Long-term serial passaging of SARS-CoV-2 reveals signatures of convergent evolution (preprint)

Measures to control the COVID-19 pandemic such as antiviral therapy and vaccination have been challenged by ongoing virus evolution under antiviral and immune pressures. Understanding viral evolutionary dynamics is crucial for responding to SARS-CoV-2, and preparing for the next pandemic, by informing prediction of virus adaptation, public health strategies, and design of broadly effective therapies. Whole-genome sequencing (WGS) of SARS-CoV-2 during the pandemic enabled fine-grained studies of virus evolution in the human population. Serial passaging in vitro offers a controlled environment to investigate the emergence and persistence of genetic variants that may confer selective advantage. Nine virus lineages, including four "variants of concern" and three former "variants under investigation" as designated by the World Health Organisation, were chosen to investigate intra- and inter-lineage evolution through long-term serial passaging in Vero E6 cells. Viruses were sampled over at least 33 passages (range 33-100) and analysed using WGS to examine evolutionary dynamics and identify key mutations with implications for virus fitness, transmissibility, and immune evasion. All passages continued to replicate in culture, despite regular accumulation of mutations. There was evidence of convergent acquisition of mutations both across passage lines and compared with contemporaneous SARS CoV-2 clinical sequences from population studies. Some of these convergent mutations are hypothesised to be important in proliferation of SARS-CoV-2 lineages, such as by evading host immune responses (e.g. S:A67V, S:H655Y). Given these mutations arose in vitro, in the absence of a multicellular host immune response, this suggests virus genome mutation resulted from stochastic events, rather than immune-driven mutation. There was a regular gain and loss of low-frequency variants during serial passaging, but some became fixed in subsequent multiple passages, suggesting either a benefit of the mutation in vitro, or at least a lack of deleterious effect. Our findings reveal valuable insights into the evolution of SARS-CoV-2 by quantitatively investigating evolutionary dynamics of the virus over the greatest number of serial passages to date. Knowledge of these evolutionary trends will be useful for public health and the development of antiviral and vaccine measures to reduce the effects of SARS CoV-2 infection on the human population.

Clinical and laboratory features of COVID-19 illness and outcomes in immunocompromised individuals during the first pandemic wave in Sydney, Australia (preprint)

People with immunocompromising conditions are at increased risk of SARS-CoV-2 infection and mortality, however early in the pandemic it was challenging to collate data on this heterogenous population. We conducted a registry study of immunocompromised individuals with polymerase chain reaction (PCR)-confirmed SARS-CoV-2 infection from March - October 2020 in Sydney, Australia to understand clinical and laboratory outcomes in this population prior to the emergence of the Delta variant. 27 participants were enrolled into the study including people with a haematologic oncologic conditions (n=12), secondary immunosuppression (N=8) and those with primary or acquired immunodeficiency (i.e. HIV; N=7). All participants had symptomatic COVID-19 with the most common features being cough (64%), fever (52%) and headache (40%). Five patients demonstrated delayed SARS-CoV-2 clearance lasting three weeks to three months. The mortality rate in this study was 7% compared to 1.3% in the state of New South Wales Australia during the same period. This study provides data from the first eight months of the pandemic on COVID-19 outcomes in at-risk patient groups.

Broadly neutralizing SARS-CoV-2 antibodies through epitope-based selection from convalescent patients (preprint)

Emerging variants of concern (VOCs) are threatening to limit the efficacy of SARS CoV-2 monoclonal antibodies and vaccines currently used in clinical practice; broadly neutralizing antibodies and strategies for their identification are therefore urgently required. Here we demonstrate that broadly neutralizing antibodies can be isolated from peripheral blood mononuclear cells (PBMCs) of convalescent patients using SARS CoV-2 receptor binding domains (RBDs) carrying epitope-specific mutations. This is exemplified by two human antibodies, GAR05, binding to epitope class 1, and GAR12, binding to a new epitope class 6 (located between class 3 and class 5). Both antibodies broadly neutralize VOCs, exceeding the potency of the clinical monoclonal sotrovimab (mAb S309) by orders of magnitude. They also provide potent prophylactic and therapeutic in vivo protection of hACE2 mice against viral challenge. Our results indicate that exposure to Wuhan SARS-CoV-2 induces antibodies that maintain potent and broad neutralization against emerging VOCs using two unique strategies: either by targeting the divergent class 1 epitope in a manner resistant to VOCs (ACE-2 mimicry, as illustrated by GAR05 and mAbs P2C-1F11/S2K14); or alternatively, by targeting rare and highly conserved epitopes, such as the new class 6 epitope identified here (as illustrated by GAR12). Our results provide guidance for next generation monoclonal antibody development and vaccine design.

