Tarigan, R. | Search bioRxiv

bioRxiv2024

Optimization of Soluble Expression of CTA1-(S14P5)4-DD and CTA1-(S21P2)4-DD Fusion Proteins as Candidates for COVID-19 Intranasal Vaccines

Developing intranasal vaccines against pandemics and devastating airborne infectious diseases is imperative. The superiority of intranasal vaccines over injectable systemic vaccines is evident, but the challenge in developing effective intranasal vaccines is more substantial. Fusing a protein antigen with the catalytic domain of cholera toxin (CTA1) and the two-domain D of staphylococcal protein A (DD) has significant potential for intranasal vaccines. In the present study, we constructed two fusion proteins containing CTA1, tandem repeat linear epitopes of the SARS-CoV-2 spike protein (S14P5 or S21P2), and DD. The in silico characteristics and solubility of the fusion proteins CTA1-(S14P5)4-DD and CTA1-(S21P2)4-DD were analyzed when overexpressed in Escherichia coli. Structural predictions indicated that each component of the fusion proteins was compatible with its origin. Both fusion proteins were predicted by computational tools to be soluble when overexpressed in E. coli. Contrary to these predictions, the constructs exhibited limited solubility. The solubility did not improve even after lowering the cultivation temperature from 37{degrees}C to 18{degrees}C. Induction with IPTG at the early log phase, instead of the usual mid-log phase growth, significantly increased soluble CTA1-(S21P2)4-DD but not CTA1-(S14P5)4-DD. The solubility of overexpressed fusion proteins significantly increased when a non-denaturing detergent (Nonidet P40, Triton X100, or Tween 20) was added to the extraction buffer. In a scale-up purification experiment, the yields were low, only 1-2 mg/L of culture, due to substantial losses during the purification stages, indicating the need for further optimization of the purification process.

immunology↗

bioRxiv2020

Heritable endogenization of an RNA virus in a mammalian species

Viruses are considered one of the driving forces for genome rearrangements via infection and endogenization into the host genome (Kazazian, 2004; Maksakova et al., 2006). In 2010, Horie and colleagues demonstrated that a non-retroviral RNA virus, Borna disease virus (BDV), could integrate into the genome in cultured somatic cells (Horie et al., 2010). However, germline transmission of viral-derived sequences using animal models is yet to be experimentally demonstrated. In this study, we reported a case of heritable endogenization using the encephalomyocarditis virus (EMCV) and laboratory mice. The EMCV is a small non-enveloped single-strand RNA virus without its own reverse transcriptase activity (Carocci et al., 2012). Here, we demonstrated that the EMCV genomic RNA was reverse transcribed into DNA fragments in the murine testes. The DNA sequence originated from the RNA genome of EMCV was also detected in the liver and earlobes of the offspring generated from the EMCV-infected father. This suggests that the exogenous sequence derived from the EMCV is transmitted into the host germline and inherited across subsequent generations. This first experimental demonstration of viral endogenization proposes reconsideration about the impact of viruses as a driving force for genome modification.

genetics↗