Evidence for the Role of a Second Fc-Binding Receptor in Placental IgG Transfer in Nonhuman Primates.

Transplacental transfer of maternal antibodies provides the fetus and newborn with passive protection against infectious diseases. While the role of the highly conserved neonatal Fc receptor (FcRn) in transfer of IgG in mammals is undisputed, recent reports have suggested that a second receptor may contribute to transport in humans. We report poor transfer efficiency of plant-expressed recombinant HIV-specific antibodies, including engineered variants with high FcRn affinity, following subcutaneous infusion into rhesus macaques close to parturition. Unexpectedly, unlike those derived from mammalian tissue culture, plant-derived antibodies were essentially unable to cross macaque placentas. This defect was associated with poor Fcγ receptor binding and altered Fc glycans and was not recapitulated in mice. These results suggest that maternal-fetal transfer of IgG across the three-layer primate placenta may require a second receptor and suggest a means of providing maternal antibody treatments during pregnancy while avoiding fetal harm. IMPORTANCE This study compared the ability of several human HIV envelope-directed monoclonal antibodies produced in plants with the same antibodies produced in mammalian cells for their ability to cross monkey and mouse placentas. We found that the two types of antibodies have comparable transfer efficiencies in mice, but they are differentially transferred across macaque placentas, consistent with a two-receptor IgG transport model in primates. Importantly, plant-produced monoclonal antibodies have excellent binding characteristics for human FcRn receptors, permitting desirable pharmacokinetics in humans. The lack of efficient transfer across the primate placenta suggests that therapeutic plant-based antibody treatments against autoimmune diseases and cancer could be provided to the mother while avoiding transfer and preventing harm to the fetus.

Phagocytosis by an HIV antibody is associated with reduced viremia irrespective of enhanced complement lysis.

Increasingly, antibodies are being used to treat and prevent viral infections. In the context of HIV, efficacy is primarily attributed to dose-dependent neutralization potency and to a lesser extent Fc-mediated effector functions. It remains unclear whether augmenting effector functions of broadly neutralizing antibodies (bNAbs) may improve their clinical potential. Here, we use bNAb 10E8v4 targeting the membrane external proximal region (MPER) to examine the role of antibody-mediated effector and complement (C') activity when administered prophylactically against SHIV challenge in rhesus macaques. With sub-protective dosing, we find a 78-88% reduction in post-acute viremia that is associated with 10E8v4-mediated phagocytosis acting at the time of challenge. Neither plasma nor tissue viremic outcomes in vivo is improved with an Fc-modified variant of 10E8v4 enhanced for C' functions as determined in vitro. These results suggest that effector functions inherent to unmodified 10E8v4 contribute to efficacy against SHIVSF162P3 in the absence of plasma neutralizing titers, while C' functions are dispensable in this setting, informing design of bNAb modifications for improving protective efficacy.

Protection of Newborn Macaques by Plant-Derived HIV Broadly Neutralizing Antibodies: a Model for Passive Immunotherapy during Breastfeeding.

Preventing human immunodeficiency virus (HIV) infection in newborns by vertical transmission remains an important unmet medical need in resource-poor areas where antiretroviral therapy (ART) is not available and mothers and infants cannot be treated prepartum or during the breastfeeding period. In the present study, the protective efficacy of the potent HIV-neutralizing antibodies PGT121 and VRC07-523, both produced in plants, were assessed in a multiple-SHIV (simian-human immunodeficiency virus)-challenge breastfeeding macaque model. Newborn macaques received either six weekly subcutaneous injections with PGT121 alone or as a cocktail of PGT121-LS plus VRC07-523-LS injected three times every 2 weeks. Viral challenge with SHIVSF162P3 was twice weekly over 5.5 weeks using 11 exposures. Despite the transient presence of plasma viral RNA either immediately after the first challenge or as single-point blips, the antibodies prevented a productive infection in all babies with no sustained plasma viremia, compared to viral loads ranging from 103 to 5 × 108 virions/ml in four untreated controls. No virus was detected in peripheral blood mononuclear cells (PBMCs), and only 3 of 159 tissue samples were weakly positive in the treated babies. Newborn macaques proved to be immunocompetent, producing transient anti-Env antibodies and anti-drug antibody (ADA), which were maintained in the circulation after passive broadly neutralizing antibody clearance. ADA responses were directed to the IgG1 Fc CH2-CH3 domains, which has not been observed to date in adult monkeys passively treated with PGT121 or VRC01. In addition, high levels of VRC07-523 anti-idiotypic antibodies in the circulation of one newborn was concomitant with the rapid elimination of VRC07. Plant-expressed antibodies show promise as passive immunoprophylaxis in a breastfeeding model in newborns. IMPORTANCE Plant-produced human neutralizing antibody prophylaxis is highly effective in preventing infection in newborn monkeys during repeated oral exposure, modeling virus in breastmilk, and offers advantages in cost of production and safety. These findings raise the possibility that anti-Env antibodies may contribute to the control of viral replication in this newborn model and that the observed immune responsiveness may be driven by the long-lived presence of immune complexes.