Post-acute COVID-19 cognitive impairment and decline uniquely associate with kynurenine pathway activation: a longitudinal observational study (preprint)

Cognitive impairment and function post-acute mild to moderate COVID-19 are poorly understood. We report findings of 128 prospectively studied SARS-CoV-2 positive patients. Cognition and olfaction were assessed at 2-, 4- and 12-months post-diagnosis. Lung function, physical and mental health were assessed at 2-month post diagnosis. Blood cytokines, neuro-biomarkers, and kynurenine pathway (KP) metabolites were measured at 2-, 4-, 8- and 12-months. Mild to moderate cognitive impairment (demographically corrected) was present in 16%, 23%, and 26%, at 2-, 4- and 12-months post diagnosis, respectively. Overall cognitive performance mildly, but significantly (p

Rapid isolation and resolution immune evasion and viral fitness across contemporary SARS-CoV-2 variants (preprint)

From late 2020 the world observed the rapid emergence of many distinct SARS-CoV-2 variants. At the same time, pandemic responses coalesced into significant global vaccine roll-out that have now significantly lowered Covid-19 hospital and mortality rates in the developed world. Over this period, we developed a rapid platform (R-20) for viral isolation and characterisation using primary remnant diagnostic swabs. This combined with quarantine testing and genomics surveillance, enabled the rapid isolation and characterisation of all major SARS-CoV-2 variants (all variants of concern and 6 variants of interest) globally with a 4-month period. This platform facilitated viral variant isolation and enabled rapid resolution of variant phenotype by allowing determining end point viral titers from primary nasopharyngeal swabs and through ranking of evasion of neutralising antibodies. In late 2021, when the Delta variant was dominating, Omicron rapidly emerged. Using this platform, we isolated and tested the first cases of this variant within Australia. In this setting we observed Omicron to diverge from other variants at two levels: Firstly, it ranks at the mots evasive to neutralisation antibodies compared to all VOCs and major VUIs. Secondly, it no longer engages TMPRSS2 during the late stages of fusion.

SARS-CoV-2 Omicron: evasion of potent humoral responses and resistance to clinical immunotherapeutics relative to viral variants of concern (preprint)

Genetically distinct viral variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been recorded since January 2020. Over this time global vaccine programs have been introduced, contributing to lowered COVID-19 hospitalisation and mortality rates, particularly in the first world. In late 2021, the Omicron (B.1.1.529) virus variant emerged, with significant genetic differences and clinical effects from other variants of concern (VOC). This variant a demonstrated higher number of polymorphisms in the gene encoding the Spike (S) protein, and there has been displacement of the dominant Delta variant. We assessed the impact of Omicron infection on the ability of: serum from vaccinated and/or previously infected individuals; concentrated human IgG from plasma donors, and licensed monoclonal antibody therapies to neutralise the virus in vitro . There was a 17 to 27-fold reduction in neutralisation titres across all donors who had a detectable neutralising antibody titre to the Omicron variant. Concentrated pooled human IgG from convalescent and vaccinated donors had greater breadth of neutralisation, although the potency was still reduced 16-fold. Of all therapeutic antibodies tested, significant neutralisation of the Omicron variant was only observed for Sotrovimab, with other monoclonal antibodies unable to neutralise Omicron in vitro . These results have implications for ongoing therapy of individuals infected with the Omicron variant.

