Mishra, R. P. | bioRxiv Science

bioRxiv2023

A broadly protective CHO cell expressed recombinant spike protein subunit based vaccine (IMT-CVAX) against SARS-CoV-2

Protective immunity induced by COVID-19 vaccines is mediated mainly by spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we report the development of a recombinant prefusion stabilized SARS-CoV-2 spike protein-subunit-based COVID-19 vaccine produced in the mammalian cell line. The gene encoding ectodomain (ECD) of the spike protein was engineered and cloned into Freedom pCHO 1.0, a mammalian expression vector, and subsequently expressed in the Chinese Hamster Ovary suspension cell line (CHO-S). The recombinant S protein ectodomain (hereafter referred to as IMT-CVAX) was purified using a combination of tangential flow filtration and liquid chromatography. Biochemical and biophysical characterization of IMT-CVAX was done to ensure its vital quality attributes. Intramuscular immunization of mice with two doses of adjuvanted IMT-CVAX elicited a strong anti-Spike IgG response. In pseudovirus-based assays, IMT-CVAX- immune mice sera exhibited a broad-spectrum neutralization of several SARS-CoV-2 variants of concern (VoCs). Golden Syrian Hamster immunized with IMT-CVAX provided excellent protection against SARS-CoV-2 infection, and, hamster immune sera neutralized the live SARS-CoV-2 virus. The adjuvanted IMT-CVAX induced robust Tfh-cells response and germinal center (GC) reaction in human ACE2 receptor-expressing transgenic mice. The findings of this study may pave the way for developing next-generation protein subunit-based vaccines to combat the existing SARS-CoV-2 and its emerging VoCs. The IMT-CVAX is produced using a scalable process and can be used for large-scale vaccine production in an industrial setup.

microbiology↗

bioRxiv2021

A Multiscale Model for Quantitative Prediction of Insulin Aggregation Nucleation Kinetics

We combined kinetic, thermodynamic, and structural information from single molecule (protein folding) and two molecule (association) explicit-solvent simulations for determination of kinetic parameters in protein aggregation nucleation with insulin as model protein. A structural bioinformatics approach was developed to account for heterogeneity of aggregation-prone species with the transition complex theory found applicable in modeling association kinetics involving non-native species. We show that a key simplification arises from presence of only a few relevant modes for non-native association kinetics. The kinetic parameters thus obtained were used in a population balance model and accurate predictions for aggregation nucleation time varying over two orders of magnitude with changes in concentration of insulin or an aggregation-inhibitor ligand were obtained while an empirical parameter set was not found to be transferable for prediction of ligand effects. This physically determined kinetic parameter set also allowed identification of the rate-limiting step in aggregation nucleation. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/431119v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@7ff3e4org.highwire.dtl.DTLVardef@653c90org.highwire.dtl.DTLVardef@6b4675org.highwire.dtl.DTLVardef@deab8a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