Virus Control in Vaccinated Rhesus Macaques Is Associated with Neutralizing and Capturing Antibodies against the SHIV Challenge Virus but Not with V1V2 Vaccine-Induced Anti-V2 Antibodies Alone.

The role of vaccine-induced anti-V2 Abs was tested in three protection experiments in rhesus macaques. In an experiment using immunogens similar to those in the RV144 vaccine trial (Anti-envelope [Env]), nine rhesus macaques were coimmunized with gp16092TH023 DNA and SIV gag and gp120A244 and gp120MN proteins. In two V2-focused experiments (Anti-V2 and Anti-V2 Mucosal), nine macaques in each group were immunized with V1V292TH023 DNA, V1V2A244 and V1V2CasaeA2 proteins, and cyclic V2CaseA2 peptide. DNA and protein immunogens, formulated in Adjuplex, were given at 0, 4, 12, and 20 weeks, followed by intrarectal SHIVBaL.P4 challenges. Peak plasma viral loads (PVL) of 106-107 copies/ml developed in all nine sham controls. Overall, PVL was undetectable in one third of immunized macaques, and two animals tightly controlled the virus with the Anti-V2 Mucosal vaccine strategy. In the Anti-Env study, Abs that captured or neutralized SHIVBaL.P4 inversely correlated with PVL. Conversely, no correlation with PVL was found in the Anti-V2 experiments with nonneutralizing plasma Abs that only captured virus weakly. Titers of Abs against eight V1V2 scaffolds and cyclic V2 peptides were comparable between controllers and noncontrollers as were Ab-dependent cellular cytotoxicity and Ab-dependent cell-mediated virus inhibition activities against SHIV-infected target cells and phagocytosis of gp120-coated beads. The Anti-Env experiment supports the role of vaccine-elicited neutralizing and nonneutralizing Abs in control of PVL. However, the two V2-focused experiments did not support a role for nonneutralizing V2 Abs alone in controlling PVL, as neither Ab-dependent cellular cytotoxicity, Ab-dependent cell-mediated virus inhibition, nor phagocytosis correlated inversely with heterologous SHIVBaL.P4 infection.

Polyfunctional Tier 2-Neutralizing Antibodies Cloned following HIV-1 Env Macaque Immunization Mirror Native Antibodies in a Human Donor.

Vaccine efforts to combat HIV are challenged by the global diversity of viral strains and shielding of neutralization epitopes on the viral envelope glycoprotein trimer. Even so, the isolation of broadly neutralizing Abs from infected individuals suggests the potential for eliciting protective Abs through vaccination. This study reports a panel of 58 mAbs cloned from a rhesus macaque (Macaca mulatta) immunized with envelope glycoprotein immunogens curated from an HIV-1 clade C-infected volunteer. Twenty mAbs showed neutralizing activity, and the strongest neutralizer displayed 92% breadth with a median IC50 of 1.35 µg/ml against a 13-virus panel. Neutralizing mAbs predominantly targeted linear epitopes in the V3 region in the cradle orientation (V3C) with others targeting the V3 ladle orientation (V3L), the CD4 binding site (CD4bs), C1, C4, or gp41. Nonneutralizing mAbs bound C1, C5, or undetermined conformational epitopes. Neutralization potency strongly correlated with the magnitude of binding to infected primary macaque splenocytes and to the level of Ab-dependent cellular cytotoxicity, but did not predict the degree of Ab-dependent cellular phagocytosis. Using an individualized germline gene database, mAbs were traced to 23 of 72 functional IgHV alleles. Neutralizing V3C Abs displayed minimal nucleotide somatic hypermutation in the H chain V region (3.77%), indicating that relatively little affinity maturation was needed to achieve in-clade neutralization breadth. Overall, this study underscores the polyfunctional nature of vaccine-elicited tier 2-neutralizing V3 Abs and demonstrates partial reproduction of the human donor's humoral immune response through nonhuman primate vaccination.

