Production of Sars-Cov-2 Membrane Protein as Better Target Protein for Diagnostic Application

Intro: The COVID-19 pandemic is caused by the SARS-CoV-2 virus, an enveloped RNA of the coronavirus family. The advancement in molecular technology and biochemistry has accelerated the development of diagnostic reagents and assays. Much attention has been focused on the S protein, but the high mutation rate in this region could lead to false negative results. Thus, a better target protein for diagnostic application is needed for accurate detection. Method(s): Nucleotide sequences encoded for membrane (M) glycoprotein gene region of SARS-CoV-2 from Malaysian isolates were extracted from GISAID, aligned, and selected accordingly. The DNA plasmid was commercially synthesized with codon optimization for Escherichia coli (E. coli), and the presence of the M gene was confirmed by PCR. The plasmid was then transformed into E. coli. Later, the expression of M glycoprotein was induced, separated on an SDS-PAGE gel, and transferred onto a nitrocellulose membrane, followed by immunostaining. Finding(s): The analysis of the M glycoprotein against the Omicron strains demonstrated that the amino acid is conserved (99.5%). The M glycoprotein was successfully expressed and detected with antibodies from SARS-CoV-2 infected patients at ~26 kDa. The protein is currently upscale for the generation of monoclonal Ab (Mab). Discussion(s): The M protein of SARS-CoV-2 is more conserved among the virus and also has been reported to confer antigenic properties. Selection of M protein perhaps a better option compared to current detection assays that use spike (S) protein, which could lead to false negative results, as this gene region particularly the ribosome-binding domain (RBD) rapidly undergoes mutations. The utilization of M protein potentially improves negative predictive value (NPV) of the diagnostic test. Conclusion(s): Further development of diagnostic reagents is needed to improve the assay's specificity. The newly developed M protein and the MAb can be used to generate a more accurate viral detection assay.Copyright © 2023

Strengthening the implementation of potentially infectious materials, poliovirus regulations in poliovirus surveillance and research programs

Introduction: Malaysia's National Polio Laboratory (NPL) has been appointed as the National Poliovirus Containment Coordinator (NPCC) in 2000 to minimize the number of facilities retaining potentially infectious materials, poliovirus (PIM). PIM is defined as respiratory, faecal, or environmental sewage samples, and derivatives such as permissible poliovirus cell culture, Oral Polio Vaccine (OPV), collected for any purpose before 31 December 2020. However, all monitoring activities were disrupted by the COVID-19 pandemic in early 2020. In addition, Polio outbreaks occurred in Sabah and WP Labuan at the same time which leads to the large-scale use of OPV to respond to widespread vaccine-derived poliovirus (VDPV) outbreaks, it could generate new PIM, which requires an update in the NPCC record database. Material(s) and Method(s): The main objective of the study is to ensure compliance of all country states to the requirement of the Global Polio Eradication Initiative (GPEI) by WHO by providing a database and evidence of action. As per the WHO Global Action Plan III (GAP III) guidelines, meetings and site visits were conducted with the government and non-government bodies that are keeping and handling PIM. In 2019, 588 survey forms were sent to 141 government hospitals, 6 government agencies (veterinary, fishery, chemical, research institutions), 19 major private laboratories, 209 private hospitals, and 16 public universities. Result(s): Out of the 588 facilities that received the survey forms, only 177 facilities have completed the survey. 173 facilities declared that they did not store any PIM, whilst 4 institutions had informed that they had completed inventories for their PIM storage and had discarded the identified materials accordingly. The other 411 facilities have informed the NPCC that they did not have any PIM and are still completing the survey forms. Conclusion(s): The NPCC actively adhered to the recommendation by GAP III guidelines for the containment activities by conducting national surveys, laboratory inspections, consultations, and inventories of records. Based on the findings, most of the laboratories that responded to the survey did not keep any PIM and follow the procedures as per the guideline. Continuous monitoring of PIM is required although the Polio outbreak was declared ended on 9th September 2021 in Malaysia. This study will be a reference for future planning by the ministry and authorities with regards of the Biosafety and Biosecurity regulations involving laboratories working with infectious materials.

Age, Disease Severity and Ethnicity Influence Humoral Responses in a Multi-Ethnic COVID-19 Cohort

The COVID-19 pandemic has affected all individuals across the globe in some way. Despite large numbers of reported seroprevalence studies, there remains a limited understanding of how the magnitude and epitope utilization of the humoral immune response to SARS-CoV-2 viral anti-gens varies within populations following natural infection. Here, we designed a quantitative, multi-epitope protein microarray comprising various nucleocapsid protein structural motifs, including two structural domains and three intrinsically disordered regions. Quantitative data from the microarray provided complete differentiation between cases and pre-pandemic controls (100% sensitivity and specificity) in a case-control cohort (n = 100). We then assessed the influence of disease severity, age, and ethnicity on the strength and breadth of the humoral response in a multi-ethnic cohort (n = 138). As expected, patients with severe disease showed significantly higher antibody titers and interestingly also had significantly broader epitope coverage. A significant increase in antibody titer and epitope coverage was observed with increasing age, in both mild and severe disease, which is promising for vaccine efficacy in older individuals. Additionally, we observed significant differences in the breadth and strength of the humoral immune response in relation to ethnicity, which may reflect differences in genetic and lifestyle factors. Furthermore, our data enabled localization of the immuno-dominant epitope to the C-terminal structural domain of the viral nucleocapsid protein in two independent cohorts. Overall, we have designed, validated, and tested an advanced serological assay that enables accurate quantitation of the humoral response post natural infection and that has revealed unexpected differences in the magnitude and epitope utilization within a population.