Identification of a Conserved, Linear Epitope on VP3 of Enterovirus A Species Recognized by a Broad-Spectrum Monoclonal Antibody.

Outbreaks of hand, foot and mouth disease (HFMD) have occurred frequently in the Asian-Pacific region over the last two decades, caused mainly by the serotypes in Enterovirus A species. High-quality monoclonal antibodies (mAbs) are needed to improve the accuracy and efficiency of the diagnosis of enteroviruses associated HFMD. In this study, a mAb 1A11 was generated using full particles of CV-A5 as an immunogen. In indirect immunofluorescence and Western blotting assays, 1A11 bound to the viral proteins of CV-A2, CV-A4, CV-A5, CV-A6, CV-A10, CV-A16, and EV-A71 of the Enterovirus A and targeted VP3. It has no cross-reactivity to strains of Enterovirus B and C. By mapping with over-lapped and truncated peptides, a minimal and linear epitope 23PILPGF28 was identified, located at the N-terminus of the VP3. A BLAST sequence search of the epitope in the NCBI genus Enterovirus (taxid: 12059) protein database indicates that the epitope sequence is highly conserved among the Enterovirus A species, but not among the other enterovirus species, first reported by us. By mutagenesis analysis, critical residues for 1A11 binding were identified for most serotypes of Enterovirus A. It may be useful for the development of a cost-effective and pan-Enterovirus A antigen detection for surveillance, early diagnosis and differentiation of infections caused by the Enterovirus A species.

Construction and immunogenicity evaluation of SARS-Co V-2 self-amplifying RNA vaccine based on Coxsackievirus-A5 replicon / 中国生物制品学杂志

@#ObjectiveTo construct self-amplifying RNA(saRNA)vaccine of severe acute respiratory syndrome coronavirus2(SARS-CoV-2)Delta mutant strain(B.1.617.2)based on Coxsackievirus-A5(CV-A5)replicon and evaluate its immunogenicity.MethodsThe recombinant plasmids pDelta-S10,pDelta-S5 and pDelta-S1(10,5 and 1 amino acid residues at the upstream of S-VP1/2A cleavage site of the fusion polyprotein respectively)were constructed by In-fusion cloning of the plasmids containing the full-length genome sequence of CV-A5 and substituting the S protein gene of SARS-CoV-2 Delta mutant for the P1 structural protein gene of CV-A5 with different lengths.Three RNA molecules,Delta-S10,Delta-S5 and Delta-S1,were obtained by in vitro transcription of linearized recombinant plasmids and transfected into HEK-293T cells respectively,which were analyzed for the expression of S protein by Western blot.The RNA molecule with the highest expression of S protein was screened out and detected for the self-amplification in HEK-293T cells by qPCR.BALB/c mice(female,6 ～ 8 weeks old and five for each group)were immunized i.m.with two doses(0.5 and 2.5 μg)of the screened Delta-S packaged with lipid nanoparticles for once on day 1 and day 14 seperately.Blood samples were collected on days 14and 28,detected for serum binding antibody titers by ELISA,and detected for neutralizing antibody titers by micro neutralization method.The spleens were harvested on day 42 and detected for the level of IFNγ secreted by mouse spleen cells by enzyme linked enzyme linked immunospot assay(ELISPOT).ResultsThe recombinant RNA molecule Delta-S10showed the highest expression of S protein and self-amplified in HEK-293T cells,which of both high and low doses induced specific binding antibody against SARS-CoV-2 Delta S1 protein in mice with obvious dose effect and enhanced immune effect;The high dose of Delta-S10 induced neutralizing antibodies and cellular immune responses in mice.ConclusionThe SARS-CoV-2 Delta mutant(B.1.617.2)saRNA vaccine Delta-S10 based on CV-A5 replicon was successfully constructed,which induced humoral and cellular immune responses in mice,laying a foundation of the further study of the construction of SARS-CoV-2 saRNA vaccine by enterovirus replication elements.

Low toxicity and high immunogenicity of an inactivated vaccine candidate against COVID-19 in different animal models.

The ongoing COVID-19 pandemic is causing huge impact on health, life, and global economy, which is characterized by rapid spreading of SARS-CoV-2, high number of confirmed cases and a fatality/case rate worldwide reported by WHO. The most effective intervention measure will be to develop safe and effective vaccines to protect the population from the disease and limit the spread of the virus. An inactivated, whole virus vaccine candidate of SARS-CoV-2 has been developed by Wuhan Institute of Biological Products and Wuhan Institute of Virology. The low toxicity, immunogenicity, and immune persistence were investigated in preclinical studies using seven different species of animals. The results showed that the vaccine candidate was well tolerated and stimulated high levels of specific IgG and neutralizing antibodies. Low or no toxicity in three species of animals was also demonstrated in preclinical study of the vaccine candidate. Biochemical analysis of structural proteins and purity analysis were performed. The inactivated, whole virion vaccine was characterized with safe double-inactivation, no use of DNases and high purity. Dosages, boosting times, adjuvants, and immunization schedules were shown to be important for stimulating a strong humoral immune response in animals tested. Preliminary observation in ongoing phase I and II clinical trials of the vaccine candidate in Wuzhi County, Henan Province, showed that the vaccine is well tolerant. The results were characterized by very low proportion and low degree of side effects, high levels of neutralizing antibodies, and seroconversion. These results consistent with the results obtained from preclinical data on the safety.

Global expression profiling reveals genetic programs underlying the developmental divergence between mouse and human embryogenesis.

BACKGROUND: Mouse has served as an excellent model for studying human development and diseases due to its similarity to human. Advances in transgenic and knockout studies in mouse have dramatically strengthened the use of this model and significantly improved our understanding of gene function during development in the past few decades. More recently, global gene expression analyses have revealed novel features in early embryogenesis up to gastrulation stages and have indeed provided molecular evidence supporting the conservation in early development in human and mouse. On the other hand, little information is known about the gene regulatory networks governing the subsequent organogenesis. Importantly, mouse and human development diverges during organogenesis. For instance, the mouse embryo is born around the end of organogenesis while in human the subsequent fetal period of ongoing growth and maturation of most organs spans more than 2/3 of human embryogenesis. While two recent studies reported the gene expression profiles during human organogenesis, no global gene expression analysis had been done for mouse organogenesis. RESULTS: Here we report a detailed analysis of the global gene expression profiles from egg to the end of organogenesis in mouse. Our studies have revealed distinct temporal regulation patterns for genes belonging to different functional (Gene Ontology or GO) categories that support their roles during organogenesis. More importantly, comparative analyses identify both conserved and divergent gene regulation programs in mouse and human organogenesis, with the latter likely responsible for the developmental divergence between the two species, and further suggest a novel developmental strategy during vertebrate evolution. CONCLUSIONS: We have reported here the first genome-wide gene expression analysis of the entire mouse embryogenesis and compared the transcriptome atlas during mouse and human embryogenesis. Given our earlier observation that genes function in a given process tends to be developmentally co-regulated during organogenesis, our microarray data here should help to identify genes associated with mouse development and/or infer the developmental functions of unknown genes. In addition, our study might be useful for invesgtigating the molecular basis of vertebrate evolution.