Broadly neutralizing antibodies against Omicron variants of SARS-CoV-2 derived from mRNA-lipid nanoparticle-immunized mice.

The COVID-19 pandemic continues to threaten human health worldwide as new variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerge. Currently, the predominant circulating strains around the world are Omicron variants, which can evade many therapeutic antibodies. Thus, the development of new broadly neutralizing antibodies remains an urgent need. In this work, we address this need by using the mRNA-lipid nanoparticle immunization method to generate a set of Omicron-targeting monoclonal antibodies. Five of our novel K-RBD-mAbs show strong binding and neutralizing activities toward all SARS-CoV-2 variants of concern (Alpha, Beta, Gamma, Delta and Omicron). Notably, the epitopes of these five K-RBD-mAbs are overlapping and localized around Y453 and F486 of the spike protein receptor binding domain (RBD). Chimeric derivatives of the five antibodies (K-RBD-chAbs) neutralize Omicron sublineages BA.1 and BA.2 with low IC50 values ranging from 5.7 to 12.9 ng/mL. Additionally, we performed antibody humanization on broadly neutralizing chimeric antibodies to create K-RBD-hAb-60 and -62, which still retain excellent neutralizing activity against Omicron. Our results collectively suggest that these five therapeutic antibodies may effectively combat current and emerging SARS-CoV-2 variants, including Omicron BA.1 and BA.2. Therefore, the antibodies can potentially be used as universal neutralizing antibodies against SARS-CoV-2.

Monoclonal antibodies against S2 subunit of spike protein exhibit broad reactivity toward SARS-CoV-2 variants.

BACKGROUND: The variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) harbor diverse spike (S) protein sequences, which can greatly influence the efficacies of therapeutics. Therefore, it would be of great value to develop neutralizing monoclonal antibodies (mAbs) that can broadly recognize multiple variants. METHODS: Using an mRNA-LNP immunization strategy, we generated several mAbs that specifically target the conserved S2 subunit of SARS-CoV-2 (B-S2-mAbs). These mAbs were assessed for their neutralizing activity with pseudotyped viruses and binding ability for SARS-CoV-2 variants. RESULTS: Among these mAbs, five exhibited strong neutralizing ability toward the Gamma variant and also recognized viral S proteins from the Wuhan, Alpha, Beta, Gamma, Delta and Omicron (BA.1, BA.2 and BA.5) variants. Furthermore, we demonstrated the broad reactivities of these B-S2-mAbs in several different applications, including immunosorbent, immunofluorescence and immunoblotting assays. In particular, B-S2-mAb-2 exhibited potent neutralization of Gamma variant (IC50 = 0.048 µg/ml) in a pseudovirus neutralization assay. The neutralizing epitope of B-S2-mAb-2 was identified by phage display as amino acid residues 1146-1152 (DSFKEEL) in the S2 subunit HR2 domain of SARS-CoV-2. CONCLUSION: Since there are not many mAbs that can bind the S2 subunit of SARS-CoV-2 variants, our set of B-S2-mAbs may provide important materials for basic research and potential clinical applications. Importantly, our study results demonstrate that the viral S2 subunit can be targeted for the production of cross-reactive antibodies, which may be used for coronavirus detection and neutralization.

SARS-CoV-2 N protein mediates intercellular nucleic acid dispersion, a feature reduced in Omicron.

The coronavirus nucleocapsid (N) protein is known to bind to nucleic acids and facilitate viral genome encapsulation. Here we report that the N protein can mediate RNA or DNA entering neighboring cells through ACE2-independent, receptor (STEAP2)-mediated endocytosis, and achieve gene expression. The effect is more pronounced for the N protein of wild-type SARS-CoV-2 than that of the Omicron variant and other human coronaviruses. This effect is enhanced by RANTES (CCL5), a chemokine induced by N protein, and lactate, a metabolite produced in hypoxia, to cause more damage. These findings might explain the clinical observations in SARS-CoV-2-infected cases. Moreover, the N protein-mediated function can be inhibited by N protein-specific monoclonal antibodies or p38 mitogen-activated protein kinase inhibitors. Since the N-protein-mediated nucleic acid endocytosis involves a receptor commonly expressed in many types of cells, our findings suggest that N protein may have an additional role in SARS-CoV-2 pathogenesis.

An efficient approach for SARS-CoV-2 monoclonal antibody production via modified mRNA-LNP immunization.

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 and markedly increased serum neutralizing activity. 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.

A critical overview of current progress for COVID-19: development of vaccines, antiviral drugs, and therapeutic antibodies.

