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.

Combined molnupiravir-nirmatrelvir treatment improves the inhibitory effect on SARS-CoV-2 in macaques.

The periodic emergence of SARS-CoV-2 variants of concern (VOCs) with unpredictable clinical severity and ability to escape preexisting immunity emphasizes the continued need for antiviral interventions. Two small molecule inhibitors, molnupiravir (MK-4482), a nucleoside analog, and nirmatrelvir (PF-07321332), a 3C-like protease inhibitor, have recently been approved as monotherapy for use in high-risk patients with COVID-19. As preclinical data are only available for rodent and ferret models, here we assessed the efficacy of MK-4482 and PF-07321332 alone and in combination against infection with the SARS-CoV-2 Delta VOC in the rhesus macaque COVID-19 model. Macaques were infected with the SARS-CoV-2 Delta variant and treated with vehicle, MK-4482, PF-07321332, or a combination of MK-4482 and PF-07321332. Clinical exams were performed at 1, 2, and 4 days postinfection to assess disease and virological parameters. Notably, use of MK-4482 and PF-07321332 in combination improved the individual inhibitory effect of both drugs, resulting in milder disease progression, stronger reduction of virus shedding from mucosal tissues of the upper respiratory tract, stronger reduction of viral replication in the lower respiratory tract, and reduced lung pathology. Our data strongly indicate superiority of combined MK-4482 and PF-07321332 treatment of SARS-CoV-2 infections as demonstrated in the closest COVID-19 surrogate model of human infection.

Rapid Protection from COVID-19 in Nonhuman Primates Vaccinated Intramuscularly but Not Intranasally with a Single Dose of a Vesicular Stomatitis Virus-Based Vaccine.

The ongoing pandemic of coronavirus (CoV) disease 2019 (COVID-19) continues to exert a significant burden on health care systems worldwide. With limited treatments available, vaccination remains an effective strategy to counter transmission of severe acute respiratory syndrome CoV 2 (SARS-CoV-2). Recent discussions concerning vaccination strategies have focused on identifying vaccine platforms, number of doses, route of administration, and time to reach peak immunity against SARS-CoV-2. Here, we generated a single-dose, fast-acting vesicular stomatitis virus (VSV)-based vaccine derived from the licensed Ebola virus (EBOV) vaccine rVSV-ZEBOV, expressing the SARS-CoV-2 spike protein and the EBOV glycoprotein (VSV-SARS2-EBOV). Rhesus macaques vaccinated intramuscularly (i.m.) with a single dose of VSV-SARS2-EBOV were protected within 10 days and did not show signs of COVID-19 pneumonia. In contrast, intranasal (i.n.) vaccination resulted in limited immunogenicity and enhanced COVID-19 pneumonia compared to results for control animals. While both i.m. and i.n. vaccination induced neutralizing antibody titers, only i.m. vaccination resulted in a significant cellular immune response. RNA sequencing data bolstered these results by revealing robust activation of the innate and adaptive immune transcriptional signatures in the lungs of i.m. vaccinated animals only. Overall, the data demonstrate that VSV-SARS2-EBOV is a potent single-dose COVID-19 vaccine candidate that offers rapid protection based on the protective efficacy observed in our study. IMPORTANCE The vesicular stomatitis virus (VSV) vaccine platform rose to fame in 2019, when a VSV-based Ebola virus (EBOV) vaccine was approved by the European Medicines Agency and the U.S. Food and Drug Administration for human use against the deadly disease. Here, we demonstrate the protective efficacy of a VSV-EBOV-based COVID-19 vaccine against challenge in nonhuman primates (NHPs). When a single dose of the VSV-SARS2-EBOV vaccine was administered intramuscularly (i.m.), the NHPs were protected from COVID-19 within 10 days. In contrast, if the vaccine was administered intranasally, there was no benefit from the vaccine and the NHPs developed pneumonia. The i.m. vaccinated NHPs quickly developed antigen-specific IgG, including neutralizing antibodies. Transcriptional analysis highlighted the development of protective innate and adaptive immune responses in the i.m. vaccination group only.

