Vaccination Of Rhesus Monkeys Research Articles

Monitoring and immunogenicity of SARS-CoV-2 vaccination of laboratory rhesus monkeys (Macaca mulatta)

The availability of effective vaccines and a high vaccination rate allowed the recent mitigation, or even withdrawal, of many protective measures for containing the SARS CoV-2 pandemic. At the same time, new and highly mutated variants of the virus are found to have significantly higher transmissibility and reduced vaccine efficacy, thus causing high infection rates during the third year of the pandemic. The combination of reduced measures and increased infectivity poses a particular risk for unvaccinated individuals, including animals susceptible to the virus. Among the latter, non-human primates (NHPs) are particularly vulnerable. They serve as important models in various fields of biomedical research and because of their cognitive capabilities, they receive particular attention in animal welfare regulations around the world. Yet, although they played an extraordinarily important role for developing and testing vaccines against SARS-CoV-2, the protection of captive rhesus monkeys against Covid-19 has rarely been discussed. We here report upon twofold mRNA vaccination of a cohort of 19 elderly rhesus monkeys (Macaca mulatta) against infection by SARS-CoV-2. All animals were closely monitored on possible side effects of vaccination, and were tested for neutralising antibodies against the virus. The data show that vaccination of rhesus monkeys is a safe and reliable measure to protect these animals against SARS-CoV-2.

Three doses of prototypic SARS-CoV-2 inactivated vaccine induce cross-protection against its variants of concern

Variants are globally emerging very quickly following pandemic prototypic SARS-CoV-2. To evaluate the cross-protection of prototypic SARS-CoV-2 vaccine against its variants, we vaccinated rhesus monkeys with three doses of prototypic SARS-CoV-2 inactivated vaccine, followed by challenging with emerging SARS-CoV-2 variants of concern (VOCs). These vaccinated animals produced neutralizing antibodies against Alpha, Beta, Delta, and Omicron variants, although there were certain declinations of geometric mean titer (GMT) as compared with prototypic SARS-CoV-2. Of note, in vivo this prototypic vaccine not only reduced the viral loads in nasal, throat and anal swabs, pulmonary tissues, but also improved the pathological changes in the lung infected by variants of Alpha, Beta, and Delta. In summary, the prototypic SARS-CoV-2 inactivated vaccine in this study protected against VOCs to certain extension, which is of great significance for prevention and control of COVID-19.

A synthetic opioid vaccine attenuates fentanyl-vs-food choice in male and female rhesus monkeys

AimOpioid-targeted vaccines are under consideration as candidate Opioid Use Disorder medications. We recently reported that a fentanyl-targeted vaccine produced a robust and long-lasting attenuation of fentanyl-vs-food choice in rats. In the current study, we evaluated an optimized fentanyl-targeted vaccine in rhesus monkeys to determine whether vaccine effectiveness to attenuate fentanyl choice translated to a species with greater phylogenetic similarity to humans. MethodsAdult male (2) and female (3) rhesus monkeys were trained to respond under a concurrent schedule of food (1 g pellets) and intravenous fentanyl (0, 0.032−1 μg/kg/injection) reinforcement during daily 2 h sessions. Fentanyl choice dose-effect functions were determined daily and 7-day buprenorphine treatments (0.0032−0.032 mg/kg/h IV; n = 4–5) were determined for comparison to vaccine effects. Subsequently, a fentanyl-CRM197 conjugate vaccine was administered at week 0, 3, 8, 15 over a 29-week experimental period during which fentanyl choice dose-effect functions continued to be determined daily. ResultsBuprenorphine significantly decreased fentanyl choice and reciprocally increased food choice. Vaccination eliminated fentanyl choice and increased food choice in four-of-the-five monkeys. A transient and less robust vaccine effect was observed in the fifth monkey. Fentanyl-specific antibody concentrations peaked after the third vaccination to approximately 50 μg/mL while anti-fentanyl antibody affinity increased to a sustained low nanomolar level. ConclusionThese results translate fentanyl vaccine effectiveness from rats to rhesus monkeys to decrease fentanyl-vs-food choice, albeit with greater individual differences observed in monkeys. These results support the potential and further clinical evaluation of this fentanyl-targeted vaccine as a candidate Opioid Use Disorder medication.

