A screening study on the detection strain of Coxsackievirus A6: the key to evaluating neutralizing antibodies in vaccines.

The increasing incidence of diseases caused by Coxsackievirus A6 (CV-A6) and the presence of various mutants in the population present significant public health challenges. Given the concurrent development of multiple vaccines in China, it is challenging to objectively and accurately evaluate the level of neutralizing antibody response to different vaccines. The choice of the detection strain is a crucial factor that influences the detection of neutralizing antibodies. In this study, the National Institutes for Food and Drug Control collected a prototype strain (Gdula), one subgenotype D1, as well as 13 CV-A6 candidate vaccine strains and candidate detection strains (subgenotype D3) from various institutions and manufacturers involved in research and development. We evaluated cross-neutralization activity using plasma from naturally infected adults (n = 30) and serum from rats immunized with the aforementioned CV-A6 strains. Although there were differences between the geometric mean titer (GMT) ranges of human plasma and murine sera, the overall trends were similar. A significant effect of each strain on the neutralizing antibody test (MAX/MIN 48.0 ∼16410.3) was observed. Among all strains, neutralization of the S112 strain by 15 different sera resulted in higher neutralizing antibody titers (GMTS112 = 132.0) and more consistent responses across different genotypic immune sera (MAX/MIN = 48.0). Therefore, S112 may serve as a detection strain for NtAb testing in various vaccines, minimizing bias and making it suitable for evaluating the immunogenicity of the CV-A6 vaccine.

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

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.

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.

Effect of freezing on recombinant hepatitis E vaccine.

Studies have revealed that vaccines are more often exposed to sub-zero temperatures during cold chain transportation than what was previously known. Such exposure might be detrimental to the potency of temperature-sensitive vaccines. The aim of this study was to evaluate the impact of exposure to freezing on the physicochemical properties and biological activities of recombinant hepatitis E (rHE) vaccine. Changes in rHE vaccine due to freezing temperatures were analyzed with regard to sedimentation rate, antigenicity, and antibody affinity and potency. The freezing temperature of rHE was measured, then rHE vaccine was exposed to freezing temperatures below -10°C.Significant increase of sedimentation rate was noted, according to shake test and massed precipitates. In addition, the binding affinity of rHE vaccine to six specific monoclonal antibodies was significantly reduced and the in vivo potency for eliciting a protective IgG response was also partially lost, especially for anti-HEV neutralizing antibodies. Altogether, our work indicates that exposure of rHE vaccine to a temperature below -10°C results in the loss of structural integrity and biological potency of rHE vaccine.

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.

A surrogate assay for measuring Coxsackievirus A6 neutralizing antibodies.

Coxsackievirus A6 (CV-A6) is one of pathogens causing hand, foot and mouth disease (HFMD) and becomes a new challenge to HFMD control. In this study, we first built a single-round pseudovirus infection system for CV-A6, and then developed a pseudovirus luciferase assay (PVLA) for anti-CV-A6 neutralizing antibody (NtAb) quantification. Since cytopahtic effect (CPE) is considered as the gold standard test for anti-enterovirus NtAb detection, a comparison study has been performed using 318 clinical serum samples, as measured both by PVLA and CPE. The sensitivity and specificity of PVLA was 94.9% (95% CI between 90.8-97.5%) and 92.7% (95% CI between 86.6-96.6%), respectively. Statistical analysis revealed that PVLA and CPE were highly correlated (spearman r = 0.931, P < 0.0001) and in good agreement (94.0%, 95% CI between 90.8-96.4%), showing that PVLA could be used as a surrogate assay for anti-CV-A6 NtAb detection and served as a valuable tool for CV-A6 vaccine evaluation and CV-A6 epidemiological surveillance.