Development of a pseudovirus-based assay for measuring neutralizing antibodies against Coxsackievirus A10

Coxsackievirus A10 (CV-A10) has recently emerged as a major pathogen of hand, foot, and mouth disease in children worldwide. Currently no effective treatments are available; development of anti-CV-A10 vaccine is a most cost-effective way for CV-A10 prevention. Robust assay to measure neutralizing antibody (NtAb) titres elicited by vaccination would greatly prompt anti-CV-A10 vaccine development. Compare to the traditional neutralization assay based on inhibition of cytopathic effects (herein after referred to as cNT) which is time-consuming and labor-intensive, in this study we developed an efficient high-throughput neutralization antibody assay based on CV-A10 pseudoviruses (herein after referred to as pNT). In the pNT, anti-CV-A10 NtAb titre was negatively corresponded with the relative luminescent unit (RLU) produced by luciferase reporter gene incorporated in pseudovirus genome. As described in this study, the NtAb against CV-A10 could be detected within 10–16 h, anti- CV-A10 NtAb in 67 human serum samples were measured in parallel with pNT and cNT assays, a good correlation (r = 0.83,p < .0001) and good agreement(97%) were shown between cNT and pNT, indicating that the pNT provides a rapid and convenient procedure for measuring NtAb production against anti-CV-A10 NtAb measurement

Mixed formulation of mRNA and protein‐based COVID‐19 vaccines triggered superior neutralizing antibody responses

Abstract Integrating different types of vaccines into a singular immunization regimen is an effective and accessible approach to strengthen and broaden the immunogenicity of existing coronavirus disease 2019 (COVID‐19) vaccine candidates. To optimize the immunization strategy of the novel mRNA‐based vaccine and recombinant protein subunit vaccine that attracted much attention in COVID‐19 vaccine development, we evaluated the immunogenicity of different combined regimens with the mRNA vaccine (RNA‐RBD) and protein subunit vaccine (PS‐RBD) in mice. Compared with homologous immunization of RNA‐RBD or PS‐RBD, heterologous prime‐boost strategies for mRNA and protein subunit vaccines failed to simultaneously enhance neutralizing antibody (NAb) and Th1 cellular response in this study, showing modestly higher serum neutralizing activity and antibody‐dependent cell‐mediated cytotoxicity for “PS‐RBD prime, RNA‐RBD boost” and robust Th1 type cellular response for “RNA‐RBD prime, PS‐RBD boost”. Interestingly, immunizing the mice with the mixed formulation of the two aforementioned vaccines in various proportions further significantly enhanced the NAb responses against ancestral, Delta, and Omicron strains and manifested increased Th1‐type responses, suggesting that a mixed formulation of mRNA and protein vaccines might be a more prospective vaccination strategy. This study provides basic research data on the combined vaccination strategies of mRNA and protein‐based COVID‐19 vaccines

A neonatal mouse model of central nervous system infections caused by Coxsackievirus B5

Abstract As one of the key members of the coxsackievirus B group, coxsackievirus B5 (CV-B5) can cause many central nervous system diseases, such as viral encephalitis, aseptic meningitis, and acute flaccid paralysis. Notably, epidemiological data indicate that outbreaks of CV-B5-associated central nervous system (CNS) diseases have been reported worldwide throughout history. In this study, which was conducted to promote CV-B5 vaccine and anti-virus drug research, a 3-day-old BALB/c mouse model was established using a CV-B5 clinical isolate (CV-B5/JS417) as the challenge strain. Mice challenged with CV-B5/JS417 exhibited a series of neural clinical symptoms and death with necrosis of neuronal cells in the cerebral cortex and the entire spinal cord, hindlimb muscles, and cardiomyocytes. The viral load of each tissue at various post-challenge time points suggested that CV-B5 replicated in the small intestine and was subsequently transmitted to various organs via viremia; the virus potentially entered the brain through the spinal axons, causing neuronal cell necrosis. In addition, this mouse model was used to evaluate the protective effect of a CV-B5 vaccine. The results indicated that both the inactivated CV-B5 vaccine and anti-CVB5 serum significantly protected mice from a lethal infection of CV-B5/JS417 by producing neutralizing antibodies. In summary, the first CV-B5 neonatal mouse model has been established and can sustain CNS infections in a manner similar to that observed in humans. This model will be a useful tool for studies on pathogenesis, vaccines, and anti-viral drug evaluations

A uniform quantitative enzyme-linked immunosorbent assay for Coxsackievirus A16 antigen in vaccine