An HIV Vaccine Targeting the V2 Region of the HIV Envelope Induces a Highly Durable Polyfunctional Fc-Mediated Antibody Response in Rhesus Macaques.

The HIV vaccine field now recognizes the potential importance of generating polyfunctional antibodies (Abs). The only clinical HIV vaccine trial to date to show significant efficacy (RV144) found that reduced infection rates correlated with the level of nonneutralizing Abs specific for the V2 region of the envelope glycoprotein. We have conducted a comprehensive preclinical reverse vaccinology-based vaccine program that has included the design and production and testing of numerous scaffolded V2 region immunogens. The most immunogenic vaccine regimen in nonhuman primates among those studied as part of this program consisted of a cocktail of three immunogens presenting V2 from different viruses and clades in the context of different scaffolds. Presently we demonstrate that the V2-specific Ab response from this regimen was highly durable and functionally diverse for the duration of the study (25 weeks after the final immunization). The total IgG binding response at this late time point exhibited only an ∼5× reduction in potency. Three immunizations appeared essential for the elicitation of a strong Ab-dependent cellular cytotoxicity (ADCC) response for all animals, as opposed to the Ab-dependent cellular phagocytosis (ADCP) and virus capture responses, which were comparably potent after only 2 immunizations. All functionalities measured were highly durable through the study period. Therefore, testing this vaccine candidate for its protective capacity is warranted.IMPORTANCE The only HIV vaccine trial for which protective efficacy was detected correlated this efficacy with V2-specific Abs that were effectively nonneutralizing. This result has fueled a decade of HIV vaccine research focused on designing an HIV vaccine capable of eliciting V2-focused, polyfunctional Abs that effectively bind HIV and trigger various leukocytes to kill the virus and restrict viral spread. From the numerous vaccine candidates designed and tested as part of our V2-focused preclinical vaccine program, we have identified immunogens and a vaccine regimen that induces a highly durable and polyfunctional V2-focused Ab response in rhesus macaques, described herein.

Adenosine deaminase-1 enhances germinal center formation and functional antibody responses to HIV-1 Envelope DNA and protein vaccines.

Adenosine deaminase-1 (ADA-1) plays both enzymatic and non-enzymatic roles in regulating immune cell function. Mutations in the ADA1 gene account for 15% of heritable severe-combined immunodeficiencies. We determined previously that ADA1 expression defines and is instrumental for the germinal center follicular helper T cell (TFH) phenotype using in vitro human assays. Herein, we tested whether ADA-1 can be used as an adjuvant to improve vaccine efficacy in vivo. In vitro, ADA-1 induced myeloid dendritic cell (mDC) maturation as measured by increased frequencies of CD40-, CD83-, CD86-, and HLA-DR-positive mDCs. ADA-1 treatment also promoted the secretion of the TFH-polarizing cytokine IL-6 from mDCs. In the context of an HIV-1 envelope (env) DNA vaccine, co-immunization with plasmid-encoded ADA-1 (pADA) enhanced humoral immunity. Animals co-immunized with env DNA and pADA had significantly increased frequencies of TFH cells in their draining lymph nodes and increased HIV-binding IgG in serum. Next, mice were co-immunized with subtype C env gp160 DNA and pADA along with simultaneous immunization with matched gp140 trimeric protein. Mice that received env gp160 DNA, pADA, and gp140 glycoprotein had significantly more heterologous HIV-specific binding IgG in their serum. Furthermore, only these mice had detectable neutralizing antibody responses. These studies support the use of ADA-1 as a vaccine adjuvant to qualitatively enhance germinal center responses and represent a novel application of an existing therapeutic agent that can be quickly translated for clinical use.

Modified Adenovirus Prime-Protein Boost Clade C HIV Vaccine Strategy Results in Reduced Viral DNA in Blood and Tissues Following Tier 2 SHIV Challenge.

