Redeveloping antigen detection kits for the diagnosis of rat hepatitis E virus.

The emergence of Rocahepevirus ratti [species HEV ratti (r HEV)] as a causative agent of hepatitis E in humans presents a new potential threat to global public health. The R. ratti genotype 1 (r-1 HEV) variant only shares 50%-60% genomic identity with Paslahepevirus balayani [species HEV balayani (b HEV)] variants, which are the main causes of hepatitis E infection in humans. Here, we report antigen diagnoses for r-1 HEV and b HEV using an enzymatic immunoassay (EIA) method. We detected recombinant virus-like particles protein (HEV 239) of r HEV and b HEV using a collection of hepatitis E virus (HEV)-specific monoclonal antibodies. Two optimal candidates, the capture antibody P#1-H4 and the detection antibodies C145 (P#1-H4*/C145#) and C158 (P#1-H4*/C158#), were selected to detect antigen in infected rat samples and r-1 HEV- or b HEV-infected human clinical samples. The two candidates showed similar diagnostic efficacy to the Wantai HEV antigen kit in b HEV-infected clinical samples. Genomic divergence resulted in low diagnostic efficacy of the Wantai HEV antigen kit (0%, 0 of 10) for detecting r-1 HEV infection. Compared with the P#1-H4*/C145# candidate (80%, 8 of 10), the P#1-H4*/C158# candidate had excellent diagnostic efficacy in r-1 HEV-infected clinical samples (100%, 10 of 10). The two candidates bind to a discrete antigenic site that is highly conserved across r HEV and b HEV. P#1-H4*/C145# and P#1-H4*/C158# are efficacious candidate antibody combinations for rat HEV antigen detection.

Case Report: Chronic hepatitis E virus Infection in an individual without evidence for immune deficiency.

Chronic hepatitis E virus (HEV) infection occurs mainly in immunosuppressed populations. We describe an investigation of chronic HEV infection of genotype 3a in an individual without evidence for immune deficiency who presented hepatitis with significant HEV viremia and viral shedding. We monitored HEV RNA in plasma and stools, and assessed anti-HEV specific immune responses. The patient was without apparent immunodeficiency based on quantified results of white blood cell, lymphocyte, neutrophilic granulocyte, CD3+ T cell, CD4+ T cell, and CD8+ T cell counts and CD4/CD8 ratio, as well as total serum IgG, IgM, and IgA, which were in the normal range. Despite HEV specific cellular response and strong humoral immunity being observed, viral shedding persisted up to 109 IU/mL. After treatment with ribavirin combined with interferon, the indicators of liver function in the patient returned to normal, accompanied by complete suppression and clearance of HEV. These results indicate that HEV chronicity can also occur in individuals without evidence of immunodeficiency.

A Secreted Form of the Hepatitis E Virus ORF2 Protein: Design Strategy, Antigenicity and Immunogenicity.

Hepatitis E virus (HEV) is an important public health burden worldwide, causing approximately 20 million infections and 70,000 deaths annually. The viral capsid protein is encoded by open reading frame 2 (ORF2) of the HEV genome. Most ORF2 protein present in body fluids is the glycosylated secreted form of the protein (ORF2S). A recent study suggested that ORF2S is not necessary for the HEV life cycle. A previously reported efficient HEV cell culture system can be used to understand the origin and life cycle of ORF2S but is not sufficient for functional research. A more rapid and productive method for yielding ORF2S could help to study its antigenicity and immunogenicity. In this study, the ORF2S (tPA) expression construct was designed as a candidate tool. A set of representative anti-HEV monoclonal antibodies was further used to map the functional antigenic sites in the candidates. ORF2S (tPA) was used to study antigenicity and immunogenicity. Indirect ELISA revealed that ORF2S (tPA) was not antigenically identical to HEV 239 antigen (p239). The ORF2S-specific antibodies were successfully induced in one-dose-vaccinated BALB/c mice. The ORF2S-specific antibody response was detected in plasma from HEV-infected patients. Recombinant ORF2S (tPA) can act as a decoy to against B cells. Altogether, our study presents a design strategy for ORF2S expression and indicates that ORF2S (tPA) can be used for functional and structural studies of the HEV life cycle.

Potential of conserved antigenic sites in development of universal SARS-like coronavirus vaccines.

Given pandemic risks of zoonotic SARS-CoV-2 variants and other SARS-like coronaviruses in the future, it is valuable to perform studies on conserved antigenic sites to design universal SARS-like coronavirus vaccines. By using antibodies obtained from convalescent COVID-19 patients, we succeeded in functional comparison of conserved antigenic sites at multiple aspects with each other, and even with SARS-CoV-2 unique antigenic sites, which promotes the cognition of process of humoral immune response to the conserved antigenic sites. The conserved antigenic sites between SARS-CoV-2 and SARS-CoV can effectively induce affinity maturation of cross-binding antibodies, finally resulting in broadly neutralizing antibodies against multiple variants of concern, which provides an important basis for universal vaccine design, however they are subdominant, putatively due to their lower accessibility relative to SARS-CoV-2 unique antigenic sites. Furthermore, we preliminarily design RBDs to improve the immunogenicity of these conserved antigenic sites. Our study focusing on conserved antigenic sites provides insights for promoting the development of universal SARS-like coronavirus vaccines, thereby enhancing our pandemic preparedness.

Potential of antibody pair targeting conserved antigenic sites in diagnosis of SARS-CoV-2 variants infection.

Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has become disaster for human society. As the pandemic becomes more regular, we should develop more rapid and accurate detection methods to achieve early diagnosis and treatment. Antigen detection methods based on spike protein has great potential, however, it has not been effectively developed, probably due to the torturing conformational complexity. By utilizing cross-blocking data, we clustered SARS-CoV-2 receptor binding domain (RBD)-specific monoclonal antibodies (mAbs) into 6 clusters. Subsequently, the antigenic sites for representative mAbs were identified by RBDs with designed residue substitutions. The sensitivity and specificity of selected antibody pairs was demonstrated using serial diluted samples of SARS-CoV-2 S protein and SARS-CoV S protein. Furthermore, pseudovirus system was constructed to determine the detection capability against SARS-CoV-2 and SARS-CoV. 6 RBD-specific mAbs, recognizing different antigenic sites, were identified as potential candidates for optimal antibody pairs for detection of SARS-CoV-2 S protein. By considering relative spatial position, accessibility and conservation of corresponding antigenic sites, affinity and the presence of competitive antibodies in clinical samples, 6H7-6G3 was rationally identified as optimal antibody pair for detection of both SARS-CoV-2 and SARS-CoV. Furthermore, our results showed that 6H7 and 6G3 effectively bind to SARS-CoV-2 variants of concern (VOCs). Taken together, we identified 6H7-6G3 antibody pair as a promising rapid antigen diagnostic tool in containing COVID-19 pandemic caused by multiple VOCs. Moreover, our results also provide an important reference in screening of antibody pairs detecting antigens with complex conformation.