Coxsackievirus A 16 infection does not interfere with the specific immune response induced by an enterovirus 71 inactivated vaccine in rhesus monkeys

Hand, foot and mouth disease is usually caused by enterovirus 71 (EV71) and coxsackievirus A 16 (CA16), which are members of the Picornaviridae family. In the present study, the characteristics of the immune response induced by an EV71 inactivated vaccine (made from human diploid cells) were explored in the presence of CA16 infection, based on the previously established neonatal rhesus monkey model. The typical clinical manifestations, including body temperature, viral viremia and virus shedding in the mouth, pharynx and feces, were characterized. A specific neutralizing antibody assay showed that the specific immune response induced by the EV71 inactivated vaccine was active against EV71 but not against CA16. No remarkable fluctuation in proinflammatory cytokine release was identified in the serum of immunized monkeys with EV71 vaccine and CA16 infections subsequently. The results showed that the specific immune response induced by the EV71 inactivated vaccine is effective against EV71 infection but is not affected by CA16 infection.

Metabolic engineering of Salmonella vaccine bacteria to boost human γδ T cell immunity (P4538)

Abstract Human Vγ2Vδ2 T cells monitor isoprenoid metabolism by recognizing foreign (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), an intermediate in the 2-C -methyl-D-erythritol-4-phosphate (MEP) pathway used by most Eubacteria and Apicomplexan parasites, and self isopentenyl pyrophosphate (IPP), an intermediate in the mevalonate pathway used by humans. Whereas microbial infections elicit prolonged expansion of memory Vγ2Vδ2 T cells, immunization with synthetic prenyl pyrophosphates or aminobisphosphonates elicit short-term expansion with rapid anergy and deletion. We hypothesized that a live attenuated bacterial vaccine metabolically engineered to overproduce HMBPP would elicit Vγ2Vδ2 T cell immunity by mimicking natural infections. Here we report on an aroA- Salmonella enterica serovar Typhimurium SL7207 strain metabolically engineered by deleting the gene for lytB (the downstream enzyme from HMBPP) and complementing with genes encoding mevalonate pathway enzymes. lytB- SL7207 had extremely high HMBPP levels, infected human cells like the wild type bacteria, and stimulated large ex vivo expansions of Vγ2Vδ2 T cells in all 6 human donors tested. Moreover, vaccination of rhesus monkeys with live lytB- SL7207 expanded Vγ2Vδ2 T cells without significant side effects. Thus, these studies provide proof-of-principle for the use of metabolic engineering to derive Salmonella vaccines that boost human γδ T cell immunity.

Adenovirus serotype 26 utilizes CD46 as primary cellular receptor and only transiently activates T lymphocytes following vaccination of rhesus monkeys

Methods Receptor usage was assessed using CD46 transgenic mouse cells, as well as by CARand CD46-specific mAb blocking studies using human PBMC. Twelve adult rhesus monkeys were inoculated with of 10 viral particles (vp) of replication-competent Ad5 and Ad26 (N=6) or saline (N=6) at weeks -8 and -4, and were vaccinated intramuscularly with 3×10 vp replication-incompetent Ad26-Gag/Pol/Env vectors. At week 2, monkeys were sacrificed to assess immunologic and inflammatory responses at mucosal surfaces.

Gag-specific cellular immunity determines in vitro viral inhibition and in vivo virologic control following SIV challenges of vaccinated monkeys

Background A vaccine for HIV-1 would ideally block HIV-1 acquisition as well as durably control viral replication in breakthrough infections. A recently published study showed that optimal SIV vaccines can reduce SIV infection risk and setpoint viral loads following SIV challenges in rhesus monkeys, and that immunization with SIV Env was required for blocking acquisition of infection (Nature 2012 482:89-93). Here we investigate whether CD8+ T lymphocytes from these vaccinated rhesus monkeys mediate viral inhibition in vitro and whether these responses predict virologic control following SIV challenge.

