Preclinical Immunological Evaluation of an Intradermally Administered Heterologous Vaccine Against SARS-CoV-2 Variants (preprint)

The recent emergence of new variants in the COVID-19 pandemic has led to new requirements for vaccines, with a focus on the capacity of vaccines to elicit high levels of neutralizing antibodies with specific recognition of S antigen variants based on the characterized vaccines licensed for use. A new strategy involving a heterologous vaccine composed of one or two doses of inactivated vaccine and a boost with the S1 protein with mutations (K-S) administered via the intradermal route was designed in this work and was found to improve immune efficacy by increasing neutralizing antibody titers and promoting specific T cell responses against 5 variants of the RBD peptide. A viral challenge test with the B.1.617.2 (Delta) variant confirmed that the both schedules of “1+1” and “2+1” administration ensured a clinical protective effect against this strain. All of these results not only suggested the feasibility of our strategy for protecting against new variants but also provided a technical pathway to enhance the anamnestic immune response in the immunized population.

Study of Ameliorating Effect of COVID-19 mRNA Vaccine in Individuals Immunized With Inactivated Vaccine

Heterologous vaccination strategy for containing COVID-19 pandemic (preprint)

An unequitable vaccine allocation and continuously emerging SARS-CoV-2 variants pose challenges to contain the pandemic, which underscores the need for licensing more vaccine candidates, increasing manufacturing capacity and implementing better immunization strategy. Here, we report data from a proof-of-concept investigation in two healthy individuals who received two doses of inactivated whole-virus COVID-19 vaccines, followed by a single heterologous boost vaccination after 7 months with an mRNA vaccine candidate (LPP-Spike-mRNA) developed by Stemirna Therapeutics. Following the boost, Spike-specific antibody (Ab), memory B cell and T cell responses were significantly increased. These findings indicate that a heterologous immunization strategy combining inactivated and mRNA vaccines can generate robust vaccine responses and therefore provide a rational and effective vaccination regimen.

An in-depth investigation of the safety and immunogenicity of an inactivated SARS-CoV-2 vaccine (preprint)

BACKGROUND In-depth investigations of the safety and immunogenicity of inactivated SARS-CoV-2 vaccines are needed. METHOD In a phase I randomized, double-blinded, and placebo-controlled trial involving 192 healthy adults 18-59 years of age, two injections of three different doses (50 EU, 100 EU and 150 EU) of an inactivated SARS-CoV-2 vaccine or the placebo were administered intramuscularly with a 2- or 4-week interval between the injections. The safety and immunogenicity of the vaccine were evaluated within 28 days. FINDING In this study, 191 subjects assigned to three doses groups or the placebo group completed the 28-day trial. There were 44 adverse reactions within the 28 days, most commonly mild pain and redness at the injection site or slight fatigue, and no abnormal variations were observed in 48 cytokines in the serum samples of immunized subjects. The serum samples diluted from 1:32 to 1:4096 and incubated with the virus did not show antibody-dependent enhancement effects (ADEs) with regard to human natural killer cells, macrophages or dendritic cells. At day 14, the seroconversion rates had reached 92%, 100% and 96% with geometric mean titers (GMTs) of 18.0, 54.5 and 37.1, and at day 28, the seroconversion rates had reached 80%, 96% and 92% with GMTs of 10.6, 15.4 and 19.6in 0, 14 and 0, 28 procedures, respectively. Seroconversion was associated with the synchronous upregulation of ELISA antibodies against the S protein, N protein and virion and a cytotoxic T lymphocyte (CTL) response. Transcriptome analysis shaped the genetic diversity of immune response induced by the vaccine. INTERPRETATION In a population aged 18-59 years, this inactivated SARS-CoV-2 vaccine was safe and immunogenic.

