Early post-infection treatment of SARS-CoV-2 infected macaques with human convalescent plasma with high neutralizing activity had no antiviral effects but moderately reduced lung inflammation.

Early in the SARS-CoV-2 pandemic, there was a high level of optimism based on observational studies and small controlled trials that treating hospitalized patients with convalescent plasma from COVID-19 survivors (CCP) would be an important immunotherapy. However, as more data from controlled trials became available, the results became disappointing, with at best moderate evidence of efficacy when CCP with high titers of neutralizing antibodies was used early in infection. To better understand the potential therapeutic efficacy of CCP, and to further validate SARS-CoV-2 infection of macaques as a reliable animal model for testing such strategies, we inoculated 12 adult rhesus macaques with SARS-CoV-2 by intratracheal and intranasal routes. One day later, 8 animals were infused with pooled human CCP with a high titer of neutralizing antibodies (RVPN NT50 value of 3,003), while 4 control animals received normal human plasma. Animals were monitored for 7 days. Animals treated with CCP had detectable but low levels of antiviral antibodies after infusion. In comparison to the control animals, CCP-treated animals had similar levels of viral RNA in upper and lower respiratory tract secretions, similar detection of viral RNA in lung tissues by in situ hybridization, but lower amounts of infectious virus in the lungs. CCP-treated animals had a moderate, but statistically significant reduction in interstitial pneumonia, as measured by comprehensive lung histology. Thus overall, therapeutic benefits of CCP were marginal and inferior to results obtained earlier with monoclonal antibodies in this animal model. By highlighting strengths and weaknesses, data of this study can help to further optimize nonhuman primate models to provide proof-of-concept of intervention strategies, and guide the future use of convalescent plasma against SARS-CoV-2 and potentially other newly emerging respiratory viruses.

Severe Acute Respiratory Syndrome Coronavirus 2 Vasculopathy in a Syrian Golden Hamster Model.

Clinical evidence of vascular dysfunction and hypercoagulability as well as pulmonary vascular damage and microthrombosis are frequently reported in severe cases of human coronavirus disease 2019 (COVID-19). Syrian golden hamsters recapitulate histopathologic pulmonary vascular lesions reported in patients with COVID-19. Herein, special staining techniques and transmission electron microscopy further define vascular pathologies in a Syrian golden hamster model of human COVID-19. The results show that regions of active pulmonary inflammation in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are characterized by ultrastructural evidence of endothelial damage with platelet marginalization and both perivascular and subendothelial macrophage infiltration. SARS-CoV-2 antigen/RNA was not detectable within affected blood vessels. Taken together, these findings suggest that the prominent microscopic vascular lesions in SARS-CoV-2-inoculated hamsters likely occur due to endothelial damage followed by platelet and macrophage infiltration.

Simian retrovirus transmission in rhesus macaques.

Historically, to generate Simian Retrovirus (SRV) positive control materials, we performed in vivo passage by inoculating uninfected rhesus macaques with whole blood from an SRV-1 infected (antibody and PCR positive) macaque. However, recent attempts using this approach have failed. This study reports observations and explores why it has become more difficult to transmit SRV via in vivo passage.

Early treatment with a combination of two potent neutralizing antibodies improves clinical outcomes and reduces virus replication and lung inflammation in SARS-CoV-2 infected macaques.

There is an urgent need for effective therapeutic interventions against SARS-CoV-2, including new variants that continue to arise. Neutralizing monoclonal antibodies have shown promise in clinical studies. We investigated the therapeutic efficacy of a combination of two potent monoclonal antibodies, C135-LS and C144-LS that carry half-life extension mutations, in the rhesus macaque model of COVID-19. Twelve young adult macaques (three groups of four animals) were inoculated intranasally and intra-tracheally with a high dose of SARS-CoV-2 and 24 hours later, treated intravenously with a high (40 mg/kg) or low (12 mg/kg) dose of the C135-LS and C144-LS antibody combination, or a control monoclonal antibody. Animals were monitored for 7 days. Compared to the control animals, animals treated with either dose of the anti-SARS-CoV-2 antibodies showed similarly improved clinical scores, lower levels of virus replication in upper and lower respiratory tract, and significantly reduced interstitial pneumonia, as measured by comprehensive lung histology. In conclusion, this study provides proof-of-concept in support of further clinical development of these monoclonal antibodies against COVID-19 during early infection.

