Development of an ELISA Assay for the Determination of SARS-CoV-2 Protein Subunit Vaccine Antigen Content.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protein subunit vaccine is one of the mainstream technology platforms for the development of COVID-19 vaccines, and most R&D units use the receptor-binding domain (RBD) or spike (S) protein as the main target antigen. The complexity of vaccine design, sequence, and expression systems makes it urgent to establish common antigen assays to facilitate vaccine development. In this study, we report the development of a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) to determine the antigen content of SARS-CoV-2 protein subunit vaccines based on the United States Pharmacopeia <1220> and ICH (international conference on harmonization) Q14 and Q2 (R2) requirements. A monoclonal antibody (mAb), 20D8, was identified as the detection antibody based on its high RBD binding activity (EC50 = 8.4 ng/mL), broad-spectrum anti-variant neutralizing activity (EC50: 2.7−9.8 ng/mL for pseudovirus and EC50: 9.6−127 ng/mL for authentic virus), good in vivo protection, and a recognized linear RBD epitope (369−379 aa). A porcine anti-RBD polyclonal antibody was selected as the coating antibody. Assay performance met the requirements of the analytical target profile with an accuracy and precision of ≥90% and adequate specificity. Within the specification range of 70−143%, the method capability index was >0.96; the misjudgment probability was <0.39%. The method successfully detected SARS-CoV-2 protein subunit vaccine antigens (RBD or S protein sequences in Alpha, Beta, Gamma, or Delta variants) obtained from five different manufacturers. Thus, we present a new robust, reliable, and general method for measuring the antigenic content of SARS-CoV-2 protein subunit vaccines. In addition to currently marketed and emergency vaccines, it is suitable for vaccines in development containing antigens derived from pre-Omicron mutant strains.

Heterologous Omicron-adapted vaccine as a secondary booster promotes neutralizing antibodies against Omicron and its sub-lineages in mice.

Over one billion people have received 2-3 dosages of an inactivated COVID-19 vaccine for basic immunization. Whether a booster dose should be delivered to protect against the Omicron variant and its sub-lineages, remains controversial. Here, we tested different vaccine platforms targeting the ancestral or Omicron strain as a secondary booster of the ancestral inactivated vaccine in mice. We found that the Omicron-adapted inactivated viral vaccine promoted a neutralizing antibody response against Omicron in mice. Furthermore, heterologous immunization with COVID-19 vaccines based on different platforms remarkably elevated the levels of cross- neutralizing antibody against Omicron and its sub-lineages. Omicron-adapted vaccines based on heterologous platforms should be prioritized in future vaccination strategies to control COVID-19.

Advances of mRNA vaccine in tumor: a maze of opportunities and challenges.

High-frequency mutations in tumor genomes could be exploited as an asset for developing tumor vaccines. In recent years, with the tremendous breakthrough in genomics, intelligence algorithm, and in-depth insight of tumor immunology, it has become possible to rapidly target genomic alterations in tumor cell and rationally select vaccine targets. Among a variety of candidate vaccine platforms, the early application of mRNA was limited by instability low efficiency and excessive immunogenicity until the successful development of mRNA vaccines against SARS-COV-2 broken of technical bottleneck in vaccine preparation, allowing tumor mRNA vaccines to be prepared rapidly in an economical way with good performance of stability and efficiency. In this review, we systematically summarized the classification and characteristics of tumor antigens, the general process and methods for screening neoantigens, the strategies of vaccine preparations and advances in clinical trials, as well as presented the main challenges in the current mRNA tumor vaccine development.

Heterologous Omicron-adapted vaccine as a secondary booster promotes neutralizing antibodies against Omicron and its sub-lineages in mice.

Over one billion people have received 2-3 dosages of an inactivated COVID-19 vaccine for basic immunization. Whether a booster dose should be delivered to protect against the Omicron variant and its sub-lineages, remains controversial. Here, we tested different vaccine platforms targeting the ancestral or Omicron strain as a secondary booster of the ancestral inactivated vaccine in mice. We found that the Omicron-adapted inactivated viral vaccine promoted a neutralizing antibody response against Omicron in mice. Furthermore, heterologous immunization with COVID-19 vaccines based on different platforms remarkably elevated the levels of cross- neutralizing antibody against Omicron and its sub-lineages. Omicron-adapted vaccines based on heterologous platforms should be prioritized in future vaccination strategies to control COVID-19.

A Short 5'triphosphate RNA nCoV-L Induces a Broad-Spectrum Antiviral Response by Activating RIG-I.