Potent SARS-CoV-2 binding and neutralization through maturation of iconic SARS-CoV-1 antibodies (preprint)

ABSTRACT Antibodies against coronavirus spike protein potently protect against infection and disease, however it remains unclear if such protection can be extended to variant coronaviruses. This is exemplified by a set of iconic and well-characterized monoclonal antibodies developed after the 2003 SARS outbreak including mAbs m396, CR3022, CR3014 and 80R, which potently neutralize SARS-CoV-1, but not SARS-CoV-2. Here we explore antibody maturation strategies to change and broaden their specificity, enabling potent binding and neutralization of SARS-CoV-2. Using targeted mutagenesis as well as light chain shuffling on phage, we identified variants with considerably increased affinity and neutralization potential. The most potent antibody, derived from the NIH-developed mAb m396, neutralized live SARS-CoV-2 virus with a half-maximal inhibitory concentration (IC 50 ) of 160 ng/ml. Intriguingly, while many of the matured clones maintained specificity of the parental antibody, new specificities were also observed, which was further confirmed by X-ray crystallography and cryo-electron microscopy, indicating that a limited set of antibodies can give rise to variants targeting diverse epitopes. Our findings open up over 15 years of antibody development efforts against SARS-CoV-1 to the SARS-CoV-2 field and outline general principles for the maturation of antibody specificity against emerging viruses.

No detectable signal for ongoing genetic recombination in SARS-CoV-2 (preprint)

The COVID-19 pandemic has led to an unprecedented global sequencing effort of its viral agent SARS-CoV-2. The first whole genome assembly of SARS-CoV-2 was published on January 5 2020. Since then, over 150,000 high-quality SARS-CoV-2 genomes have been made available. This large genomic resource has allowed tracing of the emergence and spread of mutations and phylogenetic reconstruction of SARS-CoV-2 lineages in near real time. Though, whether SARS-CoV-2 undergoes genetic recombination has been largely overlooked to date. Recombination-mediated rearrangement of variants that arose independently can be of major evolutionary importance. Moreover, the absence of recombination is a key assumption behind the application of phylogenetic inference methods. Here, we analyse the extant genomic diversity of SARS-CoV-2 and show that, to date, there is no detectable hallmark of recombination. We assess our detection power using simulations and validate our method on the related MERS-CoV for which we report evidence for widespread genetic recombination.

Genomic diversity analysis of SARS-CoV-2 genomes in Rwanda (preprint)

COVID-19 (Coronavirus disease 2019) is an emerging pneumonia-like respiratory disease of humans and is recently spreading across the globe. To analyze the genome sequence of SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) isolated from Rwanda with other viral strains from African countries. We downloaded 75 genomes sequences of clinical SARS-CoV-2 from the GISAID (global initiative on sharing all influenza data) database and we comprehensively analyzed these SARS-CoV-2 genomes sequences alongside with Wuhan SARS-CoV-2 sequences as the reference strains. We analyzed 75 genomes sequences of SARS-CoV-2 isolated in different African countries including 10 samples of SARS-CoV-2 isolated in Rwanda between July and August 2020. The phylogenetic analysis of the genome sequence of SARS-CoV-2 revealed a strong identity with reference strains between 90-95%. We identified a missense mutation in four proteins including orf1ab polyprotein, NSP2, 2'-O-ribose methyltransferase and orf1a polyprotein. The most common changes in the base are C > T. We also found that all clinically SARS-CoV-2 isolated from Rwanda had genomes belonging to clade G and lineage B.1. Tracking the genetic evolution of SARS-CoV-2 over time is important to understand viral evolution pathogenesis. These findings may help to implement public health measures in curbing COVID-19 in Rwanda.

Molnupiravir (EIDD-2801) inhibits SARS-CoV2 replication in Syrian hamsters model (preprint)

Since its emergence in Wuhan, China in December 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide resulting in a global pandemic with >1.5 million deaths until now. In the search for small molecule inhibitors of SARS-CoV-2, drug repurposing is being extensively explored. Molnupiravir (EIDD-2801) is an orally bioavailable nucleoside analog that possesses a relatively broad-spectrum antiviral activity including against coronaviruses. We here studied the effect of EIDD-2801 in a well-established Syrian hamster SARS-CoV2 infection model. Treatment of SARS-CoV-2-infected hamsters with 200 mg/kg BID of EIDD-2801 for four consecutive days, starting from the day of infection, significantly reduced infectious virus titers and viral RNA loads in the lungs and markedly improved lung histopathology. When onset of treatment was delayed until 1 or 2 days after infection, a very modest antiviral effect was observed. The potential of EIDD-2801 for the treatment and or prevention of SARS-CoV2 deserves further attention.

