Cholera toxin B scaffolded, focused SIV V2 epitope elicits antibodies that influence the risk of SIVmac251 acquisition in macaques.

Introduction: An efficacious HIV vaccine will need to elicit a complex package of innate, humoral, and cellular immune responses. This complex package of responses to vaccine candidates has been studied and yielded important results, yet it has been a recurring challenge to determine the magnitude and protective effect of specific in vivo immune responses in isolation. We therefore designed a single, viral-spike-apical, epitope-focused V2 loop immunogen to reveal individual vaccine-elicited immune factors that contribute to protection against HIV/SIV. Method: We generated a novel vaccine by incorporating the V2 loop B-cell epitope in the cholera toxin B (CTB) scaffold and compared two new immunization regimens to a historically protective 'standard' vaccine regimen (SVR) consisting of 2xDNA prime boosted with 2xALVAC-SIV and 1xΔV1gp120. We immunized a cohort of macaques with 5xCTB-V2c vaccine+alum intramuscularly simultaneously with topical intrarectal vaccination of CTB-V2c vaccine without alum (5xCTB-V2/alum). In a second group, we tested a modified version of the SVR consisting of 2xDNA prime and boosted with 1xALVAC-SIV and 2xALVAC-SIV+CTB-V2/alum, (DA/CTB-V2c/alum). Results: In the absence of any other anti-viral antibodies, V2c epitope was highly immunogenic when incorporated in the CTB scaffold and generated highly functional anti-V2c antibodies in the vaccinated animals. 5xCTB-V2c/alum vaccination mediated non-neutralizing ADCC activity and efferocytosis, but produced low avidity, trogocytosis, and no neutralization of tier 1 virus. Furthermore, DA/CTB-V2c/alum vaccination also generated lower total ADCC activity, avidity, and neutralization compared to the SVR. These data suggest that the ΔV1gp120 boost in the SVR yielded more favorable immune responses than its CTB-V2c counterpart. Vaccination with the SVR generates CCR5- α4ß7+CD4+ Th1, Th2, and Th17 cells, which are less likely to be infected by SIV/HIV and likely contributed to the protection afforded in this regimen. The 5xCTB-V2c/alum regimen likewise elicited higher circulating CCR5- α4ß7+ CD4+ T cells and mucosal α4ß7+ CD4+ T cells compared to the DA/CTB-V2c/alum regimen, whereas the first cell type was associated with reduced risk of viral acquisition. Conclusion: Taken together, these data suggest that individual viral spike B-cell epitopes can be highly immunogenic and functional as isolated immunogens, although they might not be sufficient on their own to provide full protection against HIV/SIV infection.

Mid-titer human convalescent plasma administration results in suboptimal prophylaxis against SARS-CoV-2 infection in rhesus macaques.

Introduction: ARS-CoV-2 is a respiratory pathogen currently causing a worldwide pandemic, with resulting pathology of differing severity in humans, from mild illness to severe disease and death. The rhesus macaque model of COVID-19 was utilized to evaluate the added benefit of prophylactic administration of human post-SARS-CoV-2 infection convalescent plasma (CP) on disease progression and severity. Methods: A pharmacokinetic (PK) study using CP in rhesus monkeys preceded the challenge study and revealed the optimal time of tissue distribution for maximal effect. Thereafter, CP was administered prophylactically three days prior to mucosal SARS-CoV-2 viral challenge. Results: Results show similar viral kinetics in mucosal sites over the course of infection independent of administration of CP or normal plasma, or historic controls with no plasma. No changes were noted upon necropsy via histopathology, although there were differences in levels of vRNA in tissues, with both normal and CP seemingly blunting viral loads. Discussion: Results indicate that prophylactic administration with mid-titer CP is not effective in reducing disease severity of SARS-CoV-2 infection in the rhesus COVID-19 disease model.

Recombinant Simian Varicella Virus-Simian Immunodeficiency Virus Vaccine Induces T and B Cell Functions and Provides Partial Protection against Repeated Mucosal SIV Challenges in Rhesus Macaques.