Plasma transfusion-transmission of Zika virus in mice and macaques.

BACKGROUND: Zika virus (ZIKV) epidemics with infections in pregnant women are associated with severe neurological disease in newborns. Although an arbovirus, ZIKV is also blood transfusion-transmitted (TT). Greater knowledge of the efficiency of ZIKV TT would aid decisions on testing and pathogen reduction technologies (PRT). STUDY DESIGN AND METHODS: Plasma units from ZIKV RNA-reactive blood donors were used to study infectivity in vitro, in mice, and in macaques. Furthermore, plasma units were subjected to PRT using amotosalen/ultraviolet light A (A/UVA) before transfusion. RESULTS: In vitro infectivity of ZIKV RNA-reactive plasma varied between 100 and 1000 international units (IU) of ZIKV RNA. Immunodeficient mice were more sensitive with as low as 32 IU sufficient to infect 50% of mice. 50-5500 IU of RNA led to TT in macaques using dose escalation of three different RNA-positive, seronegative plasma units. In contrast, RNA-reactive units collected postseroconversion were not infectious in macaques, even at a dose of 9 million IU RNA. After A/UVA PRT, transfusion of plasma containing up to 18 million IU was no longer infectious in vitro and did not result in ZIKV TT in macaques. CONCLUSION: Significant risks of ZIKV TT are likely confined to a relatively short viremic window before seroconversion, and that sensitive nucleic acid amplification testing likely identifies the majority of infectious plasma. PRT was demonstrated to be effective at preventing ZIKV TT. Considering that there is no approved ZIKV vaccine, these data are relevant to mitigate the risk of TT during the future ZIKV outbreaks.

A combination of two human monoclonal antibodies limits fetal damage by Zika virus in macaques.

Human infection by Zika virus (ZIKV) during pregnancy can lead to vertical transmission and fetal aberrations, including microcephaly. Prophylactic administration of antibodies can diminish or prevent ZIKV infection in animal models, but whether passive immunization can protect nonhuman primates and their fetuses during pregnancy has not been determined. Z004 and Z021 are neutralizing monoclonal antibodies to domain III of the envelope (EDIII) of ZIKV. Together the two antibodies protect nonpregnant macaques against infection even after Fc modifications to prevent antibody-dependent enhancement (ADE) in vitro and extend their half-lives. Here we report on prophylactic coadministration of the Fc-modified antibodies to pregnant rhesus macaques challenged three times with ZIKV during first and second trimester. The two antibodies did not entirely eliminate maternal viremia but limited vertical transmission, protecting the fetus from neurologic damage. Thus, maternal passive immunization with two antibodies to EDIII can shield primate fetuses from the harmful effects of ZIKV.

Sylvatic Transmission of Chikungunya Virus among Nonhuman Primates in Myanmar.

Nonhuman primates living in proximity to humans increase risks for sylvatic arbovirus transmission. We collected serum samples from nonhuman primates in Hlawga National Park near Yangon, Myanmar, and detected antibodies against chikungunya (33%) and Japanese encephalitis (4%) viruses. Buffer zones between primate and human communities might reduce cross-species arbovirus transmission.

Developing and validating SARS-CoV-2 assays for nonhuman primate surveillance.

INTRODUCTION: In early 2020, the California National Primate Research Center implemented surveillance to address the threat of SARS-CoV-2 infection in its nonhuman primate colony. MATERIALS/METHODS: To detect antiviral antibodies, multi-antigen assays were developed and validated on enzyme immunoassay and multiplex microbead immunofluorescent assay (MMIA) platforms. To detect viral RNA, RT-PCR was also performed. RESULTS/CONCLUSION: Using a 4plex, antibody was identified in 16/16 experimentally infected animals; and specificity for spike, nucleocapsid, receptor binding domain, and whole virus antigens was 95.2%, 93.8%, 94.3%, and 97.1%, respectively on surveillance samples. Six laboratories compared this MMIA favorably with nine additional laboratory-developed or commercially available assays. Using a screen and confirm algorithm, 141 of the last 2441 surveillance samples were screen-reactive requiring confirmatory testing. Although 35 samples were reactive to either nucleocapsid or spike; none were reactive to both. Over 20 000 animals have been tested and no spontaneous infections have so far been confirmed across the NIH sponsored National Primate Research Centers.

