An Efficient Approach for SARS-CoV-2 Monoclonal Antibody Production via Modified mRNA-LNP Immunization (preprint)

Throughout the COVID-19 pandemic, many prophylactic and therapeutic drugs have been evaluated and introduced. Among these treatments, monoclonal antibodies (mAbs) that bind to and neutralize SARS-CoV-2 virus have been applied as complementary and alternative treatments to vaccines. Although different methodologies have been utilized to produce mAbs, traditional hybridoma fusion technology is still commonly used for this purpose due to its unmatched performance record. In this study, we coupled the hybridoma fusion strategy with mRNA-lipid nanoparticle (LNP) immunization. This time-saving approach can circumvent biological and technical hurdles, such as difficult to express membrane proteins, antigen instability, and the lack of posttranslational modifications on recombinant antigens. We used mRNA-LNP immunization and hybridoma fusion technology to generate mAbs against the receptor binding domain (RBD) of SARS-CoV-2 spike (S) protein. Compared with traditional protein-based immunization approaches, inoculation of mice with RBD mRNA-LNP induced higher titers of serum antibodies. In addition, the mAbs we obtained can bind to SARS-CoV-2 RBDs from several variants. Notably, RBD-mAb-3 displayed particularly high binding affinities and neutralizing potencies against both Alpha and Delta variants. In addition to introducing specific mAbs against SARS-CoV-2, our data generally demonstrate that mRNA-LNP immunization may be useful to quickly generate highly functional mAbs against emerging infectious diseases.

Omicron-specific mRNA vaccine induced potent neutralizing antibody against Omicron but not other SARS-CoV-2 variants (preprint)

The emerging SARS-CoV-2 variants of concern (VOC) harbor mutations associated with increasing transmission and immune escape, hence undermine the effectiveness of current COVID-19 vaccines. In late November of 2021, the Omicron (B.1.1.529) variant was identified in South Africa and rapidly spread across the globe. It was shown to exhibit significant resistance to neutralization by serum not only from convalescent patients, but also from individuals recieving currently used COVID-19 vaccines with multiple booster shots. Therefore, there is an urgent need to develop next generation vaccines against VOCs like Omicron. In this study, we develop a panel of mRNA-LNP-based vaccines using the receptor binding domain (RBD) of Omicron and Delta variants, which are dominant in the current wave of COVID-19. In addition to the Omicron- and Delta-specific vaccines, the panel also includes a Hybrid vaccine that uses the RBD containing all 16 point-mutations shown in Omicron and Delta RBD, as well as a bivalent vaccine composed of both Omicron and Delta RBD-LNP in half dose. Interestingly, both Omicron-specific and Hybrid RBD-LNP elicited extremely high titer of neutralizing antibody against Omicron itself, but few to none neutralizing antibody against other SARS-CoV-2 variants. The bivalent RBD-LNP, on the other hand, generated antibody with broadly neutralizing activity against the wild-type virus and all variants. Surprisingly, similar cross-protection was also shown by the Delta-specifc RBD-LNP. Taken together, our data demonstrated that Omicron-specific mRNA vaccine can induce potent neutralizing antibody response against Omicron, but the inclusion of epitopes from other variants may be required for eliciting cross-protection. This study would lay a foundation for rational development of the next generation vaccines against SARS-CoV-2 VOCs.

Genetic Characteristics and Phylogeny of 969-bp S Gene Sequence of SARS-CoV-2 from Hawaii Reveals the Worldwide Emerging P681H Mutation (preprint)

COVID-19 pandemic has ravaged the world, caused over 1.8 million deaths in the first year, and severely affected the global economy. Hawaii is not spared from the transmission of SARS-CoV-2 in the local population, including high infection rates in racial and ethnic minorities. Early in the pandemic, we described in this journal various technologies used for the detection of SARS-CoV-2. Herein we characterize a 969-bp SARS-CoV-2 segment of the S gene downstream of the receptor-binding domain. At the John A. Burns School of Medicine Biocontainment Facility, RNA was extracted from an oropharyngeal swab and a nasal swab from two patients from Hawaii who were infected with the SARS-CoV-2 in August 2020. Following PCR, the two viral strains were sequenced using Sanger sequencing, and phylogenetic trees were generated using MEGAX. Phylogenetic tree results indicate that the virus has been introduced to Hawaii from multiple sources. Further, we decoded 13 single nucleotide polymorphisms across 13 unique SARS-CoV-2 genomes within this region of the S gene, with one non-synonymous mutation (P681H) found in the two Hawaii strains. The P681H mutation has unique and emerging characteristics with a significant exponential increase in worldwide frequency when compared to the plateauing of the now universal D614G mutation. The P681H mutation is also characteristic of the new SARS-CoV-2 variants from the United Kingdom and Nigeria. Additionally, several mutations resulting in cysteine residues were detected, potentially resulting in disruption of the disulfide bridges in and around the receptor-binding domain. Targeted sequence characterization is warranted to determine the origin of multiple introductions of SARS-CoV-2 circulating in Hawaii.

