A replicon RNA vaccine can induce durable protective immunity from SARS-CoV-2 in nonhuman primates after neutralizing antibodies have waned.

The global SARS-CoV-2 pandemic prompted rapid development of COVID-19 vaccines. Although several vaccines have received emergency approval through various public health agencies, the SARS-CoV-2 pandemic continues. Emergent variants of concern, waning immunity in the vaccinated, evidence that vaccines may not prevent transmission and inequity in vaccine distribution have driven continued development of vaccines against SARS-CoV-2 to address these public health needs. In this report, we evaluated a novel self-amplifying replicon RNA vaccine against SARS-CoV-2 in a pigtail macaque model of COVID-19 disease. We found that this vaccine elicited strong binding and neutralizing antibody responses against homologous virus. We also observed broad binding antibody against heterologous contemporary and ancestral strains, but neutralizing antibody responses were primarily targeted to the vaccine-homologous strain. While binding antibody responses were sustained, neutralizing antibody waned to undetectable levels in some animals after six months but were rapidly recalled and conferred protection from disease when the animals were challenged 7 months after vaccination as evident by reduced viral replication and pathology in the lower respiratory tract, reduced viral shedding in the nasal cavity and lower concentrations of pro-inflammatory cytokines in the lung. Cumulatively, our data demonstrate in pigtail macaques that a self-amplifying replicon RNA vaccine can elicit durable and protective immunity to SARS-CoV-2 infection. Furthermore, these data provide evidence that this vaccine can provide durable protective efficacy and reduce viral shedding even after neutralizing antibody responses have waned to undetectable levels.

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.

Chronic innate immune impairment and ZIKV persistence in the gastrointestinal tract during SIV infection in pigtail macaques.

Mosquito borne flaviviruses, including dengue (DENV) and Zika (ZIKV) viruses, have caused global epidemics in areas with high HIV prevalence due to the expanded geographic range of arthropod vectors. Despite the occurrence of large flavivirus outbreaks in countries with high HIV prevalence, there is little knowledge regarding the effects of flavivirus infection in people living with HIV (PLWH). Here, we use a pigtail macaque model of HIV/AIDS to investigate the impact of simian immunodeficiency virus (SIV)-induced immunosuppression on ZIKV replication and pathogenesis. Early acute SIV infection induced expansion of peripheral ZIKV cellular targets and increased innate immune activation and peripheral blood mononuclear cells (PBMC) from SIV infected macaques were less permissive to ZIKV infection in vitro. In SIV-ZIKV co-infected animals, we found increased persistence of ZIKV in the periphery and tissues corresponding to alterations in innate cellular (monocytes, neutrophils) recruitment to the blood and tissues, decreased anti-ZIKV immunity, and chronic peripheral inflammatory and innate immune gene expression. Collectively, these findings suggest that untreated SIV infection may impair cellular innate responses and create an environment of chronic immune activation that promotes prolonged ZIKV viremia and persistence in the gastrointestinal tract. These results suggest that PLWH or other immunocompromised individuals could be at a higher risk for chronic ZIKV replication, which in turn could increase the timeframe of ZIKV transmission. Thus, PLWH are important populations to target during the deployment of vaccine and treatment strategies against ZIKV.

Evaluation of the immunogenicity and efficacy of an rVSV vaccine against Zika virus infection in macaca nemestrina.

Zika virus (ZIKV) is a mosquito-borne flavivirus that causes an acute febrile illness. ZIKV can be transmitted between sexual partners and from mother to fetus. Infection is strongly associated with neurologic complications in adults, including Guillain-Barré syndrome and myelitis, and congenital ZIKV infection can result in fetal injury and congenital Zika syndrome (CZS). Development of an effective vaccine is imperative to protect against ZIKV vertical transmission and CZS. Recombinant Vesicular Stomatitis virus (rVSV) is a highly effective and safe vector for the delivery of foreign immunogens for vaccine purposes. Here, we evaluate an rVSV vaccine expressing the full length pre-membrane (prM) and ZIKV envelope (E) proteins (VSV-ZprME), shown to be immunogenic in murine models of ZIKV infection, for its capacity to induce immune responses in nonhuman primates. Moreover, we assess the efficacy of the rVSVΔM-ZprME vaccine in the protection of pigtail macaques against ZIKV infection. Administration of the rVSVΔM-ZprME vaccine was safe, but it did not induce robust anti-ZIKV T-cell responses, IgM or IgG antibodies, or neutralizing antibodies in most animals. Post ZIKV challenge, animals that received the rVSVΔM control vaccine lacking ZIKV antigen had higher levels of plasma viremia compared to animals that received the rVSVΔM-ZprME vaccine. Anti-ZIKV neutralizing Ab titers were detected in a single animal that received the rVSVΔM-ZprME vaccine that was associated with reduced plasma viremia. The overall suboptimal ZIKV-specific cellular and humoral responses post-immunization indicates the rVSVΔM-ZprME vaccine did not elicit an immune response in this pilot study. However, recall antibody response to the rVSVΔM-ZprME vaccine indicates it may be immunogenic and further developments to the vaccine construct could enhance its potential as a vaccine candidate in a nonhuman primate pre-clinical model.

