Broadly neutralizing antibody induction by non-stabilized SARS-CoV-2 Spike mRNA vaccination in nonhuman primates (preprint)

Immunization with mRNA or viral vectors encoding spike with diproline substitutions (S-2P) has provided protective immunity against severe COVID-19 disease. How immunization with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) spike elicits neutralizing antibodies (nAbs) against difficult-to-neutralize variants of concern (VOCs) remains an area of great interest. Here, we compare immunization of macaques with mRNA vaccines expressing ancestral spike either including or lacking diproline substitutions, and show the diproline substitutions were not required for protection against SARS-CoV-2 challenge or induction of broadly neutralizing B cell lineages. One group of nAbs elicited by the ancestral spike lacking diproline substitutions targeted the outer face of the receptor binding domain (RBD), neutralized all tested SARS-CoV-2 VOCs including Omicron XBB.1.5, but lacked cross-Sarbecovirus neutralization. Structural analysis showed that the macaque broad SARS-CoV-2 VOC nAbs bound to the same epitope as a human broad SARS-CoV-2 VOC nAb, DH1193. Vaccine-induced antibodies that targeted the RBD inner face neutralized multiple Sarbecoviruses, protected mice from bat CoV RsSHC014 challenge, but lacked Omicron variant neutralization. Thus, ancestral SARS-CoV-2 spike lacking proline substitutions encoded by nucleoside-modified mRNA can induce B cell lineages binding to distinct RBD sites that either broadly neutralize animal and human Sarbecoviruses or recent Omicron VOCs.

Investigation of the Impact of Clonal Hematopoiesis on Severity and Pathophysiology of COVID-19 in Rhesus Macaques (preprint)

Clinical manifestations of COVID-19 vary widely, ranging from asymptomatic to severe respiratory failure with profound inflammation. Although risk factors for severe illness have been identified, definitive determinants remain elusive. Clonal hematopoiesis (CH), the expansion of hematopoietic stem and progenitor cells bearing acquired somatic mutations, is associated with advanced age and hyperinflammation. Given the similar age range and hyperinflammatory phenotype between frequent CH and severe COVID-19, CH could impact the risk of severe COVID-19. Human cohort studies have attempted to prove this relationship, but conclusions are conflicting. Rhesus macaques (RMs) are being utilized to test vaccines and therapeutics for COVID-19. However, RMs, even other species, have not yet been reported to develop late inflammatory COVID-19 disease. Here, RMs with either spontaneous DNMT3A or engineered TET2 CH along with similarly transplanted and conditioned controls were infected with SARS-CoV-2 and monitored until 12 days post-inoculation (dpi). Although no significant differences in clinical symptoms and blood counts were noted, an aged animal with natural DNMT3A CH died on 10 dpi. CH macaques showed evidence of sustained local inflammatory responses compared to controls. Interestingly, viral loads in respiratory tracts were higher at every timepoint in the CH group. Lung sections from euthanasia showed evidence of mild inflammation in all animals, while viral antigen was more frequently detected in the lung tissues of CH macaques even at the time of autopsy. Despite the lack of striking inflammation and serious illness, our findings suggest potential pathophysiological differences in RMs with or without CH upon SARS-CoV-2 infection.

Breadth of SARS-CoV-2 Neutralization and Protection Induced by a Nanoparticle Vaccine (preprint)

Coronavirus vaccines that are highly effective against SARS-CoV-2 variants are needed to control the current pandemic. We previously reported a receptor-binding domain (RBD) sortase A-conjugated ferritin nanoparticle (RBD-scNP) vaccine that induced neutralizing antibodies against SARS-CoV-2 and pre-emergent sarbecoviruses and protected monkeys from SARS-CoV-2 WA-1 infection. Here, we demonstrate SARS-CoV-2 RBD-scNP immunization induces potent neutralizing antibodies against all eight SARS-CoV-2 variants tested including the Beta, Delta, and Omicron variants in non-human primates (NHPs). The Omicron variant was neutralized by RBD-scNP-induced serum antibodies with a mean of 4.3-fold reduction of ID50 titers compared to SARS-CoV-2 D614G. Immunization with RBD-scNPs protected NHPs from SARS-CoV-2 WA-1, Beta, and Delta variant challenge, and protected mice from challenges of SARS-CoV-2 Beta variant and two other heterologous sarbecoviruses. These results demonstrate the ability of RBD-scNPs to induce broad neutralization of SARS-CoV-2 variants and to protect NHPs and mice from multiple different SARS-related viruses. Such a vaccine could provide the needed immunity to slow the spread of and reduce disease caused by SARS-CoV-2 variants such as Delta and Omicron.

