ABSTRACT
Self-amplifying RNA replicons are promising platforms for vaccine generation. Their defects in one or more essential functions for viral replication, particle assembly, or dissemination make them highly safe as vaccines. We previously showed that the deletion of the envelope (E) gene from the Middle East respiratory syndrome coronavirus (MERS-CoV) produces a replication-competent propagation-defective RNA replicon (MERS-CoV-ΔE). Evaluation of this replicon in mice expressing human dipeptidyl peptidase 4, the virus receptor, showed that the single deletion of the E gene generated an attenuated mutant. The combined deletion of the E gene with accessory open reading frames (ORFs) 3, 4a, 4b, and 5 resulted in a highly attenuated propagation-defective RNA replicon (MERS-CoV-Δ[3,4a,4b,5,E]). This RNA replicon induced sterilizing immunity in mice after challenge with a lethal dose of a virulent MERS-CoV, as no histopathological damage or infectious virus was detected in the lungs of challenged mice. The four mutants lacking the E gene were genetically stable, did not recombine with the E gene provided in trans during their passage in cell culture, and showed a propagation-defective phenotype in vivo. In addition, immunization with MERS-CoV-Δ[3,4a,4b,5,E] induced significant levels of neutralizing antibodies, indicating that MERS-CoV RNA replicons are highly safe and promising vaccine candidates.
Subject(s)
Coronavirus Infections/prevention & control , Middle East Respiratory Syndrome Coronavirus/genetics , Middle East Respiratory Syndrome Coronavirus/immunology , RNA, Viral/administration & dosage , Replicon , Viral Vaccines/administration & dosage , Animals , Antibodies, Neutralizing/biosynthesis , Antibodies, Viral/biosynthesis , Coronavirus Infections/genetics , Coronavirus Infections/immunology , Coronavirus Infections/virology , Defective Viruses/genetics , Defective Viruses/immunology , Female , Gene Deletion , Genes, env , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Middle East Respiratory Syndrome Coronavirus/pathogenicity , RNA, Viral/genetics , RNA, Viral/immunology , Vaccines, DNA , Vaccines, Virus-Like Particle/administration & dosage , Vaccines, Virus-Like Particle/genetics , Vaccines, Virus-Like Particle/immunology , Viral Vaccines/genetics , Viral Vaccines/immunology , Virulence/genetics , Virulence/immunologyABSTRACT
We describe the timely adaption of both published WHO E-gene protocol and commercially available LightMix Modular E-gene assay to the test platform (ABI 7900 Fast real-time analyzer and TaqMan Fast One-step Virus Master Mix) available in an accredited tertiary hospital laboratory with an on-going evaluation to ensure the provision of quality service within the time constraint. The LightMix Modular E-gene was slightly more sensitive when compared to the WHO E-gene, both analytically and diagnostically. The assay was recommended for screening of SARS-CoV-2 infection. With the availability of technically competent staff through continuous training, the provision of round-the-clock service is feasible despite the test is of high complexity. The thermal cycling duration of the adapted LightMix E-gene and WHO E-gene is shortened by half and one hour respectively and allows the number of runs to double when 24-h round-the-clock service is provided. An increase in testing capacity could support surges in testing demand, which is essential to control the current SARS-CoV-2 pandemic, to prevent potential overwhelming of the healthcare system, and to optimize utilization of the isolation beds.