Multi-site proficiency testing for validation and standardization of assays to detect specific pathogen-free viruses, coronaviruses, and other agents in nonhuman primates.

In efforts to increase rigor and reproducibility, the USA National Primate Research Centers (NPRCs) have focused on qualification of reagents, cross-laboratory validations, and proficiency testing for methods to detect infectious agents and accompanying immune responses in nonhuman primates. The pathogen detection working group, comprised of laboratory scientists, colony managers, and leaders from the NPRCs, has championed the effort to produce testing that is reliable and consistent across laboratories. Through multi-year efforts with shared proficiency samples, testing percent agreement has increased from as low as 67.1% for SRV testing in 2010 to 92.1% in 2019. The 2019 average agreement for the four basic SPF agents improved to >96% (86.5% BV, 98.9 SIV, 92.1 SRV, and 97.0 STLV). As new pathogens such as SARS coronavirus type 2 emerge, these steps can now be quickly replicated to develop and implement new assays that ensure rigor, reproducibly, and quality for NHP pathogen detection.

Functional Perturbation of Mucosal Group 3 Innate Lymphoid and Natural Killer Cells in Simian-Human Immunodeficiency Virus/Simian Immunodeficiency Virus-Infected Infant Rhesus Macaques.

Mother-to-child transmission of human immunodeficiency virus type 1 (HIV-1) via breastfeeding is responsible for nearly half of new infections of children with HIV. Although innate lymphoid cells (ILC) and natural killer (NK) cells are found throughout the oral mucosae, the effects of HIV/simian-human immunodeficiency virus (SHIV) in these tissues are largely unknown. To better understand the mechanics of postnatal transmission, we performed a comprehensive study of simian immunodeficiency virus (SIV)/SHIV-infected infant rhesus macaques (RM) and tracked changes in frequency, trafficking, and function of group 3 ILC (ILC3) and NK cells using polychromatic flow cytometry and cell stimulation assays in colon, tonsil, and oral lymph node samples. Infection led to a 3-fold depletion of ILC3 in the colon and an increase in the levels of NK cells in tonsils and oral lymph nodes. ILC3 and NK cells exhibited alterations in their trafficking repertoires as a result of infection, with increased expression of CD103 in colon NK cells and curtailment of CXCR3, and a significant decrease in α4ß7 expression in colon ILC3. SPICE analyses revealed that ILC3 and NK cells displayed distinct functional profiles by tissue in naive samples. Infection perturbed these profiles, with a nearly total loss of interleukin-22 (IL-22) production in the tonsil and colon; an increase in the levels of CD107a, gamma interferon (IFN-γ), and tumor necrosis factor alpha (TNF-α) from ILC3; and an increase in the levels of CD107a, macrophage inflammatory protein 1 beta (MIP-1ß), and TNF-α from NK cells. Collectively, these data reveal that lentivirus infection alters the frequencies, receptor repertoires, and functions of innate cells in the oral and gut mucosa of infants. Further study will be required to delineate the full extent of the effect that these changes have on oral and gut homeostasis, SHIV/SIV pathogenesis, and oral opportunistic disease.IMPORTANCE Vertical transmission of HIV from mother to child accounts for many of the new cases seen worldwide. There is currently no vaccine to mitigate this transmission, and there has been limited research on the effects that lentiviral infection has on the innate immune system in oral tissues of infected children. To fill this knowledge gap, our laboratory studied infant rhesus macaques to evaluate how acute SIV/SHIV infections impacted ILC3 and NK cells, which are immune cells critical for mucosal homeostasis and antimicrobial defense. Our data revealed that SIV/SHIV infection led to a depletion of ILC3 and an increase of NK cells and to a functional shift from a homeostatic to a multifunctional proinflammatory state. Taking the results together, we describe how lentiviral infection perturbs the oral and gastrointestinal mucosae of infant macaques through alterations of resident innate immune cells giving rise to chronic inflammation and potentially exacerbating morbidity and mortality in children living with HIV.

Two Sides of a Coin: a Zika Virus Mutation Selected in Pregnant Rhesus Macaques Promotes Fetal Infection in Mice but at a Cost of Reduced Fitness in Nonpregnant Macaques and Diminished Transmissibility by Vectors.