Designing immunogens and improving delivery methods eliciting protective immunity is a paramount goal of HIV vaccine development. A comparative vaccine challenge study was performed in rhesus macaques using clade C HIV Envelope (Env) and SIV Gag antigens. One group was vaccinated using co-immunization with DNA Gag and Env expression plasmids cloned from a single timepoint and trimeric Env gp140 glycoprotein from one of these clones (DNA+Protein). The other group was a prime-boost regimen composed of two replicating simian (SAd7) adenovirus-vectored vaccines expressing Gag and one Env clone from the same timepoint as the DNA+Protein group paired with the same Env gp140 trimer (SAd7+Protein). The env genes were isolated from a single pre-peak neutralization timepoint approximately 1 year post infection in CAP257, an individual with a high degree of neutralization breadth. Both DNA+Protein and SAd7+Protein vaccine strategies elicited significant Env-specific T cell responses, lesser Gag-specific responses, and moderate frequencies of Env-specific TFH cells. Both vaccine modalities readily elicited systemic and mucosal Env-specific IgG but not IgA. There was a higher frequency and magnitude of ADCC activity in the SAd7+Protein than the DNA+Protein arm. All macaques developed moderate Tier 1 heterologous neutralizing antibodies, while neutralization of Tier 1B or Tier 2 viruses was sporadic and found primarily in macaques in the SAd7+Protein group. Neither vaccine approach provided significant protection from viral acquisition against repeated titered mucosal challenges with a heterologous Tier 2 clade C SHIV. However, lymphoid and gut tissues collected at necropsy showed that animals in both vaccine groups each had significantly lower copies of viral DNA in individual tissues compared to levels in controls. In the SAd7+Protein-vaccinated macaques, total and peak PBMC viral DNA were significantly lower compared with controls. Taken together, this heterologous Tier 2 SHIV challenge study shows that combination vaccination with SAd7+Protein was superior to combination DNA+Protein in reducing viral seeding in tissues in the absence of protection from infection, thus emphasizing the priming role of replication-competent SAd7 vector. Despite the absence of correlates of protection, because antibody responses were significantly higher in this vaccine group, we hypothesize that vaccine-elicited antibodies contribute to limiting tissue viral seeding.

Simian-Human Immunodeficiency Virus SHIV.CH505 Infection of Rhesus Macaques Results in Persistent Viral Replication and Induces Intestinal Immunopathology.

Simian-human immunodeficiency viruses (SHIVs) have been utilized to test vaccine efficacy and characterize mechanisms of viral transmission and pathogenesis. However, the majority of SHIVs currently available have significant limitations in that they were developed using sequences from chronically HIV-infected individuals or uncommon HIV subtypes or were optimized for the macaque model by serially passaging the engineered virus in vitro or in vivo Recently, a newly developed SHIV, SHIV.C.CH505.375H.dCT (SHIV.CH505), which incorporates vpu-env (gp140) sequences from a transmitted/founder HIV-1 subtype C strain, was shown to retain attributes of primary HIV-1 strains. However, a comprehensive analysis of the immunopathology that results from infection with this virus, especially in critical tissue compartments like the intestinal mucosa, has not been completed. In this study, we evaluated the viral dynamics and immunopathology of SHIV.CH505 in rhesus macaques. In line with previous findings, we found that SHIV.CH505 is capable of infecting and replicating efficiently in rhesus macaques, resulting in peripheral viral kinetics similar to that seen in pathogenic SIV and HIV infection. Furthermore, we observed significant and persistent depletions of CCR5+ and CCR6+ CD4+ T cells in mucosal tissues, decreases in CD4+ T cells producing Th17 cell-associated cytokines, CD8+ T cell dysfunction, and alterations of B cell and innate immune cell function, indicating that SHIV.CH505 elicits intestinal immunopathology typical of SIV/HIV infection. These findings suggest that SHIV.CH505 recapitulates the early viral replication dynamics and immunopathogenesis of HIV-1 infection of humans and thus can serve as a new model for HIV-1 pathogenesis, treatment, and prevention research.IMPORTANCE The development of chimeric SHIVs has been instrumental in advancing our understanding of HIV-host interactions and allowing for in vivo testing of novel treatments. However, many of the currently available SHIVs have distinct drawbacks and are unable to fully reflect the features characteristic of primary SIV and HIV strains. Here, we utilize rhesus macaques to define the immunopathogenesis of the recently developed SHIV.CH505, which was designed without many of the limitations of previous SHIVs. We observed that infection with SHIV.CH505 leads to peripheral viral kinetics and mucosal immunopathogenesis comparable with those caused by pathogenic SIV and HIV. Overall, these data provide evidence of the value of SHIV.CH505 as an effective model of SIV/HIV infection and an important tool that can be used in future studies, including preclinical testing of new therapies or prevention strategies.