SARS-CoV-2 Omicron BA.1 and BA.2 are attenuated in rhesus macaques as compared to Delta.

Since the emergence of SARS-CoV-2, five different variants of concern (VOCs) have been identified: Alpha, Beta, Gamma, Delta, and Omicron. Because of confounding factors in the human population, such as preexisting immunity, comparing severity of disease caused by different VOCs is challenging. Here, we investigate disease progression in the rhesus macaque model upon inoculation with the Delta, Omicron BA.1, and Omicron BA.2 VOCs. Disease severity in rhesus macaques inoculated with Omicron BA.1 or BA.2 was lower than those inoculated with Delta and resulted in significantly lower viral loads in nasal swabs, bronchial cytology brush samples, and lung tissue in rhesus macaques. Cytokines and chemokines were up-regulated in nasosorption samples of Delta animals compared to Omicron BA.1 and BA.2 animals. Overall, these data suggest that, in rhesus macaques, Omicron replicates to lower levels than the Delta VOC, resulting in reduced clinical disease.

Severe acute respiratory disease in American mink experimentally infected with SARS-CoV-2.

An animal model that fully recapitulates severe COVID-19 presentation in humans has been a top priority since the discovery of SARS-CoV-2 in 2019. Although multiple animal models are available for mild to moderate clinical disease, models that develop severe disease are still needed. Mink experimentally infected with SARS-CoV-2 developed severe acute respiratory disease, as evident by clinical respiratory disease, radiological, and histological changes. Virus was detected in nasal, oral, rectal, and fur swabs. Deep sequencing of SARS-CoV-2 from oral swabs and lung tissue samples showed repeated enrichment for a mutation in the gene encoding nonstructural protein 6 in open reading frame 1ab. Together, these data indicate that American mink develop clinical features characteristic of severe COVID-19 and, as such, are uniquely suited to test viral countermeasures.

Three-Week Old Pigs Are Not Susceptible to Productive Infection with SARS-COV-2.

As the COVID-19 pandemic moves into its third year, there remains a need for additional animal models better recapitulating severe COVID to study SARS-CoV-2 pathogenesis and develop countermeasures, especially treatment options. Pigs are known intermediate hosts for many viruses with zoonotic potential and are susceptible to infection with alpha, beta and delta genera of coronaviruses. Herein, we infected young (3 weeks of age) pigs with SARS-CoV-2 using a combination of respiratory and parenteral inoculation routes. Pigs did not develop clinical disease, nor macroscopic or microscopic pathologic lesions upon SARS-CoV-2 infection. Despite occasional low levels of SARS-CoV-2 genomic RNA in the respiratory tract, subgenomic RNA and infectious virus were never found, and SARS-CoV-2-specific adaptive immune responses were not detectable over the 13-day study period. We concluded that pigs are not susceptible to productive SARS-CoV-2 infection and do not serve as a SARS-CoV-2 reservoir for zoonotic transmission.

Age-related differences in immune dynamics during SARS-CoV-2 infection in rhesus macaques.

Advanced age is a key predictor of severe COVID-19. To gain insight into this relationship, we used the rhesus macaque model of SARS-CoV-2 infection. Eight older and eight younger macaques were inoculated with SARS-CoV-2. Animals were evaluated using viral RNA quantification, clinical observations, thoracic radiographs, single-cell transcriptomics, multiparameter flow cytometry, multiplex immunohistochemistry, cytokine detection, and lipidomics analysis at predefined time points in various tissues. Differences in clinical signs, pulmonary infiltrates, and virus replication were limited. Transcriptional signatures of inflammation-associated genes in bronchoalveolar lavage fluid at 3 dpi revealed efficient mounting of innate immune defenses in both cohorts. However, age-specific divergence of immune responses emerged during the post-acute phase. Older animals exhibited sustained local inflammatory innate responses, whereas local effector T-cell responses were induced earlier in the younger animals. Circulating lipid mediator and cytokine levels highlighted increased repair-associated signals in the younger animals, and persistent pro-inflammatory responses in the older animals. In summary, despite similar disease outcomes, multi-omics profiling suggests that age may delay or impair antiviral cellular immune responses and delay efficient return to immune homeostasis.

