Biological characterization of cold-adapted SARS-CoV-2 variants

Abstract

Introduction. The emergence of new epidemiologically significant variants of SARS-CoV-2 has shifted emphasis to development of a live vaccine, which would be able to provide protection against a wide range of antigenic variants of the virus. The aim of the study was to obtain SARS-CoV-2 variants attenuated through cold adaptation and to provide their biological characterization.Materials and methods. The Dubrovka laboratory strain of SARS-CoV-2 and its variants were cultured on Vero and Calu-3 cells. The virus quantification was performed by virus titration in Vero cells and by real-time reverse transcription-polymerase chain reaction. SARS-CoV-2 virions were analyzed using transmission electron microscopy. Genome sequences of the virus were identified by nanopore sequencing. The attenuation (att) phenotype of SARS-CoV-2 variants was identified using Syrian hamsters as an animal model for COVID-19. Results. Cold-adapted (ca) SARS-CoV-2 variants – Dubrovka-ca-B4 and Dubrovka-ca-D2 were produced by continued passaging of the Dubrovka strain in the Vero cell culture at the temperature being gradually decreased to 23ºC and by subsequent cloning. Up to 20 nucleotide substitutions and 18 amino acid substitutions were detected in genomes of ca-variants. Ca-variants, as distinct from the parent Dubrovka strain, actively replicated at 23ºC, while the Dubrovka-ca-D2 variant had a temperature-sensitive (ts) phenotype (did not replicate at 39ºC). Ca-variants of the virus replicated poorly at 37ºC in the Calu-3 human lung cell culture, which, along with the ts-phenotype, can be a marker of virus attenuation for humans. In the intranasally infected Syrian hamsters, ca-variants of the virus demonstrated an attenuation phenotype: they did not cause loss of appetite, fatigue, drowsiness, did not slow down weight gain, replicating much more slowly in the lungs and brain compared to the virulent Dubrovka strain. Conclusion. The obtained attenuated SARS-CoV-2 ca-variants, Dubrovka-ca-B4 and Dubrovka-ca-D2, should be studied further as candidate vaccine strains for a live attenuated vaccine against COVID-19.

Cold-adapted SARS-CoV-2 variants with different temperature sensitivity exhibit an attenuated phenotype and confer protective immunity

“…Previously, we obtained D-37, D-ca, D-B4, and D-D2 variants of SARS-CoV-2 by long-term passaging of the Dubrovka strain in Vero cells [26] ( Supplementary Table S1, Fig. S1 ).…”

“…The D strain (GenBank number MW514307.1, clade GR according to the GISAID classification, line B.1.1.317 according to the Pangolin classification), phylogenetically close to strain Wuhan-Hu-1 (GenBank number NC_045512.2), was obtained and characterized in summer 2020 in Moscow (Russia) by isolation in Vero cells from a clinical sample of a patient with COVID-19 [25] . Variants of D strain, D-37 and D-ca, were obtained by propagating of the D strain in Vero cells for 42 passages [26] . The D-37 variant was adapted to a constant cultivation temperature of 37⁰C.…”

“…The D-37 variant was adapted to a constant cultivation temperature of 37⁰C. The D-ca variant (cold-adapted) was gradually adapted from 37⁰C to 23⁰C according to the following scheme: 10 passages at a temperature of 37°C, then the cultivation temperature was lowered by 1°C every two passages, the final 6 passages were carried out at a temperature of 23°C (a total of 42 passages) [26] . D-B4 and D-D2 variants were generated by three-fold cloning of the D-ca variant at 23⁰C by limiting dilution method ( Supplementary Fig.…”

“…The SARS-CoV-2 virus titers were determined by the cytopathic effect endpoint method (CPE) in Vero cells as described earlier [26] . The virus titer was calculated as described by Ramakrishnan M.A.…”

Single intranasal immunization with attenuated Wuhan-like SARS-CoV-2 provides highly effective cross-protection against Delta and Omicron VOCs

“…An alternative solution would be to create a "universal" vaccine with cross-protective activity against different antigenic variants of the virus. One possible approach to solving this problem is to develop a live attenuated vaccine (LAV) capable of activating not only the humoral but also the cellular immune system, providing a prolonged immune response and cross-protection against different virus variants ( Earlier, by long-term passaging of Wuhan-like SARS-CoV-2 Dubrovka strain in Vero cells at lower temperature (up to 23 C), we obtained its cold-adapted (ca) variant called D-D2 strain (Faizuloev et al, 2022). D-D2 strain exhibited a temperature-sensitive (ts) phenotype (it did not reproduce at 39 C), which determined its reduced reproductive ability in the lungs of golden Syrian hamsters and, thus, an attenuative (att) phenotype.…”