Protective efficacy of rhesus adenovirus COVID-19 vaccines against mouse-adapted SARS-CoV-2 (preprint)

The global COVID-19 pandemic has sparked intense interest in the rapid development of vaccines as well as animal models to evaluate vaccine candidates and to define immune correlates of protection. We recently reported a mouse-adapted SARS-CoV-2 virus strain (MA10) with the potential to infect wild-type laboratory mice, driving high levels of viral replication in respiratory tract tissues as well as severe clinical and respiratory symptoms, aspects of COVID-19 disease in humans that are important to capture in model systems. We evaluated the immunogenicity and protective efficacy of novel rhesus adenovirus serotype 52 (RhAd52) vaccines against MA10 challenge in mice. Baseline seroprevalence is lower for rhesus adenovirus vectors than for human or chimpanzee adenovirus vectors, making these vectors attractive candidates for vaccine development. We observed that RhAd52 vaccines elicited robust binding and neutralizing antibody titers, which inversely correlated with viral replication after challenge. These data support the development of RhAd52 vaccines and the use of the MA10 challenge virus to screen novel vaccine candidates and to study the immunologic mechanisms that underscore protection from SARS-CoV-2 challenge in wild-type mice. Importance We have developed a series of SARS-CoV-2 vaccines using rhesus adenovirus serotype 52 (RhAd52) vectors, which exhibits a lower seroprevalence than human and chimpanzee vectors, supporting their development as novel vaccine vectors or as an alternative Ad vector for boosting. We sought to test these vaccines using a recently reported mouse-adapted SARS-CoV-2 (MA10) virus to i) evaluate the protective efficacy of RhAd52 vaccines and ii) further characterize this mouse-adapted challenge model and probe immune correlates of protection. We demonstrate RhAd52 vaccines elicit robust SARS-CoV-2-specific antibody responses and protect against clinical disease and viral replication in the lungs. Further, binding and neutralizing antibody titers correlated with protective efficacy. These data validate the MA10 mouse model as a useful tool to screen and study novel vaccine candidates, as well as the development of RhAd52 vaccines for COVID-19.

Acute SARS-CoV-2 Infection is Highly Cytopathic, Elicits a Robust Innate Immune Response and is Efficiently Prevented by EIDD-2801 (preprint)

All known recently emerged human coronaviruses likely originated in bats. Here, we used a single experimental platform based on human lung-only mice (LoM) to demonstrate efficient in vivo replication of all recently emerged human coronaviruses (SARS-CoV, MERS-CoV, SARS-CoV-2) and two highly relevant endogenous pre-pandemic SARS-like bat coronaviruses. Virus replication in this model occurs in bona fide human lung tissue and does not require any type of adaptation of the virus or the host. Our results indicate that bats harbor endogenous coronaviruses capable of direct transmission into humans. Further detailed analysis of pandemic SARS-CoV-2 in vivo infection of LoM human lung tissue showed predominant infection of human lung epithelial cells, including type II pneumocytes present in alveoli and ciliated airway cells. Acute SARS-CoV-2 infection was highly cytopathic and induced a robust and sustained Type I interferon and inflammatory cytokine/chemokine response. Finally, we evaluated a pre-exposure prophylaxis strategy for coronavirus infection. Our results show that prophylactic administration of EIDD-2801, an oral broad spectrum antiviral currently in phase II clinical trials for the treatment of COVID-19, dramatically prevented SARS-CoV-2 infection in vivo and thus has significant potential for the prevention and treatment of COVID-19.

Elicitation of potent neutralizing antibody responses by designed protein nanoparticle vaccines for SARS-CoV-2 (preprint)

A safe, effective, and scalable vaccine is urgently needed to halt the ongoing SARS-CoV-2 pandemic. Here, we describe the structure-based design of self-assembling protein nanoparticle immunogens that elicit potent and protective antibody responses against SARS-CoV-2 in mice. The nanoparticle vaccines display 60 copies of the SARS-CoV-2 spike (S) glycoprotein receptor-binding domain (RBD) in a highly immunogenic array and induce neutralizing antibody titers roughly ten-fold higher than the prefusion-stabilized S ectodomain trimer despite a more than five-fold lower dose. Antibodies elicited by the nanoparticle immunogens target multiple distinct epitopes on the RBD, suggesting that they may not be easily susceptible to escape mutations, and exhibit a significantly lower binding:neutralizing ratio than convalescent human sera, which may minimize the risk of vaccine-associated enhanced respiratory disease. The high yield and stability of the protein components and assembled nanoparticles, especially compared to the SARS-CoV-2 prefusion-stabilized S trimer, suggest that manufacture of the nanoparticle vaccines will be highly scalable. These results highlight the utility of robust antigen display platforms for inducing potent neutralizing antibody responses and have launched cGMP manufacturing efforts to advance the lead RBD nanoparticle vaccine into the clinic.