Subcutaneous remdesivir administration prevents interstitial pneumonia in rhesus macaques inoculated with SARS-CoV-2.

The utility of remdesivir treatment in COVID-19 patients is currently limited by the necessity to administer this antiviral intravenously, which has generally limited its use to hospitalized patients. Here, we tested a novel, subcutaneous formulation of remdesivir in the rhesus macaque model of SARS-CoV-2 infection that was previously used to establish the efficacy of remdesivir against this virus in vivo. Compared to vehicle-treated animals, macaques treated with subcutaneous remdesivir from 12 h through 6 days post inoculation showed reduced signs of respiratory disease, a reduction of virus replication in the lower respiratory tract, and an absence of interstitial pneumonia. Thus, early subcutaneous administration of remdesivir can protect from lower respiratory tract disease caused by SARS-CoV-2.

UK B.1.1.7 (Alpha) variant exhibits increased respiratory replication and shedding in nonhuman primates.

The continuing emergence of SARS-CoV-2 variants calls for regular assessment to identify differences in viral replication, shedding and associated disease. In this study, we compared African green monkeys infected intranasally with either the UK B.1.1.7 (Alpha) variant or its contemporary D614G progenitor. Both variants caused mild respiratory disease with no significant differences in clinical presentation. Significantly higher levels of viral RNA and infectious virus were found in upper and lower respiratory tract samples and tissues from B.1.1.7 infected animals. Interestingly, D614G infected animals showed significantly higher levels of viral RNA and infectious virus in rectal swabs and gastrointestinal tissues. Our results indicate that B.1.1.7 infection in African green monkeys is associated with increased respiratory replication and shedding but no disease enhancement similar to human B.1.1.7 cases.

Subtle differences in the pathogenicity of SARS-CoV-2 variants of concern B.1.1.7 and B.1.351 in rhesus macaques.

The emergence of several SARS-CoV-2 variants has caused global concerns about increased transmissibility, increased pathogenicity, and decreased efficacy of medical countermeasures. Animal models can be used to assess phenotypical changes in the absence of confounding factors. Here, we compared variants of concern (VOC) B.1.1.7 and B.1.351 to a recent B.1 SARS-CoV-2 isolate containing the D614G spike substitution in the rhesus macaque model. B.1.1.7 behaved similarly to D614G with respect to clinical disease and replication in the respiratory tract. Inoculation with B.1.351 resulted in lower clinical scores, lower lung virus titers, and less severe lung lesions. In bronchoalveolar lavages, cytokines and chemokines were up-regulated on day 4 in animals inoculated with D614G and B.1.1.7 but not with B.1.351. In nasal samples, cytokines and chemokines were up-regulated only in the B.1.1.7-inoculated animals. Together, our study suggests that circulation under diverse evolutionary pressures favors transmissibility and immune evasion rather than increased pathogenicity.

Immunogenicity of Low-Dose Prime-Boost Vaccination of mRNA Vaccine CV07050101 in Non-Human Primates.