Human natural killer cells mediate adaptive immunity to viral antigens.

Adaptive immune responses are defined as antigen sensitization-dependent and antigen-specific responses leading to establishment of long-lived immunological memory. Although natural killer (NK) cells have traditionally been considered cells of the innate immune system, mounting evidence in mice and nonhuman primates warrants reconsideration of the existing paradigm that B and T cells are the sole mediators of adaptive immunity. However, it is currently unknown whether human NK cells can exhibit adaptive immune responses. We therefore tested whether human NK cells mediate adaptive immunity to virally encoded antigens using humanized mice and human volunteers. We found that human NK cells displayed vaccination-dependent, antigen-specific recall responses in vitro, when isolated from livers of humanized mice previously vaccinated with HIV-encoded envelope protein. Furthermore, we discovered that large numbers of cytotoxic NK cells with a tissue-resident phenotype were recruited to sites of varicella-zoster virus (VZV) skin test antigen challenge in VZV-experienced human volunteers. These NK-mediated recall responses in humans occurred decades after initial VZV exposure, demonstrating that NK memory in humans is long-lived. Our data demonstrate that human NK cells exhibit adaptive immune responses upon vaccination or infection. The existence of human memory NK cells may allow for the development of vaccination-based approaches capable of establishing potent NK-mediated memory functions contributing to host protection.

Antibodies Tip the Balance Towards an HIV Cure.

Long-term drug therapy for HIV-1 infection can prevent both disease progression and virus transmission, but treatment does not eradicate the virus. A new gene therapy study (Immunity 2019;50:567-575.e5) using human antibodies shows significant promise for a functional cure in a non-human primate model.

Divergent HIV-1-Directed Immune Responses Generated by Systemic and Mucosal Immunization with Replicating Single-Cycle Adenoviruses in Rhesus Macaques.

Most human immunodeficiency virus type 1 (HIV-1) infections begin at mucosal surfaces. Providing a barrier of protection at these may assist in combating the earliest events in infection. Systemic immunization by intramuscular (i.m.) injection can drive mucosal immune responses, but there are data suggesting that mucosal immunization can better educate these mucosal immune responses. To test this, rhesus macaques were immunized with replicating single-cycle adenovirus (SC-Ad) vaccines expressing clade B HIV-1 gp160 by the intranasal (i.n.) and i.m. routes to compare mucosal and systemic routes of vaccination. SC-Ad vaccines generated significant circulating antibody titers against Env after a single i.m. immunization. Switching the route of second immunization with the same SC-Ad serotype allowed a significant boost in these antibody levels. When these animals were boosted with envelope protein, envelope-binding antibodies were amplified 100-fold, but qualitatively different immune responses were generated. Animals immunized by only the i.m. route had high peripheral T follicular helper (pTfh) cell counts in blood but low Tfh cell counts in lymph nodes. Conversely, animals immunized by the i.n. route had high Tfh cell counts in lymph nodes but low pTfh cell counts in the blood. Animals immunized by only the i.m. route had lower antibody-dependent cellular cytotoxicity (ADCC) antibody activity, whereas animals immunized by the mucosal i.n. route had higher ADCC antibody activity. When these Env-immunized animals were challenged rectally with simian-human immunodeficiency virus (SHIV) strain SF162P3 (SHIVSF162P3), they all became infected. However, mucosally SC-Ad-immunized animals had lower viral loads in their gastrointestinal tracts. These data suggest that there may be benefits in educating the immune system at mucosal sites during HIV vaccination.IMPORTANCE HIV-1 infections usually start at a mucosal surface after sexual contact. Creating a barrier of protection at these mucosal sites may be a good strategy for to protect against HIV-1 infections. While HIV-1 enters at mucosa, most vaccines are not delivered here. Most are instead injected into the muscle, a site well distant and functionally different than mucosal tissues. This study tested if delivering HIV vaccines at mucosa or in the muscle makes a difference in the quality, quantity, and location of immune responses against the virus. These data suggest that there are indeed advantages to educating the immune system at mucosal sites with an HIV-1 vaccine.