HIV vaccine mediated efficacy, using an expanded live attenuated recombinant varicella virus-vectored SIV rSVV-SIVgag/env vaccine prime with adjuvanted SIV-Env and SIV-Gag protein boosts, was evaluated in a female rhesus macaques (RM) model against repeated intravaginal SIV challenges. Vaccination induced anti-SIV IgG responses and neutralizing antibodies were found in all vaccinated RMs. Three of the eight vaccinated RM remained uninfected (vaccinated and protected, VP) after 13 repeated challenges with the pathogenic SIVmac251-CX-1. The remaining five vaccinated and infected (VI) macaques had significantly reduced plasma viral loads compared with the infected controls (IC). A significant increase in systemic central memory CD4+ T cells and mucosal CD8+ effector memory T-cell responses was detected in vaccinated RMs compared to controls. Variability in lymph node SIV-Gag and Env specific CD4+ and CD8+ T cell cytokine responses were detected in the VI RMs while all three VP RMs had more durable cytokine responses following vaccination and prior to challenge. VI RMs demonstrated predominately SIV-specific monofunctional cytokine responses while the VP RMs generated polyfunctional cytokine responses. This study demonstrates that varicella virus-vectored SIV vaccination with protein boosts induces a 37.5% efficacy rate against pathogenic SIV challenge by generating mucosal memory, virus specific neutralizing antibodies, binding antibodies, and polyfunctional T-cell responses.

Durable protection against the SARS-CoV-2 Omicron variant is induced by an adjuvanted subunit vaccine.

Despite the remarkable efficacy of COVID-19 vaccines, waning immunity and the emergence of SARS-CoV-2 variants such as Omicron represents a global health challenge. Here, we present data from a study in nonhuman primates demonstrating durable protection against the Omicron BA.1 variant induced by a subunit SARS-CoV-2 vaccine comprising the receptor binding domain of the ancestral strain (RBD-Wu) on the I53-50 nanoparticle adjuvanted with AS03, which was recently authorized for use in individuals 18 years or older. Vaccination induced neutralizing antibody (nAb) titers that were maintained at high concentrations for at least 1 year after two doses, with a pseudovirus nAb geometric mean titer (GMT) of 1978 and a live virus nAb GMT of 1331 against the ancestral strain but not against the Omicron BA.1 variant. However, a booster dose at 6 to 12 months with RBD-Wu or RBD-ß (RBD from the Beta variant) displayed on I53-50 elicited high neutralizing titers against the ancestral and Omicron variants. In addition, we observed persistent neutralization titers against a panel of sarbecoviruses, including SARS-CoV. Furthermore, there were substantial and persistent memory T and B cell responses reactive to Beta and Omicron variants. Vaccination resulted in protection against Omicron infection in the lung and suppression of viral burden in the nares at 6 weeks after the final booster immunization. Even at 6 months after vaccination, we observed protection in the lung and rapid control of virus in the nares. These results highlight the durable and cross-protective immunity elicited by the AS03-adjuvanted RBD-I53-50 nanoparticle vaccine.

Safety, immunogenicity, and protection provided by unadjuvanted and adjuvanted formulations of a recombinant plant-derived virus-like particle vaccine candidate for COVID-19 in nonhuman primates.