Gag-Specific Cellular Immunity Determines In Vitro Viral Inhibition and In Vivo Virologic Control following Simian Immunodeficiency Virus Challenges of Vaccinated Rhesus Monkeys

ABSTRACT A comprehensive vaccine for human immunodeficiency virus type 1 (HIV-1) would block HIV-1 acquisition as well as durably control viral replication in breakthrough infections. Recent studies have demonstrated that Env is required for a vaccine to protect against acquisition of simian immunodeficiency virus (SIV) in vaccinated rhesus monkeys, but the antigen requirements for virologic control remain unclear. Here, we investigate whether CD8 + T lymphocytes from vaccinated rhesus monkeys mediate viral inhibition in vitro and whether these responses predict virologic control following SIV challenge. We observed that CD8 + lymphocytes from 23 vaccinated rhesus monkeys inhibited replication of SIV in vitro . Moreover, the magnitude of inhibition prior to challenge was inversely correlated with set point SIV plasma viral loads after challenge. In addition, CD8 cell-mediated viral inhibition in vaccinated rhesus monkeys correlated significantly with Gag-specific, but not Pol- or Env-specific, CD4 + and CD8 + T lymphocyte responses. These findings demonstrate that in vitro viral inhibition following vaccination largely reflects Gag-specific cellular immune responses and correlates with in vivo virologic control following infection. These data suggest the importance of including Gag in an HIV-1 vaccine in which virologic control is desired.

Evaluation of HCMV vaccines in rhesus macaques by a novel micro-neutralization assay (113.4)

Abstract Human cytogemalovirus (HCMV) causes wide-spread infection in adult human populations. Although no symptoms observed in healthy persons, HCMV infection can cause severe and even life-threatening diseases in immune-compromised individuals. There are lines of evidence that humoral immunity can provide protection against HCMV infection. We have tested several versions of HCMV vaccines based on laboratory strain AD169 with its epithelial tropism restored in rhesus macaques. Vaccines were administered intramuscularly three times. In comparison, recombinant HCMV gB protein vaccine, which showed marginal protections in previous clinical trial, was given with adjuvant MF59. To facilitate the evaluation of these vaccines, we have developed a novel micro-neutralization assay based on the detection of a dominant HCMV antigen expressed in ARPE19 cells, using near infrared dye-labeled immune reagents, to measure HCMV neutralizing activities in serum samples of vaccinated monkeys. Our results showed that AD169 revertant vaccines induced much stronger neutralizing activities than recombinant gB vaccine in rhesus monkeys. The addition of ISCOMATRIX adjuvant (CSL, Ltd) in the vaccine formulation further enhanced its immunogenicity. There were significant boost effects after the second injection, while the third injection did not increase the neutralizing titers above those obtained post the second vaccination.

Soluble Rhesus Lymphocryptovirus gp350 Protects against Infection and Reduces Viral Loads in Animals that Become Infected with Virus after Challenge

Epstein-Barr virus (EBV) is a human lymphocryptovirus that is associated with several malignancies. Elevated EBV DNA in the blood is observed in transplant recipients prior to, and at the time of post-transplant lymphoproliferative disease; thus, a vaccine that either prevents EBV infection or lowers the viral load might reduce certain EBV malignancies. Two major approaches have been suggested for an EBV vaccine- immunization with either EBV glycoprotein 350 (gp350) or EBV latency proteins (e.g. EBV nuclear antigens [EBNAs]). No comparative trials, however, have been performed. Rhesus lymphocryptovirus (LCV) encodes a homolog for each gene in EBV and infection of monkeys reproduces the clinical, immunologic, and virologic features of both acute and latent EBV infection. We vaccinated rhesus monkeys at 0, 4 and 12 weeks with (a) soluble rhesus LCV gp350, (b) virus-like replicon particles (VRPs) expressing rhesus LCV gp350, (c) VRPs expressing rhesus LCV gp350, EBNA-3A, and EBNA-3B, or (d) PBS. Animals vaccinated with soluble gp350 produced higher levels of antibody to the glycoprotein than those vaccinated with VRPs expressing gp350. Animals vaccinated with VRPs expressing EBNA-3A and EBNA-3B developed LCV-specific CD4 and CD8 T cell immunity to these proteins, while VRPs expressing gp350 did not induce detectable T cell immunity to gp350. After challenge with rhesus LCV, animals vaccinated with soluble rhesus LCV gp350 had the best level of protection against infection based on seroconversion, viral DNA, and viral RNA in the blood after challenge. Surprisingly, animals vaccinated with gp350 that became infected had the lowest LCV DNA loads in the blood at 23 months after challenge. These studies indicate that gp350 is critical for both protection against infection with rhesus LCV and for reducing the viral load in animals that become infected after challenge. Our results suggest that additional trials with soluble EBV gp350 alone, or in combination with other EBV proteins, should be considered to reduce EBV infection or virus-associated malignancies in humans.