The SARS-CoV-2 B.1.1.529 Omicron virus causes attenuated infection and disease in mice and hamsters (preprint)

Despite the development and deployment of antibody and vaccine countermeasures, rapidly-spreading SARS-CoV-2 variants with mutations at key antigenic sites in the spike protein jeopardize their efficacy. The recent emergence of B.1.1.529, the Omicron variant1,2, which has more than 30 mutations in the spike protein, has raised concerns for escape from protection by vaccines and therapeutic antibodies. A key test for potential countermeasures against B.1.1.529 is their activity in pre-clinical rodent models of respiratory tract disease. Here, using the collaborative network of the SARS-CoV-2 Assessment of Viral Evolution (SAVE) program of the National Institute of Allergy and Infectious Diseases (NIAID), we evaluated the ability of multiple B.1.1.529 Omicron isolates to cause infection and disease in immunocompetent and human ACE2 (hACE2) expressing mice and hamsters. Despite modeling and binding data suggesting that B.1.1.529 spike can bind more avidly to murine ACE2, we observed attenuation of infection in 129, C57BL/6, and BALB/c mice as compared with previous SARS-CoV-2 variants, with limited weight loss and lower viral burden in the upper and lower respiratory tracts. Although K18-hACE2 transgenic mice sustained infection in the lungs, these animals did not lose weight. In wild-type and hACE2 transgenic hamsters, lung infection, clinical disease, and pathology with B.1.1.529 also were milder compared to historical isolates or other SARS-CoV-2 variants of concern. Overall, experiments from multiple independent laboratories of the SAVE/NIAID network with several different B.1.1.529 isolates demonstrate attenuated lung disease in rodents, which parallels preliminary human clinical data.

mRNA-1273 vaccination protects against SARS-CoV-2 elicited lung inflammation in non-human primates (preprint)

ABSTRACT Vaccine-elicited SARS-CoV-2 antibody responses are an established correlate of protection against viral infection in humans and non-human primates. However, it is less clear that vaccine-induced immunity is able to limit infection-elicited inflammation in the lower respiratory tract. To assess this, we collected bronchoalveolar lavage fluid samples post-SARS-CoV-2 strain USA-WA1/2020 challenge from rhesus macaques vaccinated with mRNA-1273 in a dose-reduction study. Single-cell transcriptomic profiling revealed a broad cellular landscape 48 hours post-challenge with distinct inflammatory signatures that correlated with viral RNA burden in the lower respiratory tract. These inflammatory signatures included phagocyte-restricted expression of chemokines such as CXCL10 (IP10) and CCL3 (MIP-1A) and the broad expression of interferon-induced genes such as MX1, ISG15 , and IFIT1 . Induction of these inflammatory profiles was suppressed by prior mRNA-1273 vaccination in a dose-dependent manner, and negatively correlated with pre-challenge serum and lung antibody titers against SARS-CoV-2 spike. These observations were replicated and validated in a second independent macaque challenge study using the B.1.351/beta-variant of SARS-CoV-2. These data support a model wherein vaccine-elicited antibody responses restrict viral replication following SARS-CoV-2 exposure, including limiting viral dissemination to the lower respiratory tract and infection-mediated inflammation and pathogenesis. One Sentence Summary Single cell RNA sequencing analysis demonstrates that mRNA-1273 vaccination limits the development of lower respiratory tract inflammation in SARS-CoV-2 challenged rhesus macaques

A SARS-CoV-2 spike ferritin nanoparticle vaccine protects against heterologous challenge with B.1.1.7 and B.1.351 virus variants in Syrian golden hamsters (preprint)

The emergence of SARS-CoV-2 variants of concern (VOC) requires adequate coverage of vaccine protection. We evaluated whether a spike ferritin nanoparticle vaccine (SpFN), adjuvanted with the Army Liposomal Formulation QS21 (ALFQ), conferred protection against the B.1.1.7 and B.1.351 VOCs in Syrian golden hamsters. SpFN-ALFQ was administered as either single or double-vaccination (0 and 4 week) regimens, using a high (10 g) or low (0.2 g) immunogen dose. Animals were intranasally challenged at week 11. Binding antibody responses were comparable between high- and low-dose groups. Neutralizing antibody titers were equivalent against WA1, B.1.1.7, and B.1.351 variants following two high dose two vaccinations. SpFN-ALFQ vaccination protected against SARS-CoV-2-induced disease and viral replication following intranasal B.1.1.7 or B.1.351 challenge, as evidenced by reduced weight loss, lung pathology, and lung and nasal turbinate viral burden. These data support the development of SpFN-ALFQ as a broadly protective, next-generation SARS-CoV-2 vaccine.