Although antivirals are important tools to control severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, effective vaccines are essential to control the current coronavirus disease 2019 (COVID-19) pandemic. Plant-derived virus-like particle (VLP) vaccine candidates have previously demonstrated immunogenicity and efficacy against influenza. Here, we report the immunogenicity and protection induced in rhesus macaques by intramuscular injections of a VLP bearing a SARS-CoV-2 spike protein (CoVLP) vaccine candidate formulated with or without Adjuvant System 03 (AS03) or cytidine-phospho-guanosine (CpG) 1018. Although a single dose of the unadjuvanted CoVLP vaccine candidate stimulated humoral and cell-mediated immune responses, booster immunization (at 28 days after priming) and adjuvant administration significantly improved both responses, with higher immunogenicity and protection provided by the AS03-adjuvanted CoVLP. Fifteen micrograms of CoVLP adjuvanted with AS03 induced a polyfunctional interleukin-2 (IL-2)-driven response and IL-4 expression in CD4 T cells. Animals were challenged by multiple routes (i.e., intratracheal, intranasal, and ocular) with a total viral dose of 106 plaque-forming units of SARS-CoV-2. Lower viral replication in nasal swabs and bronchoalveolar lavage fluid (BALF) as well as fewer SARS-CoV-2-infected cells and immune cell infiltrates in the lungs concomitant with reduced levels of proinflammatory cytokines and chemotactic factors in the BALF were observed in animals immunized with the CoVLP adjuvanted with AS03. No clinical, pathologic, or virologic evidence of vaccine-associated enhanced disease was observed in vaccinated animals. The CoVLP adjuvanted with AS03 was therefore selected for vaccine development and clinical trials.

Effective Prophylaxis of COVID-19 in Rhesus Macaques Using a Combination of Two Parenterally-Administered SARS-CoV-2 Neutralizing Antibodies.

SARS-CoV-2 is a respiratory borne pathogenic beta coronavirus that is the source of a worldwide pandemic and the cause of multiple pathologies in man. The rhesus macaque model of COVID-19 was utilized to test the added benefit of combinatory parenteral administration of two high-affinity anti-SARS-CoV-2 monoclonal antibodies (mAbs; C144-LS and C135-LS) expressly developed to neutralize the virus and modified to extend their pharmacokinetics. After completion of kinetics study of mAbs in the primate, combination treatment was administered prophylactically to mucosal viral challenge. Results showed near complete virus neutralization evidenced by no measurable titer in mucosal tissue swabs, muting of cytokine/chemokine response, and lack of any discernable pathologic sequalae. Blocking infection was a dose-related effect, cohorts receiving lower doses (6, 2 mg/kg) resulted in low grade viral infection in various mucosal sites compared to that of a fully protective dose (20 mg/kg). A subset of animals within this cohort whose infectious challenge was delayed 75 days later after mAb administration were still protected from disease. Results indicate this combination mAb effectively blocks development of COVID-19 in the rhesus disease model and accelerates the prospect of clinical studies with this effective antibody combination.

Neutralizing antibodies against Epstein-Barr virus infection of B cells can protect from oral viral challenge in the rhesus macaque animal model.

Epstein-Barr virus (EBV) and related lymphocryptoviruses (LCVs) from nonhuman primates are transmitted through oral secretions, penetrate the mucosal epithelium, and establish persistent infection in B cells. To determine whether neutralizing antibodies against epithelial or B cell infection could block oral transmission and persistent LCV infection, we use rhesus macaques, the most accurate animal model for EBV infection by faithfully reproducing acute and persistent infection in humans. Naive animals are infused with monoclonal antibodies neutralizing epithelial cell infection or B cell infection and then challenged orally with recombinant rhesus LCV. Our data show that high-titer B cell-neutralizing antibodies alone, but not epithelial cell-neutralizing antibodies, can provide complete protection of rhesus macaques from oral LCV challenge, but not in all hosts. Thus, neutralizing antibodies against B cell infection are important targets for EBV vaccine development, but they may not be sufficient.

Development of an In Vivo Probe to Track SARS-CoV-2 Infection in Rhesus Macaques.