Molecular dynamics simulations and functional studies reveal that hBD-2 binds SARS-CoV-2 spike RBD and blocks viral entry into ACE2 expressing cells (preprint)

New approaches to complement vaccination are needed to combat the spread of SARS-CoV-2 and stop COVID-19 related deaths and long-term medical complications. Human beta defensin 2 (hBD-2) is a naturally occurring epithelial cell derived host defense peptide that has antiviral properties. Our comprehensive in-silico studies demonstrate that hBD-2 binds the site on the CoV-2-RBD that docks with the ACE2 receptor. Biophysical and biochemical assays confirm that hBD-2 indeed binds to the CoV-2-receptor binding domain (RBD) (KD ~ 300 nM), preventing it from binding to ACE2 expressing cells. Importantly, hBD-2 shows specificity by blocking CoV-2/spike pseudoviral infection, but not VSV-G mediated infection, of ACE2 expressing human cells with an IC50 of 2.4+ 0.1 microM. These promising findings offer opportunities to develop hBD-2 and/or its derivatives and mimetics to safely and effectively use as novel agents to prevent SARS-CoV-2 infection.

Stable Interaction Of The UK B.1.1.7 lineage SARS-CoV-2 S1 Spike N501Y Mutant With ACE2 Revealed By Molecular Dynamics Simulation (preprint)

COVID-19 caused by SARS-CoV-2 has caused a massive health crisis across the world, and genetic variants such as the D614G gaining enhanced infectivity and competitive fitness have significantly aggravated the global concern. In this regard, the latest SARS-CoV-2 variant, B.1.1.7 lineage, reported from the United Kingdom (UK) is of great significance, in that it contains several mutations that increases its infection and transmission rates as evidenced by the increased number of clinical reports. Specifically, the N501Y mutation in the SARS-CoV-2 S1 receptor binding domain (RBD) domain has been shown to possess increased affinity for ACE2, although the basis for this not yet clear. Here, we dissect the mechanism underlying the increased affinity using molecular dynamics (MD) simulations of the available ACE2-S1-RBD complex structure (6M0J) and show a prolonged and stable interaction of the Y501 residue in the N501Y mutant S1-RBD with interfacial residues, Y41 and K353, in ACE2 as compared to the wild type S1-RBD. Additionally, we find that the N501Y mutant S1-RBD displays altered dynamics that likely aids in its enhanced interaction with ACE2. By elucidating a mechanistic basis for the increased affinity of the N501Y mutation in S1-RBD for ACE2, we believe that the results presented here will aid in developing therapeutic strategies against SARS-CoV-2 including designing drugs targeting the ACE2-S1-RBD interaction.

CpG-adjuvanted stable prefusion SARS-CoV-2 spike protein protected hamsters from SARS-CoV-2 challenge (preprint)

The COVID-19 pandemic presents an unprecedented challenge to global public health. Rapid development and deployment of safe and effective vaccines are imperative to control the pandemic. In the current study, we applied our adjuvanted stable prefusion SARS-CoV-2 spike (S-2P)-based vaccine, MVC-COV1901, to hamster models to demonstrate immunogenicity and protection from virus challenge. Golden Syrian hamsters immunized intramuscularly with two injections of 1 {micro}g or 5 {micro}g of S-2P adjuvanted with CpG 1018 and aluminum hydroxide (alum) were challenged intranasally with SARS-CoV-2. Prior to virus challenge, the vaccine induced high levels of neutralizing antibodies with 10,000-fold higher IgG level and an average of 50-fold higher pseudovirus neutralizing titers in either dose groups than vehicle or adjuvant control groups. Six days after infection, vaccinated hamsters did not display any weight loss associated with infection and had significantly reduced lung pathology and most importantly, lung viral load levels were reduced to lower than detection limit compared to unvaccinated animals. Vaccination with either 1 g or 5 g of adjuvanted S-2P produced comparable immunogenicity and protection from infection. This study builds upon our previous results to support the clinical development of MVC-COV1901 as a safe, highly immunogenic, and protective COVID-19 vaccine.

Furin Inhibitors Block SARS-CoV-2 Spike Protein Cleavage to Suppress Virus Production and Cytopathic Effects (preprint)

Development of specific antivirals is an urgent unmet need for SARS-coronavirus 2 (SARS-CoV-2) infections. This study focuses on host proteases that proteolytically activate the SARS-CoV-2 spike protein, critical for its fusion activity after binding to ACE2 receptor, as antiviral targets. We first validated cleavage at a putative furin substrate motif at SARS-CoV-2 spike by expressing it in VeroE6 cells and found prominent syncytium formation. Both cleavage and syncytium were abolished by treatment with the furin inhibitors decanoyl-RVKR-chloromethylketone (CMK) and naphthofluorescein but not by the TMPRSS2 inhibitor camostat. CMK and naphthofluorescein showed antiviral effects in SARS-CoV-2-infected cells by decreasing viral production and cytopathic effects. Further analysis revealed that, similar to camostat, CMK blocks virus entry, but it further suppresses the cleavage of spike and syncytium. Naphthofluorescein instead acts primarily by suppressing viral RNA transcription after viral entry. Therefore, furin inhibitors may become promising antivirals for prevention and treatment of SARS-CoV-2 infections.Funding: This study was supported by grants from the Ministry of Science and Technology, Taiwan (106-2622-B-002-002-CC2, 107-3017-F-002-002) and the “Center of Precision Medicine” from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan. Conflict of Interest: The authors declare no competing interests.