An interactive viral genome evolution network analysis system enabling rapid large-scale molecular tracing of SARS-CoV-2 (preprint)

Comprehensive analyses of viral genomes can provide a global picture on SARS-CoV-2 transmission and help to predict the oncoming trends of pandemic. This molecular tracing is mainly conducted through extensive phylogenetic network analyses. However, the rapid accumulation of SARS-CoV-2 genomes presents an unprecedented data size and complexity that has exceeded the capacity of existing methods in constructing evolution network through virus genotyping. Here we report a Viral genome Evolution Network Analysis System (VENAS), which uses Hamming distances adjusted by the minor allele frequency to construct viral genome evolution network. The resulting network was topologically clustered and divided using community detection algorithm, and potential evolution paths were further inferred with a network disassortativity trimming algorithm. We also employed parallel computing technology to achieve rapid processing and interactive visualization of >10,000 viral genomes, enabling accurate detection and subtyping of the viral mutations through different stages of Covid-19 pandemic. In particular, several core viral mutations can be independently identified and linked to early transmission events in Covid-19 pandemic. As a general platform for comprehensive viral genome analysis, VENAS serves as a useful computational tool in the current and future pandemics.

Cross-neutralization of SARS-CoV-2 by HIV-1 specific broadly neutralizing antibodies and polyclonal plasma (preprint)

Cross-reactive epitopes (CREs) are similar epitopes on viruses that are recognized or neutralized by same antibodies. The S protein of SARS-CoV-2, similar to type I fusion proteins of viruses such as HIV-1 envelope (Env) and influenza hemagglutinin, is heavily glycosylated. Viral Env glycans, though host derived, are distinctly processed and thereby recognized or accommodated during antibody responses. In recent years, highly potent and/or broadly neutralizing human monoclonal antibodies (bnAbs) that are generated in chronic HIV-1 infections have been defined. These bnAbs exhibit atypical features such as extensive somatic hypermutations, long complementary determining region (CDR) lengths, tyrosine sulfation and presence of insertions/deletions, enabling them to effectively neutralize diverse HIV-1 viruses despite extensive variations within the core epitopes they recognize. As some of the HIV-1 bnAbs have evolved to recognize the dense viral glycans and cross-reactive epitopes (CREs), we assessed if these bnAbs cross-react with SARS-CoV-2. Several HIV-1 bnAbs showed cross-reactivity with SARS-CoV-2 while one HIV-1 CD4 binding site bnAb, N6, neutralized SARS-CoV-2. Furthermore, neutralizing plasma antibodies of chronically HIV-1 infected children showed cross neutralizing activity against SARS-CoV-2. Collectively, our observations suggest that human monoclonal antibodies tolerating extensive epitope variability can be leveraged to neutralize pathogens with related antigenic profile. ImportanceIn the current ongoing COVID-19 pandemic, neutralizing antibodies have been shown to be a critical feature of recovered patients. HIV-1 bnAbs recognize extensively diverse cross-reactive epitopes and tolerate diversity within their core epitope. Given the unique nature of HIV-1 bnAbs and their ability to recognize and/or accommodate viral glycans, we reasoned that the glycan shield of SARS-CoV-2 spike protein can be targeted by HIV-1 specific bnAbs. Herein, we showed that HIV-1 specific antibodies cross-react and neutralize SARS-CoV-2. Understanding cross-reactive neutralization epitopes of antibodies generated in divergent viral infections will provide key evidence for engineering so called super-antibodies (antibodies that can potently neutralize diverse pathogens with similar antigenic features). Such cross-reactive antibodies can provide a blueprint upon which synthetic variants can be generated in the face of future pandemics.

SARS-CoV-2 Nsp16 activation mechanism and a cryptic pocket with pan-coronavirus antiviral potential (preprint)