Reduced Cell-Associated DNA and Improved Viral Control in Macaques following Passive Transfer of a Single Anti-V2 Monoclonal Antibody and Repeated Simian/Human Immunodeficiency Virus Challenges.

A high level of V1V2-specific IgG antibodies (Abs) in vaccinees' sera was the only independent variable that correlated with a reduced risk of human immunodeficiency virus (HIV) acquisition in the RV144 clinical trial. In contrast, IgG avidity, antibody neutralization, and antibody-dependent cellular cytotoxicity each failed as independent correlates of infection. Extended analyses of RV144 samples demonstrated the antiviral activities of V1V2-specific vaccine-induced antibodies. V2-specific antibodies have also been associated with protection from simian immunodeficiency virus (SIV), and the V2i-specific subset of human monoclonal antibodies (MAbs), while poor neutralizers, mediates Fc-dependent antiviral functions in vitro The objective of this study was to determine the protective efficacy of a V2i-specific human MAb, 830A, against mucosal simian/human immunodeficiency virus (SHIV) challenge. V2i MAb binding sites overlap the integrin binding site in the V2 region and are similar to the epitopes bound by antibodies associated with reduced HIV infection rates in RV144. Because the IgG3 subclass was a correlate of reduced infection rates in RV144, we compared passive protection by both IgG1 and IgG3 subclasses of V2i MAb 830A. This experiment represents the first in vivo test of the hypothesis emanating from RV144 and SIV studies that V2i Abs can reduce the risk of infection. The results show that passive transfer with a single V2i MAb, IgG1 830A, reduced plasma and peripheral blood mononuclear cell (PBMC) virus levels and decreased viral DNA in lymphoid tissues compared to controls, but too few animals remained uninfected to achieve significance in reducing the risk of infection. Based on these findings, we conclude that V2i antibodies can impede virus seeding following mucosal challenge, resulting in improved virus control.IMPORTANCE Since the results of the HIV RV144 clinical trial were reported, there has been significant interest in understanding how protection was mediated. Antibodies directed to a subregion of the envelope protein called V1V2 were directly correlated with a reduced risk, and surprisingly low virus neutralization was observed. To determine whether these antibodies alone could mediate protection, we used a human monoclonal antibody directed to V2 with properties similar to those elicited in the vaccine trial for passive infusions in rhesus macaques and challenge with SHIV. The single V2 antibody at the dose given did not significantly reduce the number of infections, but there was a significant reduction in the seeding of virus to the lymph nodes and a decrease in plasma viremia in the HIV antibody-infused macaques compared with the control antibody-infused animals. This finding shows that V2 antibodies mediate antiviral activities in vivo that could contribute to a protective HIV vaccine.

Functional Antibody Response Against V1V2 and V3 of HIV gp120 in the VAX003 and VAX004 Vaccine Trials.

Immunization with HIV AIDSVAX gp120 vaccines in the phase III VAX003 and VAX004 trials did not confer protection. To understand the shortcomings in antibody (Ab) responses induced by these vaccines, we evaluated the kinetics of Ab responses to the V1V2 and V3 regions of gp120 and the induction of Ab-mediated antiviral functions during the course of 7 vaccinations over a 30.5-month period. Plasma samples from VAX003 and VAX004 vaccinees and placebo recipients were measured for ELISA-binding Abs and for virus neutralization, Ab-dependent cellular phagocytosis (ADCP), and Ab-dependent cellular cytotoxicity (ADCC). Ab responses to V1V2 and V3 peaked after 3 to 4 immunizations and declined after 5 to 7 immunizations. The deteriorating responses were most evident against epitopes in the underside of the V1V2 ß-barrel and in the V3 crown. Correspondingly, vaccinees demonstrated higher neutralization against SF162 pseudovirus sensitive to anti-V1V2 and anti-V3 Abs after 3 or 4 immunizations than after 7 immunizations. Higher levels of ADCP and ADCC were also observed at early or mid-time points as compared with the final time point. Hence, VAX003 and VAX004 vaccinees generated V1V2- and V3-binding Abs and functional Abs after 3 to 4 immunizations, but subsequent boosts did not maintain these responses.

Combination Adenovirus and Protein Vaccines Prevent Infection or Reduce Viral Burden after Heterologous Clade C Simian-Human Immunodeficiency Virus Mucosal Challenge.