International seroepidemiology of adenovirus serotypes 5, 26, 35, and 48 in pediatric and adult populations

Recombinant adenovirus serotype 5 (rAd5) vaccine vectors for HIV-1 and other pathogens have been shown to be limited by high titers of Ad5 neutralizing antibodies (NAbs) in the developing world. Alternative serotype rAd vectors have therefore been constructed. Here we report Ad5, Ad26, Ad35, and Ad48 NAb titers in 4381 individuals from North America, South America, sub-Saharan Africa, and Southeast Asia. As expected, Ad5 NAb titers were both frequent and high magnitude in sub-Saharan Africa and Southeast Asia. In contrast, Ad35 NAb titers proved infrequent and low in all regions studied, and Ad48 NAbs were rare in all regions except East Africa. Ad26 NAbs were moderately common in adults in sub-Saharan Africa and Southeast Asia, but Ad26 NAb titers proved markedly lower than Ad5 NAb titers in all regions, and these relatively low Ad26 NAb titers did not detectably suppress the immunogenicity of 4×1010vp of a rAd26-Gag/Pol/Env/Nef vaccine in rhesus monkeys. These data inform the clinical development of alternative serotype rAd vaccine vectors in the developing world.

Genital Tract Sequestration of SIV following Acute Infection

We characterized the evolution of simian immunodeficiency virus (SIV) in the male genital tract by examining blood- and semen-associated virus from experimentally and sham vaccinated rhesus monkeys during primary infection. At the time of peak virus replication, SIV sequences were intermixed between the blood and semen supporting a scenario of high-level virus “spillover” into the male genital tract. However, at the time of virus set point, compartmentalization was apparent in 4 of 7 evaluated monkeys, likely as a consequence of restricted virus gene flow between anatomic compartments after the resolution of primary viremia. These findings suggest that SIV replication in the male genital tract evolves to compartmentalization after peak viremia resolves.

Vaccination Reduces Simian-Human Immunodeficiency Virus Sequence Reversion through Enhanced Viral Control

It has been suggested that vaccination prior to infection may direct the mutational evolution of human immunodeficiency virus type 1 (HIV-1) to a less fit virus, resulting in an attenuated course of disease. The present study was initiated to explore whether prior immunization might prevent the reversion of the virus to the wild-type form. Mamu-A*01 monkeys were vaccinated to generate a cytotoxic T-lymphocyte response to the immunodominant Gag p11C epitope and were then challenged with a cloned pathogenic CXCR4-tropic simian-human immunodeficiency virus (SHIV) expressing a mutant Gag p11C sequence (Δp11C SHIV). The epitopic and extraepitopic compensatory mutations introduced into gag of Δp11C SHIV resulted in attenuated replicative capacity and eventual reversions to the wild-type Gag p11C sequence in naïve rhesus monkeys. However, in vaccinated rhesus monkeys, no reversions of the challenge virus were observed, an effect that may have been a consequence of significantly decreased viral replication rather than a redirection of the mutational evolution of the virus. These findings highlight the multifactorial pressures that affect the evolution of primate immunodeficiency viruses.