Although fetal death is now understood to be a severe outcome of congenital Zika syndrome, the role of viral genetics is still unclear. We sequenced Zika virus (ZIKV) from a rhesus macaque fetus that died after inoculation and identified a single intrahost substitution, M1404I, in the ZIKV polyprotein, located in nonstructural protein 2B (NS2B). Targeted sequencing flanking position 1404 in 9 additional macaque mothers and their fetuses identified M1404I at a subconsensus frequency in the majority (5 of 9, 56%) of animals and some of their fetuses. Despite its repeated presence in pregnant macaques, M1404I has occurred rarely in humans since 2015. Since the primary ZIKV transmission cycle is human-mosquito-human, mutations in one host must be retained in the alternate host to be perpetuated. We hypothesized that ZIKV I1404 increases viral fitness in nonpregnant macaques and pregnant mice but is less efficiently transmitted by vectors, explaining its low frequency in humans during outbreaks. By examining competitive fitness relative to that of ZIKV M1404, we observed that ZIKV I1404 produced lower viremias in nonpregnant macaques and was a weaker competitor in tissues. In pregnant wild-type mice, ZIKV I1404 increased the magnitude and rate of placental infection and conferred fetal infection, in contrast to ZIKV M1404, which was not detected in fetuses. Although infection and dissemination rates were not different, Aedes aegypti mosquitoes transmitted ZIKV I1404 more poorly than ZIKV M1404. Our data highlight the complexity of arbovirus mutation-fitness dynamics and suggest that intrahost ZIKV mutations capable of augmenting fitness in pregnant vertebrates may not necessarily spread efficiently via mosquitoes during epidemics.IMPORTANCE Although Zika virus infection of pregnant women can result in congenital Zika syndrome, the factors that cause the syndrome in some but not all infected mothers are still unclear. We identified a mutation that was present in some ZIKV genomes in experimentally inoculated pregnant rhesus macaques and their fetuses. Although we did not find an association between the presence of the mutation and fetal death, we performed additional studies with ZIKV with the mutation in nonpregnant macaques, pregnant mice, and mosquitoes. We observed that the mutation increased the ability of the virus to infect mouse fetuses but decreased its capacity to produce high levels of virus in the blood of nonpregnant macaques and to be transmitted by mosquitoes. This study shows that mutations in mosquito-borne viruses like ZIKV that increase fitness in pregnant vertebrates may not spread in outbreaks when they compromise transmission via mosquitoes and fitness in nonpregnant hosts.

Reduced neuronal population in the dorsolateral prefrontal cortex in infant macaques infected with simian immunodeficiency virus (SIV).

Pediatric HIV infection remains a global health crisis with an estimated 150,000 new mother-to-child (MTCT) infections each year. Antiretroviral therapy (ART) has improved childhood survival, but only an estimated 53% of children worldwide have access to treatment. Adding to the health crisis is the neurological impact of HIV on the developing brain, in particular cognitive and executive function, which persists even when ART is available. Imaging studies suggest structural, connectivity, and functional alterations in perinatally HIV-infected youth. However, the paucity of histological data limits our ability to identify specific cortical regions that may underlie the clinical manifestations. Utilizing the pediatric simian immunodeficiency virus (SIV) infection model in infant macaques, we have previously shown that early-life SIV infection depletes the neuronal population in the hippocampus. Here, we expand on these previous studies to investigate the dorsolateral prefrontal cortex (dlPFC). A total of 11 ART-naïve infant rhesus macaques (Macaca mulatta) from previous studies were retrospectively analyzed. Infant macaques were either intravenously (IV) inoculated with highly virulent SIVmac251 at ~1 week of age and monitored for 6-10 weeks or orally challenged with SIVmac251 from week 9 of age onwards with a monitoring period of 10-23 weeks post-infection (19-34 weeks of age), and SIV-uninfected controls were euthanized at 16-17 weeks of age. Both SIV-infected groups show a significant loss of neurons along with evidence of ongoing neuronal death. Oral- and IV-infected animals showed a similar neuronal loss which was negatively correlated to chronic viremia levels as assessed by an area under the curve (AUC) analysis. The loss of dlPFC neurons may contribute to the rapid neurocognitive decline associated with pediatric HIV infection.

Dolutegravir Monotherapy of Simian Immunodeficiency Virus-Infected Macaques Selects for Several Patterns of Resistance Mutations with Variable Virological Outcomes.