Systematic discovery and functional interrogation of SARS-CoV-2 viral RNA-host protein interactions during infection (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of a pandemic with growing global mortality. There is an urgent need to understand the molecular pathways required for host infection and anti-viral immunity. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we identified 309 host proteins that bind the SARS-CoV-2 RNA during active infection. Integration of this data with viral ChIRP-MS data from three other positive-sense RNA viruses defined pan-viral and SARS-CoV-2-specific host interactions. Functional interrogation of these factors with a genome-wide CRISPR screen revealed that the vast majority of viral RNA-binding proteins protect the host from virus-induced cell death, and we identified known and novel anti-viral proteins that regulate SARS-CoV-2 pathogenicity. Finally, our RNA-centric approach demonstrated a physical connection between SARS-CoV-2 RNA and host mitochondria, which we validated with functional and electron microscopy data, providing new insights into a more general virus-specific protein logic for mitochondrial interactions. Altogether, these data provide a comprehensive catalogue of SARS-CoV-2 RNA-host protein interactions, which may inform future studies to understand the mechanisms of viral pathogenesis, as well as nominate host pathways that could be targeted for therapeutic benefit.

Autoproteolytic Products of the SARS-CoV-2 Nucleocapsid Protein are Primed for Antibody Evasion and Virus Proliferation (preprint)

The SARS-CoV-2 nucleocapsid (N) protein is the most immunogenic of the structural proteins and plays essential roles in several stages of the virus lifecycle. It is comprised of two major structural domains: the RNA binding domain, which interacts with viral and host RNA, and the oligomerization domain which assembles to form the viral core. Here, we investigate the assembly state and RNA binding properties of the full-length nucleocapsid protein using native mass spectrometry. We find that dimers, and not monomers, of full-length N protein bind RNA, implying that dimers are the functional unit of ribonucleoprotein assembly. In addition, we find that N protein binds RNA with a preference for GGG motifs which are known to form short stem loop structures. Unexpectedly, we found that N undergoes autoproteolytic processing within the linker region, separating the two major domains. This process results in the formation of at least five proteoforms that we sequenced using electron transfer dissociation, higher-energy collision induced dissociation and corroborated by peptide mapping. The cleavage sites identified are in highly conserved regions leading us to consider the potential roles of the resulting proteoforms. We found that monomers of N-terminal proteoforms bind RNA with the same preference for GGG motifs and that the oligomeric state of a C-terminal proteoform (N156-419) is sensitive to pH. We used mass spectrometry to show that N binds to a monoclonal antibody raised against full-length N. No antibody interactions were detected for N proteoforms without C-terminal residues, therefore locating antigenic regions towards the C-terminus. We then tested interactions of the proteoforms with the immunophilin cyclophilin A, a key component in coronavirus replication. We found that N1-209 and N1-273 bind directly to cyclophilin A, an interaction that is abolished by the approved immunosuppressant drug cyclosporin A. We propose that the proteoforms generated via autoproteolysis evade antibody detection through removal of the antigenic C-terminus and facilitate interactions with structured RNA or cyclophilin thereby enabling the virus to proliferate.

The strength of a NES motif in the nucleocapsid protein of human coronaviruses is related to genus, but not to pathogenic capacity (preprint)

Seven members of the Coronaviridae family infect humans, but only three (SARS-CoV, SARS-CoV-2 and MERS-CoV) cause severe disease with a high case fatality rate. Using in silico analyses (machine learning techniques and comparative genomics), several features associated to coronavirus pathogenicity have been recently proposed, including a potential increase in the strength of a predicted novel nuclear export signal (NES) motif in the nucleocapsid protein. Here, we have used a well-established nuclear export assay to experimentally establish whether the recently proposed nucleocapsid NESs are capable of mediating nuclear export, and to evaluate if their activity correlates with coronavirus pathogenicity. The six NES motifs tested were functional in our assay, but displayed wide differences in export activity. Importantly, these differences in NES strength were not related to strain pathogenicity. Rather, we found that the NESs of the strains belonging to the genus Alphacoronavirus were markedly stronger than the NESs of the strains belonging to the genus Betacoronavirus. We conclude that, while some of the genomic features recently identified in silico could be crucial contributors to coronavirus pathogenicity, this does not appear to be the case of nucleocapsid NES activity, as it is more closely related to coronavirus genus than to pathogenic capacity.