HIV vaccine development is focused on designing immunogens and delivery methods that elicit protective immunity. We evaluated a combination of adenovirus (Ad) vectors expressing HIV 1086.C (clade C) envelope glycoprotein (Env), SIV Gag p55, and human pegivirus GBV-C E2 glycoprotein. We compared replicating simian (SAd7) with nonreplicating human (Ad4) adenovirus-vectored vaccines paired with recombinant proteins in a novel prime-boost regimen in rhesus macaques, with the goal of eliciting protective immunity against SHIV challenge. In both vaccine groups, plasma and buccal Env-specific IgG, tier 1 heterologous neutralizing antibodies, and antibody-dependent cell-mediated viral inhibition were readily generated. High Env-specific T cell responses elicited in all vaccinees were significantly greater than responses targeting Gag. After three intrarectal exposures to heterologous tier 1 clade C SHIV, all 10 sham-vaccinated controls were infected, whereas 4/10 SAd7- and 3/10 Ad4-vaccinated macaques remained uninfected or maintained tightly controlled plasma viremia. Time to infection was significantly delayed in SAd7-vaccinated macaques compared to the controls. Cell-associated and plasma virus levels were significantly lower in each group of vaccinated macaques compared to controls; the lowest plasma viral burden was found in animals vaccinated with the SAd7 vectors, suggesting superior immunity conferred by the replicating simian vectors. Furthermore, higher V1V2-specific binding antibody titers correlated with viral control in the SAd7 vaccine group. Thus, recombinant Ad plus protein vaccines generated humoral and cellular immunity that was effective in either protecting from SHIV acquisition or significantly reducing viremia in animals that became infected, consequently supporting additional development of replicating Ad vectors as HIV vaccines.IMPORTANCE There is a well-acknowledged need for an effective AIDS vaccine that protects against HIV infection and limits in vivo viral replication and associated pathogenesis. Although replicating virus vectors have been advanced as HIV vaccine platforms, there have not been any direct comparisons of the replicating to the nonreplicating format. The present study directly compared the replicating SAd7 to nonreplicating Ad4 vectors in macaques and demonstrated that in the SAd7 vaccine group, the time to infection was significantly delayed compared to the control group, and V1V2 Env-specific binding antibodies correlated with viral outcomes. Viral control was significantly enhanced in vaccinated macaques compared to controls, and in infected SAd7-vaccinated macaques compared to Ad4-vaccinated macaques, suggesting that this vector may have conferred more effective immunity. Because blocking infection is so difficult with current vaccines, development of a vaccine that can limit viremia if infection occurs would be valuable. These data support further development of replicating adenovirus vectors.

Hepatocytic expression of human sodium-taurocholate cotransporting polypeptide enables hepatitis B virus infection of macaques.

Hepatitis B virus (HBV) is a major global health concern, and the development of curative therapeutics is urgently needed. Such efforts are impeded by the lack of a physiologically relevant, pre-clinical animal model of HBV infection. Here, we report that expression of the HBV entry receptor, human sodium-taurocholate cotransporting polypeptide (hNTCP), on macaque primary hepatocytes facilitates HBV infection in vitro, where all replicative intermediates including covalently closed circular DNA (cccDNA) are present. Furthermore, viral vector-mediated expression of hNTCP on hepatocytes in vivo renders rhesus macaques permissive to HBV infection. These in vivo macaque HBV infections are characterized by longitudinal HBV DNA in serum, and detection of HBV DNA, RNA, and HBV core antigen (HBcAg) in hepatocytes. Together, these results show that expressing hNTCP on macaque hepatocytes renders them susceptible to HBV infection, thereby establishing a physiologically relevant model of HBV infection to study immune clearance and test therapeutic and curative approaches.

Pre-existing neutralizing antibody mitigates B cell dysregulation and enhances the Env-specific antibody response in SHIV-infected rhesus macaques.