Variant SARS-CoV-2 mRNA vaccines confer broad neutralization as primary or booster series in mice (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of a global pandemic that has led to more than 2.8 million deaths worldwide. Safe and effective vaccines are now available, including Moderna's COVID-19 vaccine (mRNA-1273) that showed 94% efficacy in prevention of symptomatic COVID-19 disease in a phase 3 clinical study. mRNA-1273 encodes for a prefusion stabilized full length spike (S) protein of the Wuhan-Hu-1 isolate. However, the emergence of SARS-CoV-2 variants has led to concerns of viral escape from vaccine-induced immunity. Several emerging variants have shown decreased susceptibility to neutralization by vaccine induced immunity, most notably the B.1.351 variant, although the overall impact on vaccine efficacy remains to be determined. Here, we present the initial evaluation in mice of two updated COVID-19 mRNA vaccines designed to target emerging SARS-CoV-2 variants: (1) monovalent mRNA-1273.351 encodes for the S protein found in the B.1.351 lineage and (2) mRNA-1273.211 comprising a 1:1 mix of mRNA-1273 and mRNA-1273.351. Both vaccines were evaluated as a 2-dose primary series in mice; mRNA-1273.351 was also evaluated as a booster dose in animals previously vaccinated with 2-doses of mRNA-1273. The results demonstrated that a primary vaccination series of mRNA-1273.351 was effective at increasing neutralizing antibody titers against the B.1.351 lineage, while mRNA-1273.211 was most effective at providing broad cross-variant neutralization in mice. In addition, these results demonstrated a third dose of mRNA-1273.351 significantly increased both wild-type and B.1.351-specific neutralization titers. Both mRNA-1273.351 and mRNA-1273.211 are currently being evaluated in additional pre-clinical challenge models and in phase 1/2 clinical studies.

SARS-CoV-2 vaccination induces neutralizing antibodies against pandemic and pre-emergent SARS-related coronaviruses in monkeys (preprint)

Betacoronaviruses (betaCoVs) caused the severe acute respiratory syndrome (SARS) and Middle East Respiratory Syndrome (MERS) outbreaks, and now the SARS-CoV-2 pandemic. Vaccines that elicit protective immune responses against SARS-CoV-2 and betaCoVs circulating in animals have the potential to prevent future betaCoV pandemics. Here, we show that immunization of macaques with a multimeric SARS-CoV-2 receptor binding domain (RBD) nanoparticle adjuvanted with 3M-052-Alum elicited cross-neutralizing antibody responses against SARS-CoV-1, SARS-CoV-2, batCoVs and the UK B.1.1.7 SARS-CoV-2 mutant virus. Nanoparticle vaccination resulted in a SARS-CoV-2 reciprocal geometric mean neutralization titer of 47,216, and robust protection against SARS-CoV-2 in macaque upper and lower respiratory tracts. Importantly, nucleoside-modified mRNA encoding a stabilized transmembrane spike or monomeric RBD protein also induced SARS-CoV-1 and batCoV cross-neutralizing antibodies, albeit at lower titers. These results demonstrate current mRNA vaccines may provide some protection from future zoonotic betaCoV outbreaks, and provide a platform for further development of pan-betaCoV nanoparticle vaccines.

The functions of SARS-CoV-2 neutralizing and infection-enhancing antibodies in vitro and in mice and nonhuman primates (preprint)