Immunogenicity and Protective Efficacy of a Recombinant Subunit West Nile Virus Vaccine in Rhesus Monkeys

The immunogenicity and protective efficacy of a recombinant subunit West Nile virus (WNV) vaccine was evaluated in rhesus macaques (Macaca mulatta). The vaccine consisted of a recombinant envelope (E) protein truncated at the C-terminal end, resulting in a polypeptide containing 80% of the N-terminal amino acids of the native WNV protein (WN-80E), mixed with an adjuvant (GPI-0100). WN-80E was produced in a Drosophila melanogaster expression system with high yield and purified by immunoaffinity chromatography using a monoclonal antibody specific for flavivirus E proteins. Groups of monkeys were vaccinated with formulations containing 1 or 25 microg of WN-80E antigen, and both humoral and cellular immunity were assessed after vaccination. The results demonstrated potent antibody responses to vaccination, as determined by both enzyme-linked immunosorbent assay and virus-neutralizing antibody assays. All vaccinated animals responded favorably, and there was little difference in response between animals immunized with 1 or 25 microg of WN-80E. Cellular immunity was determined by lymphocyte proliferation and cytokine production assays using peripheral blood mononuclear cells from vaccinated animals stimulated in vitro with WN-80E. Cell-mediated immune responses varied from animal to animal within each group. About half of the animals responded with lymphoproliferation, cytokine production, or both. Again, there was little difference in response between animals immunized with a 1- or 25-microg dose of WN-80E in the vaccine formulations. In a separate experiment, groups of monkeys were immunized with the WN-80E/GPI-0100 vaccine or an adjuvant-only control formulation. Animals were then challenged by inoculation of wild-type WNV, and the level of viremia in each animal was monitored daily for 10 days. The results showed that whereas all animals in the control group had detectable viremia for at least 3 days after challenge, all of the vaccinated animals were negative on all days after challenge. Thus, the WN-80E vaccine was 100% efficacious in protecting monkeys against infection with WNV.

Protective Anti-Hepatitis B Virus Responses in Rhesus Monkeys Primed with a Vectored Measles Virus and Boosted with a Single Dose of Hepatitis B Surface Antigen

The widely used hepatitis B virus (HBV) vaccine is based on three doses of hepatitis B surface antigen (HBsAg) protein. We previously showed that vectored measles viruses (MV) expressing HBsAg retain measles vaccine function in monkeys but do not induce a protective anti-HBs response in all animals. We show here that a single dose of HBsAg protein following a three-dose vaccination regimen with an optimized HBsAg-expressing MV elicits protective anti-HBs responses in all four vaccinated Rhesus monkeys. Vaccination strategies coupling the effective, long-term immunity elicited by the high-coverage MV vaccine to prophylactic HBV immunity are discussed.

Hiv/Aids Vaccine: Rumors and Insights on a T-Cell-Based Vaccine

Evaluation of: Liu J, O’Brien KL, Lynch DM et al.: Immune control of an SIV challenge by a T-cell-based vaccine in rhesus monkeys. Nature 457(7225), 87–91 (2009). The generation of a vaccine against HIV/AIDS has turned out to be extremely challenging. In spite of enormous experience both in preclinical and clinical models, we still do not know what viral gene is essential for protective immunity, what the correlate(s) of protection are and what type of delivery system works better in term of safety, immunogenicity and efficacy. Recently, the STEP (double-blind, randomized trial HTVN502; Merck V520, protocol 023) vaccine failure in the scientific community raised the question as to whether a T-cell-based vaccine against HIV/AIDS is still feasible. Liu and colleagues demonstrated that a combination of serologically distinct adenoviral vectors expressing the SIV Gag protein elicited polyfunctional and broad T-cell responses that correlated with a durable but partial protection in macaques challenged with pathogenic SIV in the absence of homologous Env antigen (i.e., neutralizing antibodies). This, and other preclinical trials, are providing convincing evidence that T-cell-based vaccine strategies – while not able to elicit sterilizing immunity – could, however, be sufficient to control viral replication and lower viral transmission rate among individuals (secondary end point of HIV vaccine). These approaches, as long as they demonstrate their ability to elicit broad and antigen-specific polyfunctional activities in relevant animal models, have the potential to be studied further in clinical trials.