Coxsackievirus A16 (CV-A16), one of major etiological agents of hand, foot and mouth disease (HFMD), causes outbreaks of the disease in young children all over the world. In order to promote the prevention and control of HFMD, the research and development of CV-A16 vaccine have been carried out in China. However, due to lacking of a recognized CV-A16 antigen detection method, the evaluation and quality control (QC) of vaccine effectiveness are greatly limited. In this study, we established a quantitative enzyme-linked immunosorbent assay (Q-ELISA) to determine the antigen concentration in CV-A16 vaccines that can be applied in manufacturing in China. A neutralizing antibody 16E1 was used as a capture antibody that can bind to various CV-A16 antigens of different subgenotypes, and an antiserum from CV-A16-immunized rabbit conjugated by HRP was suitable for detecting and quantifying CV-A16 antigens. The Q-ELISA was validated for specificity, linearity, accuracy, precision and robustness by using the CV-A16 antigen national standard (NS). Furthermore, we utilized the Q-ELISA to quantify antigen contents of vaccine bulks from six manufacturers and other intermediate products from one manufacturer. The results indicated that the Q-ELISA can satisfy the requirements of QC for all manufacturers involved

A Novel Single-Stranded RNA-Based Adjuvant Improves the Immunogenicity of the SARS-CoV-2 Recombinant Protein Vaccine

The research and development (R&D) of novel adjuvants is an effective measure for improving the immunogenicity of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) recombinant protein vaccine. Toward this end, we designed a novel single-stranded RNA-based adjuvant, L2, from the SARS-CoV-2 prototype genome. L2 could initiate retinoic acid-inducible gene-I signaling pathways to effectively activate the innate immunity. ZF2001, an aluminum hydroxide (Al) adjuvanted SARS-CoV-2 recombinant receptor binding domain (RBD) subunit vaccine with emergency use authorization in China, was used for comparison. L2, with adjuvant compatibility with RBD, elevated the antibody response to a level more than that achieved with Al, CpG 7909, or poly(I:C) as adjuvants in mice. L2 plus Al with composite adjuvant compatibility with RBD markedly improved the immunogenicity of ZF2001; in particular, neutralizing antibody titers increased by about 44-fold for Omicron, and the combination also induced higher levels of antibodies than CpG 7909/poly(I:C) plus Al in mice. Moreover, L2 and L2 plus Al effectively improved the Th1 immune response, rather than the Th2 immune response. Taken together, L2, used as an adjuvant, enhanced the immune response of the SARS-CoV-2 recombinant RBD protein vaccine in mice. These findings should provide a basis for the R&D of novel RNA-based adjuvants

A potential therapeutic neutralization monoclonal antibody specifically against multi-coxsackievirus A16 strains challenge

Coxsackievirus A16 (CA16) has caused worldwide epidemics of hand, foot and mouth disease (HFMD), particularly in infants and pre-school children. Currently, there are no vaccines or antiviral drugs available for CA16-associated disease. In this study, a CA16-specific monoclonal antibody (MAb) NA11F12 was derived with an epidemic CA16 strain (GenBank no. JX127258). NA11F12 was found to have high cross-neutralization activity against different CA16 subgenotypes but not EV71 using RD cells. The neutralizing titers of NA11F12 ranged from 1:1024 to 1:12288 against A, B1, B2 and C subgenotypes of CA16 and was less than 8 against EV71 strain. In the neonatal mouse model, a single treatment of NA11F12 showed effective protection with a dose- and time-dependent relationship against lethal challenge by CA16 strain (GenBank no. JX481738). At day 1 post-infection, administering more than 0.1 μg/g of NA11F12 could protect 100% newborn mice from mobility and mortality challenged by CA16. With dose of 10 μg/g of NA11F12, a single administration fully protected mice against CA16-associated disease within 4 days post-infection. And there were 80% and 60% mice protected by administering NA11F12 at day 5 post-infection and day 6 post-infection when the control mice had shown clinical symptoms for 1- and 2-day, respectively. Immunohistochemical and histological analysis confirmed that NA11F12 significantly prohibited CA16 VP1 expression in various tissues and prevented CA16-induced necrosis. In conclusion, a CA16-specific MAb NA11F12 with high cross-neutralization activity was identified, which could effectively protect lethal CA16 challenge in mice. It could be a potential therapeutic MAb against CA16 in the future