Our central hypothesis is that protection against HIV infection will be powerfully influenced by the magnitude and quality of the B cell response. Although sterilizing immunity, mediated by pre-formed abundant and potent antibodies is the ultimate goal for B cell-targeted HIV vaccine strategies, scenarios that fall short of this may still confer beneficial defenses against viremia and disease progression. We evaluated the impact of sub-sterilizing pre-existing neutralizing antibody on the B cell response to SHIV infection. Adult male rhesus macaques received passive transfer of a sub-sterilizing amount of polyclonal neutralizing immunoglobulin (Ig) purified from previously infected animals (SHIVIG) or control Ig prior to intra-rectal challenge with SHIVSF162P4 and extensive longitudinal sampling was performed. SHIVIG treated animals exhibited significantly reduced viral load and increased de novo Env-specific plasma antibody. Dysregulation of the B cell profile was grossly apparent soon after infection in untreated animals; exemplified by a ≈50% decrease in total B cells in the blood evident 2-3 weeks post-infection which was not apparent in SHIVIG treated animals. IgD+CD5+CD21+ B cells phenotypically similar to marginal zone-like B cells were highly sensitive to SHIV infection, becoming significantly decreased as early as 3 days post-infection in control animals, while being maintained in SHIVIG treated animals, and were highly correlated with the induction of Env-specific plasma antibody. These results suggest that B cell dysregulation during the early stages of infection likely contributes to suboptimal Env-specific B cell and antibody responses, and strategies that limit this dysregulation may enhance the host's ability to eliminate HIV.

Induction of neutralizing antibodies in rhesus macaques using V3 mimotope peptides.

RV144 vaccinees with low HIV-1 Envelope-specific IgA antibodies (Abs) also had Abs directed to the hypervariable region 3 (V3) that inversely correlated with infection risk. Thus, anti-V3 HIV-1 Abs may contribute to protection from HIV-1 infection. The V3 region contains two dominant clusters of epitopes; one is preferentially recognized by mAbs encoded by VH5-51 and VL lambda genes, while the second one is recognized by mAbs encoded by other VH genes. We designed a study in rhesus macaques to induce anti-V3 Abs specific to each of these two dominant clusters of V3 epitopes to test whether the usage of the VH5-51 gene results in different characteristics of antibodies. The two C4-V3 immunogens used for immunization were each comprised of a fusion of the C4 peptide containing the T cell epitope and a V3 mimotope peptide mimicking the V3 epitope. The C4-447 peptide was designed to target B cells with several VH1-VH4 genes, the C4-VH5-51 peptide was designed to specifically target B cells with the VH5-51 gene. Six animals in two groups were immunized five times with these two immunogens, and screening of 10 sequential plasma samples post immunization demonstrated that C4-447 induced higher titers of plasma anti-V3 Abs and significantly more potent neutralizing activities against tier 1 and some tier 2 pseudoviruses than C4-VH5-51. Levels of anti-V3 Abs in buccal secretions were significantly higher in sequential samples derived from C4-447- than from C4-VH5-51-immunized animals. The titers of anti-V3 Abs in plasma strongly correlated with their levels in mucosal secretions. The results show that high titers of vaccine-induced anti-V3 Abs in plasma determine the potency and breadth of neutralization, as well as the rate of transduction of Abs to mucosal tissues, where they can play a role in preventing HIV-1 infection.

Vaccine delivery to the oral cavity using coated microneedles induces systemic and mucosal immunity.

PURPOSE: The objective of this study is to evaluate the feasibility of using coated microneedles to deliver vaccines into the oral cavity to induce systemic and mucosal immune responses. METHOD: Microneedles were coated with sulforhodamine, ovalbumin and two HIV antigens. Coated microneedles were inserted into the inner lower lip and dorsal surface of the tongue of rabbits. Histology was used to confirm microneedle insertion, and systemic and mucosal immune responses were characterized by measuring antigen-specific immunoglobulin G (IgG) in serum and immunoglobulin A (IgA) in saliva, respectively. RESULTS: Histological evaluation of tissues shows that coated microneedles can penetrate the lip and tongue to deliver coatings. Using ovalbumin as a model antigen it was found that the lip and the tongue are equally immunogenic sites for vaccination. Importantly, both sites also induced a significant (p < 0.05) secretory IgA in saliva compared to pre-immune saliva. Microneedle-based oral cavity vaccination was also compared to the intramuscular route using two HIV antigens, a virus-like particle and a DNA vaccine. Microneedle-based delivery to the oral cavity and the intramuscular route exhibited similar (p > 0.05) yet significant (p < 0.05) levels of antigen-specific IgG in serum. However, only the microneedle-based oral cavity vaccination group stimulated a significantly higher (p < 0.05) antigen-specific IgA response in saliva, but not intramuscular injection. CONCLUSION: In conclusion, this study provides a novel method using microneedles to induce systemic IgG and secretory IgA in saliva, and could offer a versatile technique for oral mucosal vaccination.