Comparative immunogenicity evaluations of influenza A virus M2 peptide as recombinant virus like particle or conjugate vaccines in mice and monkeys

Immunization against M2 peptide, also called M2e, from influenza A virus is an innovative vaccine approach for induction of cross-strain protective immunity. Two promising M2 vaccine compositions reported to date are M2 peptide chemically conjugated to carrier proteins or M2 peptide recombinantly expressed on the surface of virus like particles (VLPs) of hepatitis B virus core antigen (HBVc). To conduct a head-to-head comparison of these approaches, we constructed two recombinant HBVc VLPs expressing M2 peptide and prepared two conjugate vaccines with M2 peptide chemically coupled to Neisseria meningitidis outer membrane complex (OMPC) or HBVc VLP, respectively. Here, we showed superior immunogenicity of M2 peptide conjugated to OMPC and M2 peptide expressed on the surface of HBVc antigen based on dose–titration responses in mice. Surprisingly, HBVc expressing M2 peptide was an inferior vaccine in rhesus monkeys, whether as a primary vaccine or as a booster vaccine, when compared with M2-OMPC conjugate vaccine.

Immune control of an SIV challenge by a T-cell-based vaccine in rhesus monkeys.

A recombinant adenovirus serotype 5 (rAd5) vector-based vaccine for HIV-1 has recently failed in a phase 2b efficacy study in humans1, 2. Consistent with these results, preclinical studies have demonstrated that rAd5 vectors expressing SIV Gag failed to reduce peak or setpoint viral loads following SIV challenge of rhesus monkeys that lacked the protective MHC class I allele Mamu-A*013. Here we show that an improved T cell-based vaccine regimen utilizing two serologically distinct adenovirus vectors afforded substantially improved protective efficacy in this stringent challenge model. In particular, a heterologous rAd26 prime, rAd5 boost vaccine regimen expressing SIV Gag elicited cellular immune responses with augmented magnitude, breadth, and polyfunctionality as compared with the homologous rAd5 regimen. Following SIVmac251 challenge, monkeys vaccinated with the heterologous rAd26/rAd5 regimen exhibited a 1.4 log reduction of peak and a 2.4 log reduction of setpoint viral loads as well as decreased AIDS-related mortality as compared with control animals. These data demonstrate that durable partial immune control of a pathogenic SIV challenge for over 500 days can be achieved by a T cell-based vaccine in Mamu-A*01-negative rhesus monkeys in the absence of a homologous Env antigen. These findings have important implications for the development of next generation T cell-based vaccine candidates for HIV-1.

Immunogenicity and safety of a novel formalin-inactivated and alum-adjuvanted candidate subunit vaccine for mumps

While an attenuated vaccine against mumps has played a role in controlling the epidemic of this disease worldwide, some problems with efficacy and safety of the vaccine are still present. In the work described here, a novel mumps vaccine with good immunity and safety was developed by selecting an antigen component of the mumps virus. The results suggest that this purified antigen vaccine is immunogenic in animals and is capable of inducing a specific neutralizing antibody response against viral HN, but not against other viral proteins. The clinical and pathological observations after challenge of vaccinated rhesus monkeys indicated further that the immune response induced by this vaccine provided complete protection from wild-type virus infection. Furthermore, the immunological analysis and clinical observation of experimental monkeys that were immunized with the vaccine, followed by infection with wild-type virus, suggest that no abnormal changes in expression of inflammatory cytokines and no clinical allergic reaction were found at 1 month after challenge.

Clinical Measles after Measles Virus Challenge in Simian Immunodeficiency Virus–Infected Measles Virus–Vaccinated Rhesus Monkeys

Understanding the impact of human immunodeficiency virus (HIV) infection on the clinical manifestations and kinetics of measles virus (MV) replication in MV-vaccinated and unvaccinated individuals is important for developing successful vaccine strategies for measles eradication. To model the pathogenesis of MV infection in MV-vaccinated and unvaccinated individuals infected with HIV, previously vaccinated and unvaccinated rhesus monkeys infected with simian immunodeficiency virus (SIV) were challenged with MV and monitored for clinical, virologic, and immunologic sequelae of infection. The magnitude and duration of MV viremia were unchanged by SIV infection. Nevertheless, clinical manifestations of MV infection were altered in animals with significant CD4(+) T lymphocyte loss. Importantly, 2 of the 3 SIV-infected monkeys with high titers of vaccine-induced MV-neutralizing antibody developed clinical evidence of MV infection. Thus, in this experimental animal model, a high-titer vaccine-induced MV-neutralizing antibody response does not protect against clinical manifestations of measles in the setting of a chronic acquired immunodeficiency syndrome virus infection.