The novel coronavirus disease (COVID-19) pandemic remains a global public health crisis, presenting a broad range of challenges. To help address some of the main problems, the scientific community has designed vaccines, diagnostic tools and therapeutics for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The rapid pace of technology development, especially with regard to vaccines, represents a stunning and historic scientific achievement. Nevertheless, many challenges remain to be overcome, such as improving vaccine and drug treatment efficacies for emergent mutant strains of SARS-CoV-2. Outbreaks of more infectious variants continue to diminish the utility of available vaccines and drugs. Thus, the effectiveness of vaccines and drugs against the most current variants is a primary consideration in the continual analyses of clinical data that supports updated regulatory decisions. The first two vaccines granted Emergency Use Authorizations (EUAs), BNT162b2 and mRNA-1273, still show more than 60% protection efficacy against the most widespread current SARS-CoV-2 variant, Omicron. This variant carries more than 30 mutations in the spike protein, which has largely abrogated the neutralizing effects of therapeutic antibodies. Fortunately, some neutralizing antibodies and antiviral COVID-19 drugs treatments have shown continued clinical benefits. In this review, we provide a framework for understanding the ongoing development efforts for different types of vaccines and therapeutics, including small molecule and antibody drugs. The ripple effects of newly emergent variants, including updates to vaccines and drug repurposing efforts, are summarized. In addition, we summarize the clinical trials supporting the development and distribution of vaccines, small molecule drugs, and therapeutic antibodies with broad-spectrum activity against SARS-CoV-2 strains.

A booster dose of Delta × Omicron hybrid mRNA vaccine produced broadly neutralizing antibody against Omicron and other SARS-CoV-2 variants.

BACKGROUND: With the continuous emergence of new SARS-CoV-2 variants that feature increased transmission and immune escape, there is an urgent demand for a better vaccine design that will provide broader neutralizing efficacy. METHODS: We report an mRNA-based vaccine using an engineered "hybrid" receptor binding domain (RBD) that contains all 16 point-mutations shown in the currently prevailing Omicron and Delta variants. RESULTS: A booster dose of hybrid vaccine in mice previously immunized with wild-type RBD vaccine induced high titers of broadly neutralizing antibodies against all tested SARS-CoV-2 variants of concern (VOCs). In naïve mice, hybrid vaccine generated strong Omicron-specific neutralizing antibodies as well as low but significant titers against other VOCs. Hybrid vaccine also elicited CD8+/IFN-γ+ T cell responses against a conserved T cell epitope present in wild type and all VOCs. CONCLUSIONS: These results demonstrate that inclusion of different antigenic mutations from various SARS-CoV-2 variants is a feasible approach to develop cross-protective vaccines.

Antibody cocktail effective against variants of SARS-CoV-2.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an RNA virus with a high mutation rate. Importantly, several currently circulating SARS-CoV-2 variants are associated with loss of efficacy for both vaccines and neutralizing antibodies. METHODS: We analyzed the binding activity of six highly potent antibodies to the spike proteins of SARS-CoV-2 variants, assessed their neutralizing abilities with pseudovirus and authentic SARS-CoV-2 variants and evaluate efficacy of antibody cocktail in Delta SARS-CoV-2-infected hamster models as prophylactic and post-infection treatments. RESULTS: The tested RBD-chAbs, except RBD-chAb-25, maintained binding ability to spike proteins from SARS-CoV-2 variants. However, only RBD-chAb-45 and -51 retained neutralizing activities; RBD-chAb-1, -15, -25 and -28 exhibited diminished neutralization for all SARS-CoV-2 variants. Notably, several cocktails of our antibodies showed low IC50 values (3.35-27.06 ng/ml) against the SARS-CoV-2 variant pseudoviruses including United Kingdom variant B.1.1.7 (Alpha), South Africa variant B.1.351 (Beta), Brazil variant P1 (Gamma), California variant B.1.429 (Epsilon), New York variant B.1.526 (Iota), and India variants, B.1.617.1 (Kappa) and B.1.617.2 (Delta). RBD-chAb-45, and -51 showed PRNT50 values 4.93-37.54 ng/ml when used as single treatments or in combination with RBD-chAb-15 or -28, according to plaque assays with authentic Alpha, Gamma and Delta SARS-CoV-2 variants. Furthermore, the antibody cocktail of RBD-chAb-15 and -45 exhibited potent prophylactic and therapeutic effects in Delta SARS-CoV-2 variant-infected hamsters. CONCLUSIONS: The cocktail of RBD-chAbs exhibited potent neutralizing activities against SARS-CoV-2 variants. These antibody cocktails are highly promising candidate tools for controlling new SARS-CoV-2 variants, including Delta.

Monoclonal Antibodies against Nucleocapsid Protein of SARS-CoV-2 Variants for Detection of COVID-19.

Mitigation strategies of the coronavirus disease 2019 (COVID-19) pandemic have been greatly hindered by the continuous emergence of SARS-CoV-2 variants. New sensitive, rapid diagnostic tests for the wide-spectrum detection of viral variants are needed. We generated a panel of 41 monoclonal antibodies against the SARS-CoV-2 nucleocapsid protein (NP) by using mice hybridoma techniques. Of these mAbs, nine exhibited high binding activities and were applied in latex-based lateral flow immunoassays (LFIAs). The LFIAs utilizing NP-mAb-7 and -40 had the best sensitivity and lowest limit of detection: 8 pg for purified NP and 625 TCID50/mL for the authentic virus (hCoV-19/Taiwan/4/2020). The specificity tests showed that the NP-mAb-40/7 LFIA strips did not cross-react with five human coronavirus strains or 20 other common respiratory pathogens. Importantly, we found that 10 NP mutants, including alpha (B.1.1.7), beta (B.1.351), gamma (P.1), and delta (B.1.617.2) variants, could be detected by NP-mAb-40/7 LFIA strips. A clinical study (n = 60) of the NP-mAb-40/7 LFIA strips demonstrated a specificity of 100% and sensitivity of 90% in infected individuals with cycle threshold (Ct) values < 29.5. These anti-NP mAbs have strong potential for use in the clinical detection of SARS-CoV-2 infection, whether the virus is wild-type or a variant of concern.