SARS-CoV-2 neutralizing antibodies (NAbs) protect against COVID-19, making them a focus of vaccine design. A safety concern regarding SARS-CoV-2 antibodies is whether they mediate disease enhancement. Here, we isolated potent NAbs against the receptor-binding domain (RBD) and the N-terminal domain (NTD) of SARS-CoV-2 spike protein from individuals with acute or convalescent SARS-CoV-2 or a history of SARS-CoV-1 infection. Cryo-electron microscopy of RBD and NTD antibodies demonstrated function-specific modes of antibody binding. Select RBD NAbs also demonstrated Fc receptor-{gamma} (Fc{gamma}R)-mediated enhancement of virus infection in vitro, while five non-neutralizing NTD antibodies mediated Fc{gamma}R-independent in vitro infection enhancement. However, both in vitro neutralizing and infection-enhancing RBD or infection-enhancing NTD antibodies protected from SARS-CoV-2 challenge in non-human primates and mice. One of 30 monkeys infused with enhancing antibodies had lung pathology and bronchoalveolar lavage cytokine evidence suggestive of enhanced disease. Thus, these in vitro assessments of enhanced antibody-mediated infection do not necessarily indicate biologically relevant in vivo infection enhancement.

Comprehensive analysis of the host-virus interactome of SARS-CoV-2 (preprint)

Host-virus protein-protein interaction is the key component of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lifecycle. We conducted a comprehensive interactome study between the virus and host cells using tandem affinity purification and proximity labeling strategies and identified 437 human proteins as the high-confidence interacting proteins. Functional characterization and further validation of these interactions elucidated how distinct SARS-CoV-2 viral proteins participate in its lifecycle, and discovered potential drug targets to the treatment of COVID-19. The interactomes of two key SARS-CoV-2 encoded viral proteins, NSP1 and N protein, were compared with the interactomes of their counterparts in other human coronaviruses. These comparisons not only revealed common host pathways these viruses manipulate for their survival, but also showed divergent protein-protein interactions that may explain differences in disease pathology. This comprehensive interactome of coronavirus disease-2019 provides valuable resources for understanding and treating this disease.

Unbuttoning the impact of N501Y mutant RBD on viral entry mechanism: A computational insight (preprint)

The ongoing coronavirus disease 2019 (COVID-19) pandemic has become a serious global threat. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the virus responsible for this pandemic has imposed a severe burden on the medical settings. The spike (S) protein of SARS-CoV-2 is an important structural protein playing a key role in the viral entry. This protein is responsible for the receptor recognition and cell membrane fusion process. The recent reports of the appearance and spread of new SARS-CoV-2 strain has raised alarms. It was reported that this new variant containing the prominent active site mutation in the RBD (N501Y) was rapidly spreading within the population. The reported N501Y mutation within the spike's essential part, known as the receptor-binding domain has raised several questions. Here in this study we have tried to explore the effect of N501Y mutation within the spike protein using several in silico approaches

Rapid inactivation of SARS-CoV-2 on copper touch surfaces determined using a cell culture infectivity assay (preprint)

COVID-19, caused by SARS-CoV-2, was first reported in China in 2019 and has transmitted rapidly around the world, currently responsible for 83 million reported cases and over 1.8 million deaths. The mode of transmission is believed principally to be airborne exposure to respiratory droplets from symptomatic and asymptomatic patients but there is also a risk of the droplets contaminating fomites such as touch surfaces including door handles, stair rails etc, leading to hand pick up and transfer to eyes, nose and mouth. We have previously shown that human coronavirus 229E survives for more than 5 days on inanimate surfaces and another laboratory reproduced this for SARS-CoV-2 this year. However, we showed rapid inactivation of Hu-CoV-229E within 10 minutes on different copper surfaces while the other laboratory indicated this took 4 hours for SARS-CoV-2. So why the difference? We have repeated our work with SARS-CoV-2 and can confirm that this coronavirus can be inactivated on copper surfaces in as little as 1 minute. We discuss why the 4 hour result may be technically flawed.

Alveolar type II cells harbouring SARS-CoV-2 show senescence with a proinflammatory phenotype (preprint)

SARS-CoV-2 infection of the respiratory system can evolve to a multi-system disease. Excessive levels of proinflammatory cytokines, known as a "cytokine storm" are associated with high mortality rates especially in the elderly and in patients with age-related morbidities. Senescent cells, characterized by secretion of such cytokines (Senescence Associated Secretory Phenotype - SASP), are known to occur in this context as well as upon a variety of stressogenic insults. Applying both: i) a novel "in house" antibody against the spike protein of SARS-CoV-2 and ii) a unique senescence detecting methodology, we identified for the first time in lung tissue from COVID-19 patients alveolar cells acquiring senescent features harboring also SARS-CoV-2. Moreover, using the same detection workflow we demonstrated the inflammatory properties of these cells. Our findings justify the application of senotherapeutics for the treatment or prevention of COVID-19 patients.