Coronaviruses have caused multiple epidemics in the past two decades, in addition to the current COVID-19 pandemic that is severely damaging global health and the economy. Coronaviruses employ between twenty and thirty proteins to carry out their viral replication cycle including infection, immune evasion, and replication. Among these, nonstructural protein 16 (Nsp16), a 2-O-methyltransferase, plays an essential role in immune evasion. Nsp16 achieves this by mimicking its human homolog, CMTr1, which methylates mRNA to enhance translation efficiency and distinguish self from other. Unlike human CMTr1, Nsp16 requires a binding partner, Nsp10, to activate its enzymatic activity. The requirement of this binding partner presents two questions that we investigate in this manuscript. First, how does Nsp10 activate Nsp16? While experimentally-derived structures of the active Nsp16/Nsp10 complex exist, structures of inactive, monomeric Nsp16 have yet to be solved. Therefore, it is unclear how Nsp10 activates Nsp16. Using over one millisecond of molecular dynamics simulations of both Nsp16 and its complex with Nsp10, we investigate how the presence of Nsp10 shifts Nsp16s conformational ensemble in order to activate it. Second, guided by this activation mechanism and Markov state models (MSMs), we investigate if Nsp16 adopts inactive structures with cryptic pockets that, if targeted with a small molecule, could inhibit Nsp16 by stabilizing its inactive state. After identifying such a pocket in SARS-CoV-2 Nsp16, we show that this cryptic pocket also opens in SARS-CoV-1 and MERS, but not in human CMTr1. Therefore, it may be possible to develop pan-coronavirus antivirals that target this cryptic pocket. Statement of SignificanceCoronaviruses are a major threat to human health. These viruses employ molecular machines, called proteins, to infect host cells and replicate. Characterizing the structure and dynamics of these proteins could provide a basis for designing small molecule antivirals. In this work, we use computer simulations to understand the moving parts of an essential SARS-CoV-2 protein, understand how a binding partner turns it on and off, and identify a novel pocket that antivirals could target to shut this protein off. The pocket is also present in other coronaviruses but not in the related human protein, so it could be a valuable target for pan-coronavirus antivirals.

A single dose, BCG-adjuvanted SARS-CoV-2 vaccine induces Th1-polarized immunity and high-titre neutralizing antibodies in mice (preprint)

Next-generation vaccines that are safe, effective and with equitable access globally are required to prevent SARS-CoV-2 transmission at a population level. One strategy that has gained significant interest is to repurpose existing licensed vaccines for use against COVID-19. In this report, we have exploited the immunostimulatory properties of bacille Calmette-Guerin (BCG), the vaccine for tuberculosis, to develop a SARS-CoV-2-specific and highly immunogenic vaccine candidate. Combination of BCG with a stabilized, trimeric form of the SARS-CoV-2 spike antigen promoted rapid development of virus-specific IgG antibodies in the sera of vaccinated mice, which could be further augmented by the addition of alum. This vaccine formulation, termed BCG:CoVac, induced a Th1-biased response both in terms of IgG antibody subclass and cytokine release by vaccine-specific CD4+ and CD8+ T cells. A single dose of BCG:CoVac was sufficient to induce high-titre SARS-CoV-2 neutralizing antibodies (NAbs) that were detectable as early as 2 weeks post-vaccination; NAb levels were greater than that seen in the sera of SARS-CoV-2-infected individuals. Boosting of BCG:CoVac-primed mice with a heterologous vaccine combination (spike protein plus alum) could further increase SARS-CoV-2 spike protein-specific antibody response. BCG:CoVac would be broadly applicable for all populations susceptible to SARS-CoV-2 infection and in particular could be readily incorporated into current vaccine schedules in countries where BCG is currently used. ImportanceEffective distribution of vaccine to low- and middle-income countries is critical for the control of the COVID-19 pandemic. To achieve this, vaccines must offer effective protective immunity yet should be cheap to manufacture and meet cold chain management requirements. This study describes a unique COVID-19 vaccine candidate, termed BCG:CoVac, that when delivered as a single dose induces potent SARS-CoV-2 specific immunity in mice, particularly through generation of high-titre, anti-viral neutralising antibodies. BCG:CoVac is built on safe and well-characterised vaccine components: 1) the BCG vaccine, used for control of tuberculosis since 1921 which also has remarkable off target effects, protecting children and the elderly against diverse respiratory viral infections; 2) Alhydrogel adjuvant (Alum), a low cost, globally accessible vaccine adjuvant with an excellent safety record in humans (part of >20 licensed human vaccines and in use >70 years); 3) Stabilized, trimeric SARS-CoV-2 spike protein, which stimulates immune specificity for COVID-19. Further assessment in humans will determine if BCG:CoVac can impart protective immunity against not only SARS-CoV-2, but also other respiratory infections where BCG has known efficacy.