Structure-guided antibody cocktail for prevention and treatment of COVID-19.

Development of effective therapeutics for mitigating the COVID-19 pandemic is a pressing global need. Neutralizing antibodies are known to be effective antivirals, as they can be rapidly deployed to prevent disease progression and can accelerate patient recovery without the need for fully developed host immunity. Here, we report the generation and characterization of a series of chimeric antibodies against the receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. Some of these antibodies exhibit exceptionally potent neutralization activities in vitro and in vivo, and the most potent of our antibodies target three distinct non-overlapping epitopes within the RBD. Cryo-electron microscopy analyses of two highly potent antibodies in complex with the SARS-CoV-2 spike protein suggested they may be particularly useful when combined in a cocktail therapy. The efficacy of this antibody cocktail was confirmed in SARS-CoV-2-infected mouse and hamster models as prophylactic and post-infection treatments. With the emergence of more contagious variants of SARS-CoV-2, cocktail antibody therapies hold great promise to control disease and prevent drug resistance.

Effect of SARS-CoV-2 B.1.1.7 mutations on spike protein structure and function.

The B.1.1.7 variant of SARS-CoV-2 first detected in the UK harbors amino-acid substitutions and deletions in the spike protein that potentially enhance host angiotensin conversion enzyme 2 (ACE2) receptor binding and viral immune evasion. Here we report cryo-EM structures of the spike protein of B.1.1.7 in the apo and ACE2-bound forms. The apo form showed one or two receptor-binding domains (RBDs) in the open conformation, without populating the fully closed state. All three RBDs were engaged in ACE2 binding. The B.1.1.7-specific A570D mutation introduces a molecular switch that could modulate the opening and closing of the RBD. The N501Y mutation introduces a π-π interaction that enhances RBD binding to ACE2 and abolishes binding of a potent neutralizing antibody (nAb). Cryo-EM also revealed how a cocktail of two nAbs simultaneously bind to all three RBDs, and demonstrated the potency of the nAb cocktail to neutralize different SARS-CoV-2 pseudovirus strains, including B.1.1.7.

Rapid generation of mouse model for emerging infectious disease with the case of severe COVID-19.

Since the pandemic of COVID-19 has intensely struck human society, small animal model for this infectious disease is in urgent need for basic and pharmaceutical research. Although several COVID-19 animal models have been identified, many of them show either minimal or inadequate pathophysiology after SARS-CoV-2 challenge. Here, we describe a new and versatile strategy to rapidly establish a mouse model for emerging infectious diseases in one month by multi-route, multi-serotype transduction with recombinant adeno-associated virus (AAV) vectors expressing viral receptor. In this study, the proposed approach enables profound and enduring systemic expression of SARS-CoV-2-receptor hACE2 in wild-type mice and renders them vulnerable to SARS-CoV-2 infection. Upon virus challenge, generated AAV/hACE2 mice showed pathophysiology closely mimicking the patients with severe COVID-19. The efficacy of a novel therapeutic antibody cocktail RBD-chAbs for COVID-19 was tested and confirmed by using this AAV/hACE2 mouse model, further demonstrating its successful application in drug development.

Identification of COVID-19 B-cell epitopes with phage-displayed peptide library.

BACKGROUND: Coronavirus disease 19 (COVID-19) first appeared in the city of Wuhan, in the Hubei province of China. Since its emergence, the COVID-19-causing virus, SARS-CoV-2, has been rapidly transmitted around the globe, overwhelming the medical care systems in many countries and leading to more than 3.3 million deaths. Identification of immunological epitopes on the virus would be highly useful for the development of diagnostic tools and vaccines that will be critical to limiting further spread of COVID-19. METHODS: To find disease-specific B-cell epitopes that correspond to or mimic natural epitopes, we used phage display technology to determine the targets of specific antibodies present in the sera of immune-responsive COVID-19 patients. Enzyme-linked immunosorbent assays were further applied to assess competitive antibody binding and serological detection. VaxiJen, BepiPred-2.0 and DiscoTope 2.0 were utilized for B-cell epitope prediction. PyMOL was used for protein structural analysis. RESULTS: 36 enriched peptides were identified by biopanning with antibodies from two COVID-19 patients; the peptides 4 motifs with consensus residues corresponding to two potential B-cell epitopes on SARS-CoV-2 viral proteins. The putative epitopes and hit peptides were then synthesized for validation by competitive antibody binding and serological detection. CONCLUSIONS: The identified B-cell epitopes on SARS-CoV-2 may aid investigations into COVID-19 pathogenesis and facilitate the development of epitope-based serological diagnostics and vaccines.