Infection with the novel coronavirus, SARS-CoV-2, results in pneumonia and other respiratory symptoms as well as pathologies at diverse anatomical sites. An outstanding question is whether these diverse pathologies are due to replication of the virus in these anatomical compartments and how and when the virus reaches those sites. To answer these outstanding questions and study the spatiotemporal dynamics of SARS-CoV-2 infection a method for tracking viral spread in vivo is needed. We developed a novel, fluorescently labeled, antibody-based in vivo probe system using the anti-spike monoclonal antibody CR3022 and demonstrated that it could successfully identify sites of SARS-CoV-2 infection in a rhesus macaque model of COVID-19. Our results showed that the fluorescent signal from our antibody-based probe could differentiate whole lungs of macaques infected for 9 days from those infected for 2 or 3 days. Additionally, the probe signal corroborated the frequency and density of infected cells in individual tissue blocks from infected macaques. These results provide proof of concept for the use of in vivo antibody-based probes to study SARS-CoV-2 infection dynamics in rhesus macaques.

Effects of Social Housing Changes on Immunity and Vaccine-Specific Immune Responses in Adolescent Male Rhesus Macaques.

Nonhuman primates (NHPs) in research institutions may be housed in a variety of social settings, such as group housing, pair housing or single housing based on the needs of studies. Furthermore, housing may change over the course of studies. The effects of housing and changes in housing on cell activation and vaccine mediated immune responses are not well documented. We hypothesized that animals moved indoors from group to single housing (GH-SH) would experience more stress than those separated from groups into pair housing (GH-PH), or those placed briefly into pair housing and separated 5 weeks later into single housing (GH-PH-SH). We also compared the effects of separation from group to pair housing with the separation from pair to single housing. Eighteen male rhesus macaques were followed over the course of changes in housing condition over 10-14 weeks, as well as prior to and after primary vaccination with a commercially available measles vaccine. We identified two phenotypic biomarkers, namely total CD8 population and proliferating B cells, that differed significantly across treatment groups over time. At 10 weeks post-separation, levels of proliferating B cells were higher in GH-SH subjects compared to GH-PH subjects, and in the latter, levels were lower at 10 weeks than prior to removal from group housing. At 2 weeks post-separation from group to single housing, the frequency of CD8+ T cells was higher in GH-SH subjects compared to one week post separation from pair into single housing in the GH-PH-SH subjects. Comparing the same elapsed time since the most recent separation activated CD20 populations were persistently higher in the GH-SH animals than the GH-PH-SH animals. Housing configuration did not influence vaccine-mediated responses. Overall, our study found benefits of pair housing over single housing, suggesting that perturbations in immune function will be more severe following separation from group to single housing than from pair to single housing, and supporting the use of short-duration pair housing even when animals must subsequently be separated. These findings are useful for planning the housing configurations of research NHPs used for vaccine studies and other studies where immune response is being assessed.

Polyclonal HIV envelope-specific breast milk antibodies limit founder SHIV acquisition and cell-associated virus loads in infant rhesus monkeys.

Breast milk HIV-1 transmission is currently the predominant contributor to pediatric HIV infections. Yet, only ~10% of breastfeeding infants born to untreated HIV-infected mothers become infected. This study assessed the protective capacity of natural HIV envelope-specific antibodies isolated from the milk of HIV-infected women in an infant rhesus monkey (RM), tier 2 SHIV oral challenge model. To mimic placental and milk maternal antibody transfer, infant RMs were i.v. infused and orally treated at the time of challenge with a single weakly neutralizing milk monoclonal antibody (mAb), a tri-mAb cocktail with weakly neutralizing and ADCC functionalities, or an anti-influenza control mAb. Of these groups, the fewest tri-mAb-treated infants had SHIV detectable in plasma or tissues (2/6, 5/6, and 7/8 animals infected in tri-mAb, single-mAb, and control-mAb groups, respectively). Tri-mAb-treated infants demonstrated significantly fewer plasma transmitted/founder variants and reduced peripheral CD4+ T cell proviral loads at 8 weeks post-challenge compared to control mAb-treated infants. Abortive infection was observed as detectable CD4+ T cell provirus in non-viremic control mAb- and single mAb-, but not in tri-mAb-treated animals. These results suggest that polyfunctional milk antibodies contribute to the natural inefficiency of HIV-1 transmission through breastfeeding and infant vaccinations eliciting non-neutralizing antibody responses could reduce postnatal HIV transmission.