Pre-existing T cell-mediated cross-reactivity to SARS-CoV-2 cannot solely be explained by prior exposure to endemic human coronaviruses (preprint)

Several studies have reported the presence of pre-existing humoral or cell-mediated cross-reactivity to SARS-CoV-2 peptides in healthy individuals unexposed to SARS-CoV-2. In particular, the current literature suggests that this pre-existing cross-reactivity could, in part, derive from prior exposure to common cold endemic human coronaviruses (HCoVs). In this study, we characterised the sequence homology of SARS-CoV-2-derived T-cell epitopes reported in the literature across the entire diversity of the Coronaviridae family. Slightly over half (54.8%) of the tested epitopes did not have noticeable homology to any of the human endemic coronaviruses (HKU1, OC43, NL63 and 229E), suggesting prior exposure to these viruses cannot explain the full cross-reactive profiles observed in healthy unexposed individuals. Further, we find that the proportion of cross-reactive SARS-CoV-2 epitopes with noticeable sequence homology is extremely well predicted by the phylogenetic distance to SARS-CoV-2 (R2 = 96.6%). None of the coronaviruses sequenced to date showed a statistically significant excess of T-cell epitope homology relative to the proportion of expected random matches given the sequence similarity of their core genome to SARS-CoV-2. Taken together, our results suggest that the repertoire of cross-reactive epitopes reported in healthy adults cannot be primarily explained by prior exposure to any coronavirus known to date, or any related yet-uncharacterised coronavirus.

Conformational diversity of CDR region during affinity maturation determines the affinity and stability of Sars-Cov-1 VHH-72 nanobody (preprint)

The affinity maturation of Sars-Cov-1 VHH-72 nanobody from its germline predecessor has been studied at the molecular level. The effect of somatic mutations accumulated during affinity maturation process on flexibility, stability and affinity of the germline and affinity matured nanobody was studied. Affinity maturation results in loss of local flexibility in CDR of H3 and this resulted in a gain of affinity towards the antigen. Further affinity maturation was found to destabilize the nanobody. Mechanistically the loss of flexibility of the CDR H3 is due to the redistribution of hydrogen bond network due to somatic mutation A50T, also this contributes significantly to the destability of the nanobody. Unlike antibody, in nanobody the framework region is highly conserved and structural diversity in CDR is the determining factor in diverse antigen binding and also a factor contributing to the stability. This study provide insights into the interrelationship between flexibility, stability and affinity during affinity maturation in a nanobody.

Single dose immunization with a COVID-19 DNA vaccine encoding a chimeric homodimeric protein targeting receptor binding domain (RBD) to antigen-presenting cells induces rapid, strong and long-lasting neutralizing IgG, Th1 dominated CD4+ T cells and strong CD8+ T cell responses in mice (preprint)

The pandemic caused by the SARS-CoV-2 virus in 2020 has led to a global public health emergency, and non-pharmaceutical interventions required to limit the viral spread are severely affecting health and economies across the world. A vaccine providing rapid and persistent protection across populations is urgently needed to prevent disease and transmission. We here describe the development of novel COVID-19 DNA plasmid vaccines encoding homodimers consisting of a targeting unit that binds chemokine receptors on antigen-presenting cells (human MIP-1 /LD78{beta}), a dimerization unit (derived from the hinge and CH3 exons of human IgG3), and an antigenic unit (Spike or the receptor-binding domain (RBD) from SARS-CoV-2). The candidate encoding the longest RBD variant (VB2060) demonstrated high secretion of a functional protein and induced rapid and dose-dependent RBD IgG antibody responses that persisted up to at least 3 months after a single dose of the vaccine in mice. Neutralizing antibody (nAb) titers against the live virus were detected from day 7 after one dose. All tested dose regimens reached titers that were higher or comparable to those seen in sera from human convalescent COVID-19 patients from day 28. T cell responses were detected already at day 7, and were subsequently characterized to be multifunctional CD8+ and Th1 dominated CD4+ T cells. Responses remained at sustained high levels until at least 3 months after a single vaccination, being further strongly boosted by a second vaccination at day 89. These findings, together with the simplicity and scalability of plasmid DNA manufacturing, safety data on the vaccine platform in clinical trials, low cost of goods, data indicating potential long term storage at +2{degrees} to 8{degrees}C and simple administration, suggests the VB2060 candidate is a promising second generation candidate to prevent COVID-19.