A replicon RNA vaccine induces durable protective immunity from SARS-CoV-2 in nonhuman primates after neutralizing antibodies have waned.

The global SARS-CoV-2 pandemic prompted rapid development of COVID-19 vaccines. Although several vaccines have received emergency approval through various public health agencies, the SARS-CoV-2 pandemic continues. Emergent variants of concern, waning immunity in the vaccinated, evidence that vaccines may not prevent transmission and inequity in vaccine distribution have driven continued development of vaccines against SARS-CoV-2 to address these public health needs. In this report, we evaluated a novel self-amplifying replicon RNA vaccine against SARS-CoV-2 in a pigtail macaque model of COVID-19 disease. We found that this vaccine elicited strong binding and neutralizing antibody responses. While binding antibody responses were sustained, neutralizing antibody waned to undetectable levels after six months but were rapidly recalled and conferred protection from disease when the animals were challenged 7 months after vaccination as evident by reduced viral replication and pathology in the lower respiratory tract, reduced viral shedding in the nasal cavity and lower concentrations of pro-inflammatory cytokines in the lung. Cumulatively, our data demonstrate in pigtail macaques that a self-amplifying replicon RNA vaccine can elicit durable and protective immunity to SARS-CoV-2 infection. Furthermore, these data provide evidence that this vaccine can provide durable protective efficacy and reduce viral shedding even after neutralizing antibody responses have waned to undetectable levels.

A Single Dose SARS-CoV-2 Replicon RNA Vaccine Induces Cellular and Humoral Immune Responses in Simian Immunodeficiency Virus Infected and Uninfected Pigtail Macaques.

The ongoing COVID-19 vaccine rollout is critical for reducing SARS-CoV-2 infections, hospitalizations, and deaths worldwide. Unfortunately, massive disparities exist in getting vaccines to vulnerable populations, including people living with HIV. Preliminary studies indicate that COVID-19 mRNA vaccines are safe and immunogenic in people living with HIV that are virally suppressed with potent antiretroviral therapy but may be less efficacious in immunocompromised individuals. This raises the concern that COVID-19 vaccines may be less effective in resource poor settings with limited access to antiretroviral therapy. Here, we evaluated the immunogenicity of a single dose COVID-19 replicon RNA vaccine expressing Spike protein (A.1) from SARS-CoV-2 (repRNA-CoV2S) in immunocompromised, SIV infected and immune competent, naïve pigtail macaques. Moderate vaccine-specific cellular Th1 T-cell responses and binding and neutralizing antibodies were induced by repRNA-CoV2S in SIV infected animals and naïve animals. Furthermore, vaccine immunogenicity was elicited even among the animals with the highest SIV viral burden or lowest peripheral CD4 counts prior to immunization. This study provides evidence that a SARS-CoV-2 repRNA vaccine could be employed to induce strong immunity against COVID-19 in HIV infected and other immunocompromised individuals.

Effects of persistent modulation of intestinal microbiota on SIV/HIV vaccination in rhesus macaques.

An effective vaccine to prevent HIV transmission has not yet been achieved. Modulation of the microbiome via probiotic therapy has been suggested to result in enhanced mucosal immunity. Here, we evaluated whether probiotic therapy could improve the immunogenicity and protective efficacy of SIV/HIV vaccination. Rhesus macaques were co-immunized with an SIV/HIV DNA vaccine via particle-mediated epidermal delivery and an HIV protein vaccine administered intramuscularly with Adjuplex™ adjuvant, while receiving daily oral Visbiome® probiotics. Probiotic therapy alone led to reduced frequencies of colonic CCR5+ and CCR6+ CD4+ T cells. Probiotics with SIV/HIV vaccination led to similar reductions in colonic CCR5+ CD4+ T cell frequencies. SIV/HIV-specific T cell and antibody responses were readily detected in the periphery of vaccinated animals but were not enhanced with probiotic treatment. Combination probiotics and vaccination did not impact rectal SIV/HIV target populations or reduce the rate of heterologous SHIV acquisition during the intrarectal challenge. Finally, post-infection viral kinetics were similar between all groups. Thus, although probiotics were well-tolerated when administered with SIV/HIV vaccination, vaccine-specific responses were not significantly enhanced. Additional work will be necessary to develop more effective strategies of microbiome modulation in order to enhance mucosal vaccine immunogenicity and improve protective immune responses.