Small molecular nucleic acid drugs produce antiviral effects by activating pattern recognition receptors (PRRs). In this study, a small molecular nucleotide containing 5'triphosphoric acid (5'PPP) and possessing a double-stranded structure was designed and named nCoV-L. nCoV-L was found to specifically activate RIG-I, induce interferon responses, and inhibit duplication of four RNA viruses (Human enterovirus 71, Human poliovirus 1, Human coxsackievirus B5 and Influenza A virus) in cells. In vivo, nCoV-L quickly induced interferon responses and protected BALB/c suckling mice from a lethal dose of the enterovirus 71. Additionally, prophylactic administration of nCoV-L was found to reduce mouse death and relieve morbidity symptoms in a K18-hACE2 mouse lethal model of SARS-CoV-2. In summary, these findings indicate that nCoV-L activates RIG-I and quickly induces effective antiviral signals. Thus, it has potential as a broad-spectrum antiviral drug.

B.1.351 SARS-CoV-2 Variant Exhibits Higher Virulence but Less Viral Shedding than That of the Ancestral Strain in Young Nonhuman Primates.

We investigated the distribution, virulence, and pathogenic characteristics of mutated SARS-CoV-2 to clarify the association between virulence and the viral spreading ability of current and future circulating strains. Chinese rhesus macaques were infected with ancestral SARS-CoV-2 strain GD108 and Beta variant B.1.351 (B.1.351) and assessed for clinical signs, viral distribution, pathological changes, and pulmonary inflammation. We found that GD108 replicated more efficiently in the upper respiratory tract, whereas B.1.351 replicated more efficiently in the lower respiratory tract and lung tissue, implying a reduced viral shedding and spreading ability of B.1.351 compared with that of GD108. Importantly, B.1.351 caused more severe lung injury and dramatically elevated the level of inflammatory cytokines compared with those observed after infection with GD108. Moreover, both B.1.351 and GD108 induced spike-specific T-cell responses at an early stage of infection, with higher levels of interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) in the B.1.351 group and higher levels of interleukin 17 (IL-17) in the GD108 group, indicating a divergent pattern in the T-cell-mediated inflammatory "cytokine storm." This study provides a basis for exploring the pathogenesis of SARS-CoV-2 variants of concern (VOCs) and establishes an applicable animal model for evaluating the efficacy and safety of vaccines and drugs. IMPORTANCE One of the priorities of the current SARS-CoV-2 vaccine and drug research strategy is to determine the changes in transmission ability, virulence, and pathogenic characteristics of SARS-CoV-2 variants. In addition, nonhuman primates (NHPs) are suitable animal models for the study of the pathogenic characteristics of SARS-CoV-2 and could contribute to the understanding of pathogenicity and transmission mechanisms. As SARS-CoV-2 variants continually emerge and the viral biological characteristics change frequently, the establishment of NHP infection models for different VOCs is urgently needed. In the study, the virulence and tissue distribution of B.1.351 and GD108 were comprehensively studied in NHPs. We concluded that the B.1.351 strain was more virulent but exhibited less viral shedding than the latter. This study provides a basis for determining the pathogenic characteristics of SARS-CoV-2 and establishes an applicable animal model for evaluating the efficacy and safety of vaccines and drugs.

How SARS-CoV-2 dodges immune surveillance and facilitates infection: an analytical review.

INTRODUCTION: Effective treatments for the ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic are limited. The virus has evolved strategies to evade the immune system or hijack immune responses to facilitate infection and escape immune surveillance. Mechanistically, SARS-CoV-2 takes advantage of TLR4 and cytokine-induced integrins to promote its entrance into the cell. Furthermore, the activation of pattern recognition receptors (PRR)-mediated signaling pathways is compromised by SARS-CoV-2 non-structural proteins (NSPs), accessory protein open reading frames (ORFs), and structural proteins upon infection, contributing to viral infection and replication. Host factors necessary for cellular protein synthesis, metabolism, and viral replication can also be inhibited by the SARS-CoV-2 proteins. Exploring specific mechanisms would optimize the therapy methods and benefit drug research and development. AREAS COVERED: We describe pathways and mechanisms by which SARS-CoV-2 evades immune system; these include the mechanisms that operate during virus entry, signaling pathways involved, and processes at RNA and protein levels. EXPERT OPINION: Increased understanding of how viruses interfere with immune responses would provide more evidence for drug development. Drugs targeting conserved viral proteins to inhibit their replication or host factors to enhance immune responses would minimize the impact of virus mutations and prepare for future coronavirus outbreaks.

Research progress on vaccine efficacy against SARS-CoV-2 variants of concern.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to circulate worldwide and a variety of variants have emerged. Variants of concern (VOC) designated by the World Health Organization (WHO) have triggered epidemic waves due to their strong infectivity or pathogenicity and potential immune escape, among other reasons. Although large-scale vaccination campaigns undertaken globally have contributed to the improved control of SARS-CoV-2, the efficacies of current vaccines against VOCs have declined to various degrees. In particular, the highly infectious Delta and Omicron variants have caused recent epidemics and prompted concerns about control measures. This review summarizes current VOCs, the protective efficacy of vaccines against VOCs, and the shortcomings in methods for evaluating vaccine efficacy. In addition, strategies for responding to variants are proposed for future epidemic prevention and control as well as for vaccine research and development.