Many different vaccine candidates against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19, are currently approved and under development. Vaccine platforms vary from mRNA vaccines to viral-vectored vaccines, and several candidates have been shown to produce humoral and cellular responses in small animal models, non-human primates, and human volunteers. In this study, six non-human primates received a prime-boost intramuscular vaccination with 4 µg of mRNA vaccine candidate CV07050101, which encodes a pre-fusion stabilized spike (S) protein of SARS-CoV-2. Boost vaccination was performed 28 days post prime vaccination. As a control, six animals were similarly injected with PBS. Humoral and cellular immune responses were investigated at time of vaccination, and two weeks afterwards. No antibodies could be detected at two and four weeks after prime vaccination. Two weeks after boost vaccination, binding but no neutralizing antibodies were detected in four out of six non-human primates. SARS-CoV-2 S protein-specific T cell responses were detected in these four animals. In conclusion, prime-boost vaccination with 4 µg of vaccine candidate CV07050101 resulted in limited immune responses in four out of six non-human primates.

Intranasal ChAdOx1 nCoV-19/AZD1222 vaccination reduces viral shedding after SARS-CoV-2 D614G challenge in preclinical models.

ChAdOx1 nCoV-19/AZD1222 is an approved adenovirus-based vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) currently being deployed globally. Previous studies in rhesus macaques revealed that intramuscular vaccination with ChAdOx1 nCoV-19/AZD1222 provided protection against pneumonia but did not reduce shedding of SARS-CoV-2 from the upper respiratory tract. Here, we investigated whether intranasally administered ChAdOx1 nCoV-19 reduces detection of virus in nasal swabs after challenging vaccinated macaques and hamsters with SARS-CoV-2 carrying a D614G mutation in the spike protein. Viral loads in swabs obtained from intranasally vaccinated hamsters were decreased compared to control hamsters, and no viral RNA or infectious virus was found in lung tissue after a direct challenge or after direct contact with infected hamsters. Intranasal vaccination of rhesus macaques resulted in reduced virus concentrations in nasal swabs and a reduction in viral loads in bronchoalveolar lavage and lower respiratory tract tissue. Intranasal vaccination with ChAdOx1 nCoV-19/AZD1222 reduced virus concentrations in nasal swabs in two different SARS-CoV-2 animal models, warranting further investigation as a potential vaccination route for COVID-19 vaccines.

Recovery from Acute SARS-CoV-2 Infection and Development of Anamnestic Immune Responses in T Cell-Depleted Rhesus Macaques.

Severe coronavirus disease 2019 (COVID-19) has been associated with T cell lymphopenia, but no causal effect of T cell deficiency on disease severity has been established. To investigate the specific role of T cells in recovery from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, we studied rhesus macaques that were depleted of either CD4+, CD8+, or both T cell subsets prior to infection. Peak virus loads were similar in all groups, but the resolution of virus in the T cell-depleted animals was slightly delayed compared to that in controls. The T cell-depleted groups developed virus-neutralizing antibody responses and class switched to IgG. When reinfected 6 weeks later, the T cell-depleted animals showed anamnestic immune responses characterized by rapid induction of high-titer virus-neutralizing antibodies, faster control of virus loads, and reduced clinical signs. These results indicate that while T cells play a role in the recovery of rhesus macaques from acute SARS-CoV-2 infections, their depletion does not induce severe disease, and T cells do not account for the natural resistance of rhesus macaques to severe COVID-19. Neither primed CD4+ nor CD8+ T cells appeared critical for immunoglobulin class switching, the development of immunological memory, or protection from a second infection. IMPORTANCE Patients with severe COVID-19 often have decreased numbers of T cells, a cell type important in fighting most viral infections. However, it is not known whether the loss of T cells contributes to severe COVID-19 or is a consequence of it. We studied rhesus macaques, which develop only mild COVID-19, similar to most humans. Experimental depletion of T cells slightly prolonged their clearance of virus, but there was no increase in disease severity. Furthermore, they were able to develop protection from a second infection and produced antibodies capable of neutralizing the virus. They also developed immunological memory, which allows a much stronger and more rapid response upon a second infection. These results suggest that T cells are not critical for recovery from acute SARS-CoV-2 infections in this model and point toward B cell responses and antibodies as the essential mediators of protection from re-exposure.