Cross-Antigenicity between EV71 Sub-Genotypes: Implications for Vaccine Efficacy

Enterovirus A-71 (EV71) is a global, highly contagkkious pathogen responsible for severe cases of hand-food-mouth-disease (HFMD). The use of vaccines eliciting cross neutralizing antibodies (NTAbs) against the different circulating EV71 sub-genotypes is important for preventing HFMD outbreaks. Here, we tested the cross-neutralizing activities induced by EV71 genotype/sub-genotype A, B0-B4, C1, C2, C4, and C5 viruses using rats. Differences were noted in the cross-neutralization of the 10 sub-genotypes tested but there were generally good levels of cross-neutralization except against genotype A virus, against which neutralization antibody titres (NTAb) where the lowest with NTAbs being the highest against sub-genotype B4. Moreover, NTAb responses induced by C4, B4, C1, and C2 viruses were homogenous, with values of maximum/minimum NTAb ratios (MAX/MIN) against all B and C viruses ranging between 4.0 and 6.0, whereas MAX/MIN values against B3 and A viruses were highly variable, 48.0 and 256.0, respectively. We then dissected the cross-neutralizing ability of sera from infants and children and rats immunized with C4 EV71 vaccines. Cross-neutralizing titers against the 10 sub-genotypes were good in both vaccinated infants and children and rats with the MAX/MIN ranging from 1.8–3.4 and 5.1–7.1, respectively, which were similar to those found in naturally infected patients (2.8). Therefore, we conclude that C4 EV71 vaccines can provide global protection to infants and children against HFMD caused by different sub-genotypes

Evaluation of the cross-neutralization activities elicited by Coxsackievirus A10 vaccine strains

Increased severity of diseases caused by Coxsackievirus A10 (CV-A10) as well as a large number of mutants and recombinants circulating in the population are a cause of concern for public health. A vaccine with broad-spectrum and homogenous protective capacity is needed to prevent outbreaks of CV-A10. Here, we evaluated cross-neutralization of prototype strain and 17 CV-A10 strains from related manufacturers in mainland China in vitro using 30 samples of plasma collected from naturally infected human adults and 18 sera samples from murine immunized with the above strains of CV-A10. Both human plasma and murine sera exhibited varying degrees of cross-neutralizing activities. Prototype A/Kowalik and sub-genotype C3/S113 were most difficult to neutralize. Among all strains tested, neutralization of S102 and S108 strains by 18 different sera was the most uniform, suggesting their suitability for detection of NtAb titers of different vaccines for avoiding biases introduced by detection strain. Furthermore, among all immune-sera, cross-neutralization of the 18 strains of CV-A10 by anti-S110 and anti-S102 was the most homogenous. Anti-S102 exhibiting higher geometric mean titer (GMT) in vitro was evaluated for its cross-protection capacity in vivo. Remarkably, administration of anti-S102 protected mice from lethal dosage of eight strains of CV-A10. These results provide a framework for formulating strategies for the R&D of vaccines targeting CV-A10 infections

Non-small cell lung cancers (NSCLCs) oncolysis using coxsackievirus B5 and synergistic DNA-damage response inhibitors

Abstract With the continuous in-depth study of the interaction mechanism between viruses and hosts, the virus has become a promising tool in cancer treatment. In fact, many oncolytic viruses with selectivity and effectiveness have been used in cancer therapy. Human enterovirus is one of the most convenient sources to generate oncolytic viruses, however, the high seroprevalence of some enteroviruses limits its application which urges to exploit more oncolytic enteroviruses. In this study, coxsackievirus B5/Faulkner (CV-B5/F) was screened for its potential oncolytic effect against non-small cell lung cancers (NSCLCs) through inducing apoptosis and autophagy. For refractory NSCLCs, DNA-dependent protein kinase (DNA-PK) or ataxia telangiectasia mutated protein (ATM) inhibitors can synergize with CV-B5/F to promote refractory cell death. Here, we showed that viral infection triggered endoplasmic reticulum (ER) stress-related pro-apoptosis and autophagy signals, whereas repair for double-stranded DNA breaks (DSBs) contributed to cell survival which can be antagonized by inhibitor-induced cell death, manifesting exacerbated DSBs, apoptosis, and autophagy. Mechanistically, PERK pathway was activated by the combination of CV-B5/F and inhibitor, and the irreversible ER stress-induced exacerbated cell death. Furthermore, the degradation of activated STING by ERphagy promoted viral replication. Meanwhile, no treatment-related deaths due to CV-B5/F and/or inhibitors occurred. Conclusively, our study identifies an oncolytic CV-B5/F and the synergistic effects of inhibitors of DNA-PK or ATM, which is a potential therapy for NSCLCs