Drug resistance remains a major concern for human immunodeficiency virus (HIV) treatment. To date, very few resistance mutations have emerged in first-line combination therapy that includes the integrase strand transfer inhibitor (INSTI) dolutegravir (DTG). In vitro, DTG selects for several primary mutations that induce low-level DTG resistance; secondary mutations, while increasing the level of resistance, however, further impair replication fitness, which raised the idea that DTG monotherapy may be feasible. The simian immunodeficiency virus (SIV) rhesus macaque model of HIV infection can be useful to explore this concept. Nine macaques were infected with virulent SIVmac251 and started on DTG monotherapy during either acute (n = 2) or chronic infection (n = 7). Within 4 weeks of treatment, all animals demonstrated a reduction in viremia of 0.8 to 3.5 log RNA copies/ml plasma. Continued treatment led to overall sustained benefits, but the outcome after 10 to 50 weeks of treatment was highly variable and ranged from viral rebound to near pretreatment levels to sustained suppression, with viremia being 0.5 to 5 logs lower than expected based on pretreatment viremia. A variety of mutations previously described to confer low-level resistance of HIV-1 to DTG or other INSTI were detected, and these were sometimes followed by mutations believed to be compensatory. Some mutations, such as G118R, previously shown to severely impair the replication capacity in vitro, were associated with more sustained virological and immunological benefits of continued DTG therapy, while other mutations, such as E92Q and G140A/Q148K, were associated with more variable outcomes. The observed variability of the outcomes in macaques warrants avoidance of DTG monotherapy in HIV-infected people.IMPORTANCE A growing number of anti-HIV drug combinations are effective in suppressing virus replication in HIV-infected persons. However, to reduce their cost and risk for toxicity, there is considerable interest in simplifying drug regimens. A major concern with single-drug regimens is the emergence of drug-resistant viral mutants. It has been speculated that DTG monotherapy may be a feasible option, because DTG may have a higher genetic barrier for the development of drug resistance than other commonly used antiretrovirals. To explore treatment initiation with DTG monotherapy, we started SIV-infected macaques on DTG during either acute or chronic infection. Although DTG initially reduced virus replication, continued treatment led to the emergence of a variety of viral mutations previously described to confer low-level resistance of HIV-1 to DTG, and this was associated with variable clinical outcomes. This unpredictability of mutational pathways and outcomes warns against using DTG monotherapy as initial treatment for HIV-infected people.

Simian-Human Immunodeficiency Virus SHIV.CH505-Infected Infant and Adult Rhesus Macaques Exhibit Similar Env-Specific Antibody Kinetics, despite Distinct T-Follicular Helper and Germinal Center B Cell Landscapes.

Global elimination of pediatric human immunodeficiency virus (HIV) infections will require the development of novel immune-based approaches, and understanding infant immunity to HIV is critical to guide the rational design of these intervention strategies. Despite their immunological immaturity, chronically HIV-infected children develop broadly neutralizing antibodies (bnAbs) more frequently and earlier than adults do. However, the ontogeny of humoral responses during acute HIV infection is poorly defined in infants and challenging to study in human cohorts due to the presence of maternal antibodies. To further our understanding of age-related differences in the development of HIV-specific immunity during acute infection, we evaluated the generation of virus-specific humoral immune responses in infant (n = 6) and adult (n = 12) rhesus macaques (RMs) infected with a transmitted/founder (T/F) simian-human immunodeficiency virus (SHIV) (SHIV.C.CH505 [CH505]). The plasma HIV envelope-specific IgG antibody kinetics were similar in SHIV-infected infant and adult RMs, with no significant differences in the magnitude or breadth of these responses. Interestingly, autologous tier 2 virus neutralization responses also developed with similar frequencies and kinetics in infant and adult RMs, despite infants exhibiting significantly higher follicular T helper cell (Tfh) and germinal center B cell frequencies than adults. Finally, we show that plasma viral load was the strongest predictor of the development of autologous virus neutralization in both age groups. Our results indicate that the humoral immune response to SHIV infection develops with similar kinetics among infant and adult RMs, suggesting that the early-life immune system is equipped to respond to HIV-1 and promote the production of neutralizing HIV antibodies.IMPORTANCE There is a lack of understanding of how the maturation of the infant immune system influences immunity to HIV infection or how these responses differ from those of adults. Improving our knowledge of infant HIV immunity will help guide antiviral intervention strategies that take advantage of the unique infant immune environment to successfully elicit protective immune responses. We utilized a rhesus macaque model of SHIV infection as a tool to distinguish the differences in HIV humoral immunity in infants versus adults. Here, we demonstrate that the kinetics and quality of the infant humoral immune response to HIV are highly comparable to those of adults during the early phase of infection, despite distinct differences in their Tfh responses, indicating that slightly different mechanisms may drive infant and adult humoral immunity.