Intradermal delivery of a synthetic DNA vaccine protects macaques from Middle East respiratory syndrome coronavirus.

Emerging coronaviruses from zoonotic reservoirs, including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have been associated with human-to-human transmission and significant morbidity and mortality. Here, we study both intradermal and intramuscular 2-dose delivery regimens of an advanced synthetic DNA vaccine candidate encoding a full-length MERS-CoV spike (S) protein, which induced potent binding and neutralizing antibodies as well as cellular immune responses in rhesus macaques. In a MERS-CoV challenge, all immunized rhesus macaques exhibited reduced clinical symptoms, lowered viral lung load, and decreased severity of pathological signs of disease compared with controls. Intradermal vaccination was dose sparing and more effective in this model at protecting animals from disease. The data support the further study of this vaccine for preventing MERS-CoV infection and transmission, including investigation of such vaccines and simplified delivery routes against emerging coronaviruses.

A single intranasal dose of chimpanzee adenovirus-vectored vaccine protects against SARS-CoV-2 infection in rhesus macaques.

The deployment of a vaccine that limits transmission and disease likely will be required to end the coronavirus disease 2019 (COVID-19) pandemic. We recently described the protective activity of an intranasally administered chimpanzee adenovirus-vectored vaccine encoding a pre-fusion stabilized spike (S) protein (ChAd-SARS-CoV-2-S [chimpanzee adenovirus-severe acute respiratory syndrome-coronavirus-2-S]) in the upper and lower respiratory tracts of mice expressing the human angiotensin-converting enzyme 2 (ACE2) receptor. Here, we show the immunogenicity and protective efficacy of this vaccine in non-human primates. Rhesus macaques were immunized with ChAd-Control or ChAd-SARS-CoV-2-S and challenged 1 month later by combined intranasal and intrabronchial routes with SARS-CoV-2. A single intranasal dose of ChAd-SARS-CoV-2-S induces neutralizing antibodies and T cell responses and limits or prevents infection in the upper and lower respiratory tracts after SARS-CoV-2 challenge. As ChAd-SARS-CoV-2-S confers protection in non-human primates, it is a promising candidate for limiting SARS-CoV-2 infection and transmission in humans.

Single-cell RNA sequencing reveals SARS-CoV-2 infection dynamics in lungs of African green monkeys.

Detailed knowledge about the dynamics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is important for uncovering the viral and host factors that contribute to coronavirus disease 2019 (COVID-19) pathogenesis. Old-World nonhuman primates recapitulate mild to moderate cases of COVID-19, thereby serving as important pathogenesis models. We compared African green monkeys inoculated with infectious SARS-CoV-2 or irradiated, inactivated virus to study the dynamics of virus replication throughout the respiratory tract. Genomic RNA from the animals inoculated with the irradiated virus was found to be highly stable, whereas subgenomic RNA, an indicator of viral replication, was found to degrade quickly. We combined this information with single-cell RNA sequencing of cells isolated from the lung and lung-draining mediastinal lymph nodes and developed new analysis methods for unbiased targeting of important cells in the host response to SARS-CoV-2 infection. Through detection of reads to the viral genome, we were able to determine that replication of the virus in the lungs appeared to occur mainly in pneumocytes, whereas macrophages drove the inflammatory response. Monocyte-derived macrophages recruited to the lungs, rather than tissue-resident alveolar macrophages, were most likely to be responsible for phagocytosis of infected cells and cellular debris early in infection, with their roles switching during clearance of infection. Together, our dataset provides a detailed view of the dynamics of virus replication and host responses over the course of mild COVID-19 and serves as a valuable resource to identify therapeutic targets.

Hydroxychloroquine prophylaxis and treatment is ineffective in macaque and hamster SARS-CoV-2 disease models.