Structural basis for repurposing a 100-years-old drug suramin for treating COVID-19 (preprint)

The COVID-19 pandemic by non-stop infections of SARS-CoV-2 has continued to ravage many countries worldwide. Here we report the discovery of suramin, a 100-year-old drug, as a potent inhibitor of the SARS-CoV-2 RNA dependent RNA polymerase (RdRp) through blocking the binding of RNA to the enzyme. In biochemical assays, suramin and its derivatives are at least 20-fold more potent than remdesivir, the currently approved nucleotide drug for COVID-19. The 2.6 [A] cryo-EM structure of the viral RdRp bound to suramin reveals two binding sites of suramin, with one site directly blocking the binding of the RNA template strand and the other site clash with the RNA primer strand near the RdRp catalytic active site, therefore inhibiting the viral RNA replication. Furthermore, suramin potently inhibits SARS-CoV-2 duplication in Vero E6 cells. These results provide a structural mechanism for the first non-nucleotide inhibitor of the SARS-CoV-2 RdRp and a rationale for repurposing suramin for treating COVID-19.

SARS-CoV-2 infection damages airway motile cilia and impairs mucociliary clearance (preprint)

Understanding how SARS-CoV-2 spreads within the respiratory tract is important to define the parameters controlling the severity of COVID-19. We examined the functional and structural consequences of SARS-CoV-2 infection in a reconstituted human bronchial epithelium model. SARS-CoV-2 replication caused a transient decrease in epithelial barrier function and disruption of tight junctions, though viral particle crossing remained limited. Rather, SARS-CoV-2 replication led to a rapid loss of the ciliary layer, characterized at the ultrastructural level by axoneme loss and misorientation of remaining basal bodies. The motile cilia function was compromised, as measured in a mucociliary clearance assay. Epithelial defense mechanisms, including basal cell mobilization and interferon-lambda induction, ramped up only after the initiation of cilia damage. Analysis of SARS-CoV-2 infection in Syrian hamsters further demonstrated the loss of motile cilia in vivo. This study identifies cilia damage as a pathogenic mechanism that could facilitate SARS-CoV-2 spread to the deeper lung parenchyma.

Sub-second heat inactivation of coronavirus (preprint)

Heat treatment denatures viral proteins that comprise the virion, making virus incapable of infecting a host. Coronavirus (CoV) virions contain single-stranded RNA genomes with a lipid envelope and 4 proteins, 3 of which are associated with the lipid envelope and thus are thought to be easily denatured by heat or surfactant-type chemicals. Prior studies have shown that a temperature of as low as 75 oC and treatment duration of 15 min can effectively inactivate CoV. The applicability of a CoV heat inactivation method greatly depends on the length of time of a heat treatment and the temperature needed to inactivate the virus. With the goal of finding conditions where sub-second heat exposure of CoV can sufficiently inactivate CoV, we designed and developed a simple system that can measure sub-second heat inactivation of CoV. The system is composed of capillary stainless-steel tubing immersed in a temperature-controlled oil bath followed by an ice bath, through which virus solution can be flowed at various speeds. Flowing virus solution at different speeds, along with a real-time temperature monitoring system, allows the virus to be accurately exposed to a desired temperature for various durations of time. Using mouse hepatitis virus (MHV), a beta-coronavirus, as a model system, we identified that 85.2 oC for 0.48 s exposure is sufficient to obtain > 5 Log10 reduction in viral titer (starting titer: 5 x 107 PFU/mL), and that when exposed to 83.4 oC for 0.95 s, the virus was completely inactivated (zero titer, > 6 Log10 reduction).

A genetic variant protective for COVID-19 is inherited from Neanderthals (preprint)

It was recently shown that the major genetic risk factor associated with becoming severely ill with COVID-19 when infected by SARS-CoV-2 is inherited from Neandertals. Thanks to new genetic association studies additional risk factors are now being discovered. Using data from a recent genome-wide associations from the Genetics of Mortality in Critical Care (GenOMICC) consortium, we show that a haplotype at a region associated with requiring intensive care is inherited from Neandertals. It encodes proteins that activate enzymes that are important during infections with RNA viruses. As compared to the previously described Neandertal risk haplotype, this Neandertal haplotype is protective against severe COVID-19, is of more moderate effect, and is found at substantial frequencies in all regions of the world outside Africa.