TLR7/8 adjuvant overcomes newborn hyporesponsiveness to pneumococcal conjugate vaccine at birth.

Infection is the most common cause of mortality in early life, and immunization is the most promising biomedical intervention to reduce this burden. However, newborns fail to respond optimally to most vaccines. Adjuvantation is a key approach to enhancing vaccine immunogenicity, but responses of human newborn leukocytes to most candidate adjuvants, including most TLR agonists, are functionally distinct. Herein, we demonstrate that 3M-052 is a locally acting lipidated imidazoquinoline TLR7/8 agonist adjuvant in mice, which, when properly formulated, can induce robust Th1 cytokine production by human newborn leukocytes in vitro, both alone and in synergy with the alum-adjuvanted pneumococcal conjugate vaccine 13 (PCV13). When admixed with PCV13 and administered i.m. on the first day of life to rhesus macaques, 3M-052 dramatically enhanced generation of Th1 CRM-197-specific neonatal CD4+ cells, activation of newborn and infant Streptococcus pneumoniae polysaccharide-specific (PnPS-specific) B cells as well as serotype-specific antibody titers, and opsonophagocytic killing. Remarkably, a single dose at birth of PCV13 plus 0.1 mg/kg 3M-052 induced PnPS-specific IgG responses that were approximately 10-100 times greater than a single birth dose of PCV13 alone, rapidly exceeding the serologic correlate of protection, as early as 28 days of life. This potent immunization strategy, potentially effective with one birth dose, could represent a new paradigm in early life vaccine development.

Aerosol vaccination with AERAS-402 elicits robust cellular immune responses in the lungs of rhesus macaques but fails to protect against high-dose Mycobacterium tuberculosis challenge.

Development of a vaccine against pulmonary tuberculosis may require immunization strategies that induce a high frequency of Ag-specific CD4 and CD8 T cells in the lung. The nonhuman primate model is essential for testing such approaches because it has predictive value for how vaccines elicit responses in humans. In this study, we used an aerosol vaccination strategy to administer AERAS-402, a replication-defective recombinant adenovirus (rAd) type 35 expressing Mycobacterium tuberculosis Ags Ag85A, Ag85B, and TB10.4, in bacillus Calmette-Guérin (BCG)-primed or unprimed rhesus macaques. Immunization with BCG generated low purified protein derivative-specific CD4 T cell responses in blood and bronchoalveolar lavage. In contrast, aerosolized AERAS-402 alone or following BCG induced potent and stable Ag85A/b-specific CD4 and CD8 effector T cells in bronchoalveolar lavage that largely produced IFN-γ, as well as TNF and IL-2. Such responses induced by BCG, AERAS-402, or both failed to confer overall protection following challenge with 275 CFUs M. tuberculosis Erdman, although vaccine-induced responses associated with reduced pathology were observed in some animals. Anamnestic T cell responses to Ag85A/b were not detected in blood of immunized animals after challenge. Overall, our data suggest that a high M. tuberculosis challenge dose may be a critical factor in limiting vaccine efficacy in this model. However, the ability of aerosol rAd immunization to generate potent cellular immunity in the lung suggests that using different or more immunogens, alternative rAd serotypes with enhanced immunogenicity, and a physiological challenge dose may achieve protection against M. tuberculosis.

Efficacy and safety of live attenuated persistent and rapidly cleared Mycobacterium tuberculosis vaccine candidates in non-human primates.