Gene Transfer in Adeno-Associated Virus Seropositive Rhesus Macaques Following Rapamycin Treatment and Subcutaneous Delivery of AAV6, but Not Retargeted AAV6 Vectors.

Adeno-associated virus (AAV) vectors such as AAV6, which shows tropism for primary human CD4+ T cells in vitro, are being explored for delivery of anti-HIV therapeutic modalities in vivo. However, pre-existing immunity and sequestration in nontarget organs can significantly hinder their performance. To overcome these challenges, we investigated whether immunosuppression would allow gene delivery by AAV6 or targeted AAV6 derivatives in seropositive rhesus macaques. Animals were immune suppressed with rapamycin before intravenous (IV) or subcutaneous (SC) delivery of AAV, and we monitored vector biodistribution, gene transfer, and safety. Macaques received phosphate-buffered saline, AAV6 alone, or an equal dose of AAV6 and an AAV6-55.2 vector retargeted to CD4 through a direct ankyrin repeat protein (DARPin). AAV6 and AAV6-55.2 vector genomes were found in peripheral blood mononuclear cells and most organs up to 28 days postadministration, with the highest levels seen in liver, spleen, lymph nodes (LNs), and muscle, suggesting that retargeting did not prevent vector sequestration. Despite vector genome detection, gene expression from AAV6-55.2 was not detected in any tissue. SC injection of AAV6 facilitated efficient gene expression in muscle adjacent to the injection site, plus low-level gene expression in spleen, LNs, and liver, whereas gene expression following IV injection of AAV6 was predominantly seen in the spleen. AAV vectors were well tolerated, although elevated liver enzymes were detected in three of four AAV-treated animals 14 days after rapamycin withdrawal. One SC-injected animal had muscle inflammation proximal to the injection site, plus detectable T cell responses against transgene and AAV6 capsid at study finish. Overall, our data suggest that rapamycin treatment may offer a possible strategy to express anti-HIV therapeutics such as broadly neutralizing antibodies from muscle. This study provides important safety and efficacy data that will aid study design for future anti-HIV gene therapies.

Antibiotic-induced microbiome perturbations are associated with significant alterations to colonic mucosal immunity in rhesus macaques.

The diverse bacterial communities that colonize the gastrointestinal tract play an essential role in maintaining immune homeostasis through the production of critical metabolites such as short-chain fatty acids (SCFAs) and this can be disrupted by antibiotic use. However, few studies have addressed the effects of specific antibiotics longitudinally on the microbiome and immunity. We evaluated the effects of four specific antibiotics: enrofloxacin, cephalexin, paromomycin, and clindamycin, in healthy female rhesus macaques. All antibiotics disrupted the microbiome, including reduced abundances of fermentative bacteria and increased abundances of potentially pathogenic bacteria, including Enterobacteriaceae in the stool, and decreased Helicobacteraceae in the colon. This was associated with decreased SCFAs, indicating altered bacterial metabolism. Importantly, antibiotic use also substantially altered local immune responses, including increased neutrophils and Th17 cells in the colon. Furthermore, we observed increased soluble CD14 in plasma, indicating microbial translocation. These data provide a longitudinal evaluation of antibiotic-induced changes to the composition and function of colonic bacterial communities associated with specific alterations in mucosal and systemic immunity.

Correction: Antibiotic-induced microbiome perturbations are associated with significant alterations to colonic mucosal immunity in rhesus macaques.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Correction: Laparoscopic Technique for Serial Collection of Para-Colonic, Left Colic, and Inferior Mesenteric Lymph Nodes in Macaques.

[This corrects the article DOI: 10.1371/journal.pone.0157535.].

Evidence for persistence of the SHIV reservoir early after MHC haploidentical hematopoietic stem cell transplantation.

Allogeneic transplantation (allo-HCT) has led to the cure of HIV in one individual, raising the question of whether transplantation can eradicate the HIV reservoir. To test this, we here present a model of allo-HCT in SHIV-infected, cART-suppressed nonhuman primates. We infect rhesus macaques with SHIV-1157ipd3N4, suppress them with cART, then transplant them using MHC-haploidentical allogeneic donors during continuous cART. Transplant results in ~100% myeloid donor chimerism, and up to 100% T-cell chimerism. Between 9 and 47 days post-transplant, terminal analysis shows that while cell-associated SHIV DNA levels are reduced in the blood and in lymphoid organs post-transplant, the SHIV reservoir persists in multiple organs, including the brain. Sorting of donor-vs.-recipient cells reveals that this reservoir resides in recipient cells. Moreover, tetramer analysis indicates a lack of virus-specific donor immunity post-transplant during continuous cART. These results suggest that early post-transplant, allo-HCT is insufficient for recipient reservoir eradication despite high-level donor chimerism and GVHD.

