogens. We show that it can be used repeatedly to generate mucosal humoral, CD4, and CD8 T-cell responses and as such may be applicable to other mucosally transmitted pathogens such as HIV. Indeed, in a lethal challenge model, we show that aerosolized recombinant adenoviral immunization completely protects ferrets against H5N1 highly pathogenic avian influenza virus. Thus, genetic immunization in the lung offers a powerful platform approach to generating protective immune responses against respiratory pathogens.

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.

Sutterella and its metabolic pathways positively correlate with vaccine-elicited antibody responses in infant rhesus macaques.

Introduction: It is becoming clearer that the microbiota helps drive responses to vaccines; however, little is known about the underlying mechanism. In this study, we aimed to identify microbial features that are associated with vaccine immunogenicity in infant rhesus macaques. Methods: We analyzed 16S rRNA gene sequencing data of 215 fecal samples collected at multiple timepoints from 64 nursery-reared infant macaques that received various HIV vaccine regimens. PERMANOVA tests were performed to determine factors affecting composition of the gut microbiota throughout the first eight months of life in these monkeys. We used DESeq2 to identify differentially abundant bacterial taxa, PICRUSt2 to impute metagenomic information, and mass spectrophotometry to determine levels of fecal short-chain fatty acids and bile acids. Results: Composition of the early-life gut microbial communities in nursery-reared rhesus macaques from the same animal care facility was driven by age, birth year, and vaccination status. We identified a Sutterella and a Rodentibacter species that positively correlated with vaccine-elicited antibody responses, with the Sutterella species exhibiting more robust findings. Analysis of Sutterella-related metagenomic data revealed five metabolic pathways that significantly correlated with improved antibody responses following HIV vaccination. Given these pathways have been associated with short-chain fatty acids and bile acids, we quantified the fecal concentration of these metabolites and found several that correlated with higher levels of HIV immunogen-elicited plasma IgG. Discussion: Our findings highlight an intricate bidirectional relationship between the microbiota and vaccines, where multiple aspects of the vaccination regimen modulate the microbiota and specific microbial features facilitate vaccine responses. An improved understanding of this microbiota-vaccine interplay will help develop more effective vaccines, particularly those that are tailored for early life.

In Vivo Treatment with Insulin-like Growth Factor 1 Reduces CCR5 Expression on Vaccine-Induced Activated CD4+ T-Cells.

At the heart of the DNA/ALVAC/gp120/alum vaccine's efficacy in the absence of neutralizing antibodies is a delicate balance of pro- and anti-inflammatory immune responses that effectively decreases the risk of SIVmac251 acquisition in macaques. Vaccine efficacy is linked to antibodies recognizing the V2 helical conformation, DC-10 tolerogenic dendritic cells eliciting the clearance of apoptotic cells via efferocytosis, and CCR5 downregulation on vaccine-induced gut homing CD4+ cells. RAS activation is also linked to vaccine efficacy, which prompted the testing of IGF-1, a potent inducer of RAS activation with vaccination. We found that IGF-1 changed the hierarchy of V1/V2 epitope recognition and decreased both ADCC specific for helical V2 and efferocytosis. Remarkably, IGF-1 also reduced the expression of CCR5 on vaccine-induced CD4+ gut-homing T-cells, compensating for its negative effect on ADCC and efferocytosis and resulting in equivalent vaccine efficacy (71% with IGF-1 and 69% without).