GNS561 exhibits potent in vitro antiviral activity against SARS-CoV-2 through autophagy inhibition (preprint)

Since December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2/2019-nCoV) has spread quickly worldwide, with more than 29 million cases and 920,000 deaths. Interestingly, coronaviruses were found to subvert and hijack the autophagic process to allow their viral replication. One of the spotlights had been focused on the autophagy inhibitors as a target mechanism effective in the inhibition of SARS-CoV-2 infection. Consequently, chloroquine (CQ) and hydroxychloroquine (HCQ), a derivative of CQ, was suggested as the first potentially be therapeutic strategies as they are known to be autophagy inhibitors. Then, they were used as therapeutics in SARS-CoV-2 infection along with remdesivir, for which the FDA approved emergency use authorization. Here, we investigated the antiviral activity and associated mechanism of GNS561, a small basic lipophilic molecule inhibitor of late-stage autophagy, against SARS-CoV-2. Our data indicated that GNS561 showed the highest antiviral effect for two SARS-CoV-2 strains compared to CQ and remdesivir. Focusing on the autophagy mechanism, we showed that GNS561, located in LAMP2-positive lysosomes, together with SARS-CoV-2, blocked autophagy by increasing the size of LC3-II spots and the accumulation of autophagic vacuoles in the cytoplasm with the presence of multilamellar bodies characteristic of a complexed autophagy. Finally, our study revealed that the combination of GNS561 and remdesivir was associated with a strong synergistic antiviral effect against SARS-CoV-2. Overall, our study highlights GNS561 as a powerful drug in SARS-CoV-2 infection and supports that the hypothesis that autophagy inhibitors could be an alternative strategy for SARS-CoV-2 infection.

Multi-Clonal Live SARS-CoV-2 In Vitro Neutralization by Antibodies Isolated from Severe COVID-19 Convalescent Donors (preprint)

The interactions between antibodies, SARS-CoV-2 and immune cells contribute to the pathogenesis of COVID-19 and protective immunity. To understand the differences between antibody responses in mild versus severe cases of COVID-19, we analyzed the B cell responses in patients 1.5 months post SARS-CoV-2 infection. Severe and not mild infection correlated with high titers of IgG against Spike receptor binding domain (RBD) that were capable of viral inhibition. B cell receptor (BCR) sequencing revealed two VH genes, VH3-38 and VH3-53, that were enriched during severe infection. Of the 22 antibodies cloned from two severe donors, six exhibited potent neutralization against live SARS-CoV-2, and inhibited syncytia formation. Using peptide libraries, competition ELISA and RBD mutagenesis, we mapped the epitopes of the neutralizing antibodies (nAbs) to three different sites on the Spike. Finally, we used combinations of nAbs targeting different immune-sites to efficiently block SARS-CoV-2 infection. Analysis of 49 healthy BCR repertoires revealed that the nAbs germline VHJH precursors comprise up to 2.7% of all VHJHs. We demonstrate that severe COVID-19 is associated with unique BCR signatures and multi-clonal neutralizing responses that are relatively frequent in the population. Moreover, our data support the use of combination antibody therapy to prevent and treat COVID-19.

A valid protective immune response elicited in rhesus macaques by an inactivated vaccine is capable of defending against SARS-CoV-2 infection (preprint)

With the relatively serious global epidemic outbreak of SARS-CoV-2 infection, public concerns focus on not only clinical therapeutic measures and public quarantine for this disease but also the development of vaccines. The technical design of our SARS-CoV-2 inactivated vaccine provides a viral antigen that enables the exposure of more than one structural protein based upon the antibody composition of COVID-19 patients convalescent serum. This design led to valid immunity with increasing neutralizing antibody titers and a CTL response detected post-immunization of this vaccine by two injections in rhesus macaques. Further, this elicited immunoprotection in macaques enables not only to restrain completely viral replication in tissues of immunized animals, compared to the adjuvant control and those immunized by an RBD peptide vaccine, but also to significantly alleviate inflammatory lesion in lung tissues in histo-pathologic detection, compared to the adjuvant control with developed interstitial pneumonia. The data obtained from these macaques immunized with the inactivated vaccine or RBD peptide vaccine suggest that immunity with a clinically protective effect against SARS-CoV-2 infection should include not only specific neutralizing antibodies but also specific CTL responses against at least the S and N antigens.