Laparoscopic Technique for Serial Collection of Liver and Mesenteric Lymph Nodes in Macaques.

The mesenteric lymph nodes (MLN) and the liver are exposed to microbes and microbial products from the gastrointestinal (GI) tract, making them immunologically unique. The GI tract and associated MLN are sites of early viral replication in human immunodeficiency virus (HIV) infection and the MLN are likely important reservoir sites that harbor latently-infected cells even after prolonged antiretroviral therapy (ART). The liver has been shown to play a significant role in immune responses to lentiviruses and appears to play a significant role in clearance of virus from circulation. Nonhuman primate (NHP) models for HIV and Acquired Immunodeficiency Syndrome (AIDS) closely mimic these aspects of HIV infection and serial longitudinal sampling of primary sites of viral replication and the associated immune responses in this model will help to elucidate critical events in infection, pathogenesis, and the impact of various intervention strategies on these events. Current published techniques to sample liver and MLN together involve major surgery and/or necropsy, which limits the ability to investigate these important sites in a serial fashion in the same animal. We have previously described a laparoscopic technique for collection of MLN. Here, we describe a minimally invasive laparoscopic technique for serial longitudinal sampling of liver and MLN through the same two port locations required for the collection of MLN. The use of the same two ports minimizes the impact to the animals as no additional incisions are required. This technique can be used with increased sampling frequency compared to major abdominal surgery and reduces the potential for surgical complications and associated local and systemic inflammatory responses that could complicate interpretation of results. This procedure has potential to facilitate studies involving NHP models while improving animal welfare.

Minimally Invasive Lumbar Port System for the Collection of Cerebrospinal Fluid from Rhesus Macaques (Macaca mulatta).

Biomedical translational research frequently incorporates collection of CSF from NHP, because CSF drug levels are used as a surrogate for CNS tissue penetration in pharmacokinetic and dynamic studies. Surgical placement of a CNS ventricular catheter reservoir for CSF collection is an intensive model to create and maintain and thus may not be feasible or practical for short-term studies. Furthermore, previous NHP lumbar port models require laminectomy for catheter placement. The new model uses a minimally invasive technique for percutaneous placement of a lumbar catheter to create a closed, subcutaneous system for effective, repeated CSF sample collection. None of the rhesus macaques (Macaca mulatta; n = 10) implanted with our minimally invasive lumbar port (MILP) system experienced neurologic deficits, postoperative infection of the surgical site, or skin erosion around the port throughout the 21.7-mo study. Functional MILP systems were maintained in 70% of the macaques, with multiple, high-quality, 0.5- to 1.0-mL samples of CSF collected for an average of 3 mo by using aspiration or gravitational flow. Among these macaques, 57% had continuous functionality for a mean of 19.2 mo; 50% of the cohort required surgical repair for port repositioning and replacement during the study. The MILP was unsuccessful in 2 macaques, at an average of 9.5 d after surgery. Nonpatency in these animals was attributed to the position of the lumbar catheter. The MILP system is an appropriate replacement for temporary catheterization and previous models requiring laminectomy and is a short-term alternative for ventricular CSF collection systems in NHP.

Laparoscopic Technique for Serial Collection of Para-Colonic, Left Colic, and Inferior Mesenteric Lymph Nodes in Macaques.

Unlike peripheral lymph nodes (PLN), the mesenteric lymph nodes (MLN) draining the gastrointestinal (GI) tract are exposed to microbes and microbial products from the intestines and as such, are immunologically distinct. GI draining (MLN) have also been shown to be sites of early viral replication and likely impact early events that determine the course of HIV infection. They also are important reservoir sites that harbor latently-infected cells and from which the virus can emerge even after prolonged combination antiretroviral therapy (cART). Changes in the microbial flora and increased permeability of the GI epithelium associated with lentiviral infection can impact the gut associated lymphoid tissue (GALT) and induce changes to secondary lymphoid organs limiting immune reconstitution with cART. Nonhuman primate models for AIDS closely model HIV infection in humans and serial sampling of the GALT and associated secondary lymphoid organs in this model is crucial to gain a better understanding of the critical early events in infection, pathogenesis, and the role of immune responses or drugs in controlling virus at these sites. However, current techniques to sample GI draining (MLN) involve major surgery and/or necropsy, which have, to date, limited the ability to investigate mechanisms mediating the initiation, persistence and control of infection in this compartment. Here, we describe a minimally invasive laparoscopic technique for serial sampling of these sites that can be used with increased sampling frequency, yields greater cell numbers and immune cell subsets than current non-invasive techniques of the GALT and reduces the potential for surgical complications that could complicate interpretation of the results. This procedure has potential to facilitate studies of pathogenesis and evaluation of preventive and treatment interventions, reducing sampling variables that can influence experimental results, and improving animal welfare.