Coadministration of CH31 Broadly Neutralizing Antibody Does Not Affect Development of Vaccine-Induced Anti-HIV-1 Envelope Antibody Responses in Infant Rhesus Macaques.

Prevention of mother-to-child transmission (MTCT) is an indispensable component in combatting the global AIDS epidemic. A combination of passive broadly neutralizing antibody (bnAb) infusion and active vaccination promises to provide protection of infants against MTCT from birth through the breastfeeding period and could prime the immune system for lifelong immunity. In this study, we investigate the impact of a single infusion of CD4 binding site (CD4bs) bnAb administered at birth on de novo antibody responses elicited by concurrent active HIV envelope vaccination. Four groups of infant macaques received active immunizations with subunit Env protein or modified vaccinia Ankara (MVA)-vectored Env and subunit Env protein, with or without a single intravenous coadministration of CH31 bnAb at birth. Vaccinated animals were monitored to evaluate binding and functional antibody responses elicited by the active vaccinations. Despite achieving plasma concentrations that were able to neutralize tier 2 viruses, coadministration of CH31 did not have a large impact on the kinetics, magnitude, specificity, or avidity of vaccine-elicited binding or functional antibody responses, including epitope specificity, the development of CD4bs antibodies, neutralization, binding to infected cells, or antibody-dependent cell-mediated cytotoxicity (ADCC). We conclude that infusion of CD4bs bnAb CH31 at birth does not interfere with de novo antibody responses to active vaccination and that a combination of passive bnAb infusion and active HIV-1 Env vaccination is a viable strategy for immediate and prolonged protection against MTCT.IMPORTANCE Our study is the first to evaluate the impact of passive infusion of a broadly neutralizing antibody in newborns on the de novo development of antibody responses following active vaccinations in infancy. We demonstrated the safety and the feasibility of bnAb administration to achieve biologically relevant levels of the antibody and showed that the passive infusion did not impair the de novo antibody production following HIV-1 Env vaccination. Our study paves the way for further investigations of the combination strategy using passive plus active immunization to provide protection of infants born to HIV-1-positive mothers over the entire period of risk for mother-to-child transmission.

SARS-CoV-2 surveillance for a non-human primate breeding research facility.

BACKGROUND: The emergence of SARS-CoV-2 and the ensuing COVID-19 pandemic prompted the need for a surveillance program to determine the viral status of the California National Primate Research Center non-human primate breeding colony, both for reasons of maintaining colony health and minimizing the risk of interference in COVID-19 and other research studies. METHODS: We collected biological samples from 10% of the rhesus macaque population for systematic testing to detect SARS-CoV-2 virus by RT-PCR and host antibody response by ELISA. Testing required the development and validation of new assays and an algorithm using in laboratory-developed and commercially available reagents and protocols. RESULTS AND CONCLUSIONS: No SARS-CoV-2 RNA or antibody was detected in this study; therefore, we have proposed a modified testing algorithm for sentinel surveillance to monitor for any future transmissions. As additional reagents and controls become available, assay development and validation will continue, leading to the enhanced sensitivity, specificity, accuracy, and efficiency of testing.

Adjuvant-Dependent Enhancement of HIV Env-Specific Antibody Responses in Infant Rhesus Macaques.