Tuberculosis (TB) remains a global health burden for which safe vaccines are needed. BCG has limitations as a TB vaccine so we have focused on live attenuated Mycobacterium tuberculosis mutants as vaccine candidates. Prior to human studies, however, it is necessary to demonstrate safety in non-human primates (NHP). In this study, we evaluate the safety and efficacy of two live attenuated M. tuberculosis double deletion vaccine strains mc(2)6020 (DeltalysA DeltapanCD) and mc(2)6030 (DeltaRD1 DeltapanCD) in cynomolgus macaques. In murine models, mc(2)6020 is rapidly cleared while mc(2)6030 persists. Both mc(2)6020 and mc(2)6030 were safe and well tolerated in cynomolgus macaques. Following a high-dose intrabronchial challenge with virulent M. tuberculosis, mc(2)6020-vaccinates were afforded a level of protection intermediate between that elicited by BCG vaccination and no vaccination. BCG vaccinates had reduced tuberculosis-associated pathology and improved clinical scores as compared to saline and mc(2)6030 vaccinates, but survival did not differ among the groups.

The macaque gut microbiome in health, lentiviral infection, and chronic enterocolitis.

The vertebrate gut harbors a vast community of bacterial mutualists, the composition of which is modulated by the host immune system. Many gastrointestinal (GI) diseases are expected to be associated with disruptions of host-bacterial interactions, but relatively few comprehensive studies have been reported. We have used the rhesus macaque model to investigate forces shaping GI bacterial communities. We used DNA bar coding and pyrosequencing to characterize 141,000 sequences of 16S rRNA genes obtained from 100 uncultured GI bacterial samples, allowing quantitative analysis of community composition in health and disease. Microbial communities of macaques were distinct from those of mice and humans in both abundance and types of taxa present. The macaque communities differed among samples from intestinal mucosa, colonic contents, and stool, paralleling studies of humans. Communities also differed among animals, over time within individual animals, and between males and females. To investigate changes associated with disease, samples of colonic contents taken at necropsy were compared between healthy animals and animals with colitis and undergoing antibiotic therapy. Communities from diseased and healthy animals also differed significantly in composition. This work provides comprehensive data and improved methods for studying the role of commensal microbiota in macaque models of GI diseases and provides a model for the large-scale screening of the human gut microbiome.

Measurement of plasma-derived substance P: biological, methodological, and statistical considerations.

The undecapeptide substance P (SP) is a member of the tachykinin family of neurotransmitters, which has a pivotal role in the regulation of inflammatory and immune responses. One of the major barriers to the study of the in vivo role of SP in a number of immune disorders is the accurate measurement of SP in fluids. This is reflected in the variability of reported SP levels in serum and plasma of humans in both healthy and diseased states. This study was initiated in order to identify sources of variability by the comparative evaluation of the influences of sample preparation and analytical detection methods on the measurement of SP in plasma. The results indicate that sample preparation (peptide extraction versus no extraction) and the choice of analytical method for SP quantitation may yield significantly different values and may contribute to the variability in SP values reported in the literature. These results further emphasize the need for careful consideration in the selection of methods for SP quantitation, as well as caution in the interpretation and comparison of data reported in the literature.

Immunogenicity study of glycoprotein-deficient rabies virus expressing simian/human immunodeficiency virus SHIV89.6P envelope in a rhesus macaque.

Rabies virus (RV) has recently been developed as a novel vaccine candidate for human immunodeficiency virus type 1 (HIV-1). The RV glycoprotein (G) can be functionally replaced by HIV-1 envelope glycoprotein (Env) if the gp160 cytoplasmic domain (CD) of HIV-1 Env is replaced by that of RV G. Here, we describe a pilot study of the in vivo replication and immunogenicity of an RV with a deletion of G (DeltaG) expressing a simian/human immunodeficiency virus SHIV(89.6P) Env ectodomain and transmembrane domain fused to the RV G CD (DeltaG-89.6P-RVG) in a rhesus macaque. An animal vaccinated with DeltaG-89.6P-RVG developed SHIV(89.6P) virus-neutralizing antibodies and SHIV(89.6P)-specific cellular immune responses after challenge with SHIV(89.6P). There was no evidence of CD4(+) T-cell loss, and plasma viremia was controlled to undetectable levels by 6 weeks postchallenge and has remained suppressed out to 22 weeks postchallenge.