We remain largely without effective prophylactic/therapeutic interventions for COVID-19. Although many human COVID-19 clinical trials are ongoing, there remains a deficiency of supportive preclinical drug efficacy studies to help guide decisions. Here we assessed the prophylactic/therapeutic efficacy of hydroxychloroquine (HCQ), a drug of interest for COVID-19 management, in 2 animal disease models. The standard human malaria HCQ prophylaxis (6.5 mg/kg given weekly) and treatment (6.5 mg/kg given daily) did not significantly benefit clinical outcome, nor did it reduce SARS-CoV-2 replication/shedding in the upper and lower respiratory tract in the rhesus macaque disease model. Similarly, when used for prophylaxis or treatment, neither the standard human malaria dose (6.5 mg/kg) nor a high dose (50 mg/kg) of HCQ had any beneficial effect on clinical disease or SARS-CoV-2 kinetics (replication/shedding) in the Syrian hamster disease model. Results from these 2 preclinical animal models may prove helpful in guiding clinical use of HCQ for prophylaxis/treatment of COVID-19.

ChAdOx1 nCoV-19 vaccine prevents SARS-CoV-2 pneumonia in rhesus macaques.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in December 20191,2 and is responsible for the coronavirus disease 2019 (COVID-19) pandemic3. Vaccines are an essential countermeasure and are urgently needed to control the pandemic4. Here we show that the adenovirus-vector-based vaccine ChAdOx1 nCoV-19, which encodes the spike protein of SARS-CoV-2, is immunogenic in mice and elicites a robust humoral and cell-mediated response. This response was predominantly mediated by type-1 T helper cells, as demonstrated by the profiling of the IgG subclass and the expression of cytokines. Vaccination with ChAdOx1 nCoV-19 (using either a prime-only or a prime-boost regimen) induced a balanced humoral and cellular immune response of type-1 and type-2 T helper cells in rhesus macaques. We observed a significantly reduced viral load in the bronchoalveolar lavage fluid and lower respiratory tract tissue of vaccinated rhesus macaques that were challenged with SARS-CoV-2 compared with control animals, and no pneumonia was observed in vaccinated SARS-CoV-2-infected animals. However, there was no difference in nasal shedding between vaccinated and control SARS-CoV-2-infected macaques. Notably, we found no evidence of immune-enhanced disease after viral challenge in vaccinated SARS-CoV-2-infected animals. The safety, immunogenicity and efficacy profiles of ChAdOx1 nCoV-19 against symptomatic PCR-positive COVID-19 disease will now be assessed in randomized controlled clinical trials in humans.

Clinical benefit of remdesivir in rhesus macaques infected with SARS-CoV-2.

Effective therapies to treat coronavirus disease 2019 (COVID-19) are urgently needed. While many investigational, approved, and repurposed drugs have been suggested as potential treatments, preclinical data from animal models can guide the search for effective treatments by ruling out those that lack efficacy in vivo. Remdesivir (GS-5734) is a nucleotide analogue prodrug with broad antiviral activity1,2 that is currently being investigated in COVID-19 clinical trials and recently received Emergency Use Authorization from the US Food and Drug Administration3,4. In animal models, remdesivir was effective against infection with Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV)2,5,6. In vitro, remdesivir inhibited replication of SARS-CoV-27,8. Here we investigate the efficacy of remdesivir in a rhesus macaque model of SARS-CoV-2 infection9. Unlike vehicle-treated animals, macaques treated with remdesivir did not show signs of respiratory disease; they also showed reduced pulmonary infiltrates on radiographs and reduced virus titres in bronchoalveolar lavages twelve hours after the first dose. Virus shedding from the upper respiratory tract was not reduced by remdesivir treatment. At necropsy, remdesivir-treated animals had lower lung viral loads and reduced lung damage. Thus, treatment with remdesivir initiated early during infection had a clinical benefit in rhesus macaques infected with SARS-CoV-2. Although the rhesus macaque model does not represent the severe disease observed in some patients with COVID-19, our data support the early initiation of remdesivir treatment in patients with COVID-19 to prevent progression to pneumonia.