Newcastle disease virus (NDV) expressing the spike protein of SARS-CoV-2 as vaccine candidate (preprint)

Due to the lack of protective immunity of humans towards the newly emerged SARS-CoV-2, this virus has caused a massive pandemic across the world resulting in hundreds of thousands of deaths. Thus, a vaccine is urgently needed to contain the spread of the virus. Here, we describe Newcastle disease virus (NDV) vector vaccines expressing the spike protein of SARS-CoV-2 in its wild type or a pre-fusion membrane anchored format. All described NDV vector vaccines grow to high titers in embryonated chicken eggs. In a proof of principle mouse study, we report that the NDV vector vaccines elicit high levels of antibodies that are neutralizing when the vaccine is given intramuscularly. Importantly, these COVID-19 vaccine candidates protect mice from a mouse-adapted SARS-CoV-2 challenge with no detectable viral titer and viral antigen in the lungs. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSThe spike (S) protein of the SARS-CoV-2 is the major antigen that notably induces neutralizing antibodies to block viral entry. Many COVID-19 vaccines are under development, among them viral vectors expressing the S protein of SARS-CoV-2 exhibit many benefits. Viral vector vaccines have the potential of being used as both live or inactivated vaccines and they can induce Th1 and Th2-based immune responses following different immunization regimens. Additionally, viral vector vaccines can be handled under BSL-2 conditions and they grow to high titers in cell cultures or other species restricted-hosts. For a SARS-CoV-2 vaccine, several viral vectors are being tested, such as adenovirus, measles virus and Modified vaccinia Ankara. Added value of this studyThe NDV vector vaccine against SARS-CoV-2 described in this study has advantages similar to those of other viral vector vaccines. But the NDV vector can be amplified in embryonated chicken eggs, which allows for high yields and low costs per dose. Also, the NDV vector is not a human pathogen, therefore the delivery of the foreign antigen would not be compromised by any pre-existing immunity in humans. Finally, NDV has a very good safety record in humans, as it has been used in many oncolytic virus trials. This study provides an important option for a cost-effective SARS-CoV-2 vaccine. Implications of all the available evidenceThis study informs of the value of a viral vector vaccine against SARS-CoV-2. Specifically, for this NDV based SARS-CoV-2 vaccine, the existing egg-based influenza virus vaccine manufactures in the U.S. and worldwide would have the capacity to rapidly produce hundreds of millions of doses to mitigate the consequences of the ongoing COVID-19 pandemic.

Remdesivir Potently Inhibits SARS-CoV-2 in Human Lung Cells and Chimeric SARS-CoV Expressing the SARS-CoV-2 RNA Polymerase in Mice (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in 2019 as the causative agent of the novel pandemic viral disease COVID-19. With no approved therapies, this pandemic illustrates the urgent need for safe, broad-spectrum antiviral countermeasures against SARS-CoV-2 and future emerging CoVs. We report that remdesivir (RDV), a monophosphoramidate prodrug of an adenosine analog, potently inhibits SARS-CoV-2 replication in human lung cells and primary human airway epithelial cultures (EC50 = 0.01 µM). Weaker activity was observed in Vero E6 cells (EC50 = 1.65 µM) due to their low capacity to metabolize RDV. To rapidly evaluate in vivo efficacy, we engineered a chimeric SARS-CoV encoding the viral target of RDV, the RNA-dependent RNA polymerase, of SARS-CoV-2. In mice infected with chimeric virus, therapeutic RDV administration diminished lung viral load and improved pulmonary function as compared to vehicle treated animals. These data provide evidence that RDV is potently active against SARS-CoV-2 in vitro and in vivo, supporting its further clinical testing for treatment of COVID-19.Funding: This project was funded in part by the National Institute of Allergy and Infectious Diseases, National 284 Institutes of Health, Department of Health and Human Service awards: 1U19AI142759 (Antiviral Drug 285 Discovery and Development Center awarded to M.R.D. and R.S.B); 5R01AI132178 awarded to T.P.S. 286 and R.S.B.; and 5R01AI108197 awarded to M.R.D. and R.S.B. D.R.M was funded by T32 AI007151 and 287 a Burroughs Wellcome Fund Postdoctoral Enrichment Program Award. The Marsico Lung Institute 288 Tissue Procurement and Cell Culture Core is supported by NIH grant DK065988 and Cystic Fibrosis 289 Foundation grant BOUCHE15RO. We also are grateful for support from the Dolly Parton COVID-19 290 Research Fund, the VUMC Office of Research, and the Elizabeth B. Lamb Center for Pediatric Research 291 at Vanderbilt University. Conflict of Interest: The authors affiliated with Gilead Sciences, Inc. are employees of the company and own company stock. The other authors have no conflict of interest to report.Ethical Approval: Human tracheobronchial epithelial cells provided by Dr. Scott Randell were obtained from airway specimens resected from patients undergoing surgery under University of North Carolina Institutional Review Board-approved protocols (#03-1396) by the Cystic Fibrosis Center Tissue Culture Core.

An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 and multiple endemic, epidemic and bat coronavirus (preprint)

Coronaviruses (CoVs) traffic frequently between species resulting in novel disease outbreaks, most recently exemplified by the newly emerged SARS-CoV-2. Herein, we show that the ribonucleoside analog {beta}-D-N4-hydroxycytidine (NHC, EIDD-1931) has broad spectrum antiviral activity against SARS-CoV 2, MERS-CoV, SARS-CoV, and related zoonotic group 2b or 2c Bat-CoVs, as well as increased potency against a coronavirus bearing resistance mutations to another nucleoside analog inhibitor. In mice infected with SARS-CoV or MERS-CoV, both prophylactic and therapeutic administration of EIDD-2801, an orally bioavailable NHC-prodrug (b-D-N4-hydroxycytidine-5-isopropyl ester), improved pulmonary function, and reduced virus titer and body weight loss. Decreased MERS-CoV yields in vitro and in vivo were associated with increased transition mutation frequency in viral but not host cell RNA, supporting a mechanism of lethal mutagenesis. The potency of NHC/EIDD-2801 against multiple coronaviruses, its therapeutic efficacy, and oral bioavailability in vivo, all highlight its potential utility as an effective antiviral against SARS-CoV-2 and other future zoonotic coronaviruses.