Toward the goal of developing an effective HIV vaccine that can be administered in infancy to protect against postnatal and lifelong sexual HIV transmission risks, the current pilot study was designed to compare the effect of novel adjuvants on the induction of HIV Env-specific antibody responses in infant macaques. Aligning our studies with the adjuvanted proteins evaluated in a prime-boost schedule with ALVAC in the ongoing HVTN (HIV Vaccine Trials Network) 702 efficacy trial, we selected the bivalent clade C Env immunogens gp120 C.1086 and gp120 TV1 in combination with the MF59 adjuvant. However, we hypothesized that the adjuvant system AS01, that is included in the pediatric RTS,S malaria vaccine, would promote Env-specific antibody responses superior to those of the oil-in-water MF59 emulsion adjuvant. In a second study arm, we compared two emulsions, glucopyranosyl lipid adjuvant formulated in a stable emulsion (GLA-SE) and 3M-052-SE, containing Toll-like receptor 4 (TLR4) and TLR7/TLR8 (TLR7/8) ligand, respectively. The latter adjuvant had been previously demonstrated to be especially effective in activating neonatal antigen-presenting cells. Our results demonstrate that different adjuvants drive quantitatively or qualitatively distinct responses to the bivalent Env vaccine. AS01 induced higher Env-specific plasma IgG antibody levels than the antigen in MF59 and promoted improved antibody function in infants, and 3M-052-SE outperformed GLA-SE by inducing the highest breadth and functionality of antibody responses. Thus, distinct adjuvants are likely to be required for maximizing vaccine-elicited immune responses in infants, particularly when immunization in infancy aims to elicit both perinatal and lifelong immunity against challenging pathogens such as HIV.IMPORTANCE Alum remains the adjuvant of choice for pediatric vaccines. Yet the distinct nature of the developing immune system in infants likely requires novel adjuvants targeted specifically at the pediatric population to reach maximal vaccine efficacy with an acceptable safety profile. The current study supports the idea that additional adjuvants for pediatric vaccines should be, and need to be, tested in infants for their potential to enhance immune responses. Using an infant macaque model, our results suggest that both AS01 and 3M-052-SE can significantly improve and better sustain HIV Env-specific antibody responses than alum. Despite the limited number of animals, the results revealed interesting differences that warrant further testing of promising novel adjuvant candidates in larger preclinical and clinical studies to define the mechanisms leading to adjuvant-improved antibody responses and to identify targets for adjuvant and vaccine optimization.

Interferon-Gamma test for the detection of Mycobacterium tuberculosis complex infection in Macaca mulatta and other non-human primates.

We have formatted an assay to detect Mycobacterium tuberculosis complex infections of non-human primates. Commercially available reagents were used to elicit a specific immune response that was measured by interferon-gamma release. Initial evaluation using blood samples from Rhesus macaques experimentally infected with M tuberculosis distinguished infected versus uninfected animals.

HIV DNA-Adenovirus Multiclade Envelope Vaccine Induces gp41 Antibody Immunodominance in Rhesus Macaques.

Dominant antibody responses in vaccinees who received the HIV-1 multiclade (A, B, and C) envelope (Env) DNA/recombinant adenovirus virus type 5 (rAd5) vaccine studied in HIV-1 Vaccine Trials Network (HVTN) efficacy trial 505 (HVTN 505) targeted Env gp41 and cross-reacted with microbial antigens. In this study, we asked if the DNA/rAd5 vaccine induced a similar antibody response in rhesus macaques (RMs), which are commonly used as an animal model for human HIV-1 infections and for testing candidate HIV-1 vaccines. We also asked if gp41 immunodominance could be avoided by immunization of neonatal RMs during the early stages of microbial colonization. We found that the DNA/rAd5 vaccine elicited a higher frequency of gp41-reactive memory B cells than gp120-memory B cells in adult and neonatal RMs. Analysis of the vaccine-induced Env-reactive B cell repertoire revealed that the majority of HIV-1 Env-reactive antibodies in both adult and neonatal RMs were targeted to gp41. Interestingly, a subset of gp41-reactive antibodies isolated from RMs cross-reacted with host antigens, including autologous intestinal microbiota. Thus, gp41-containing DNA/rAd5 vaccine induced dominant gp41-microbiota cross-reactive antibodies derived from blood memory B cells in RMs as observed in the HVTN 505 vaccine efficacy trial. These data demonstrated that RMs can be used to investigate gp41 immunodominance in candidate HIV-1 vaccines. Moreover, colonization of neonatal RMs occurred within the first week of life, and immunization of neonatal RMs during this time also induced a dominant gp41-reactive antibody response.IMPORTANCE Our results are critical to current work in the HIV-1 vaccine field evaluating the phenomenon of gp41 immunodominance induced by HIV-1 Env gp140 in RMs and humans. Our data demonstrate that RMs are an appropriate animal model to study this phenomenon and to determine the immunogenicity in new HIV-1 Env trimer vaccine designs. The demonstration of gp41 immunodominance in memory B cells of both adult and neonatal RMs indicated that early vaccination could not overcome gp41 dominant responses.