Rapid isolation and resolution immune evasion and viral tness across contemporary SARS-CoV-2 variants

From late 2020 the world observed the rapid emergence of many distinct SARS-CoV-2 variants. At the same time, pandemic responses coalesced into significant global vaccine rollout that have now significantly lowered COVID-19 hospitalisation and mortality rates in the developed world. Over this period, we developed a rapid platform (R-20) for viral isolation and characterisation using primary remnant diagnostic swabs. This combined with quarantine testing and genomics surveillance, enabled the rapid isolation and characterisation of all major SARS-CoV-2 variants (all variants of concern (VOC) and six variants of interest (VUI)) globally within a four-month period. This platform facilitated viral variant isolation and enabled rapid resolution of variant phenotype by determining end-point viral titre from primary nasopharyngeal swabs and through ranking of evasion of neutralising antibodies. In late 2021, when the Delta variant was dominating, Omicron rapidly emerged. Using this platform, we isolated and tested the first cases of this variant within Australia. In this setting we observed Omicron to diverge from other variants at two levels: Firstly, it ranks as the most evasive to neutralisation antibodies compared to all VOCs and major VUIs. Secondly, it no longer engages TMPRSS2 during late stages of fusion. Introduction As of December 2021, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, has accounted for more than 250 million infections and more than 5 million deaths worldwide (https://covid19.who.int/). Within the first 12 months of the pandemic the virus accumulated polymorphisms primarily within its Spike glycoprotein that have initially driven higher levels of transmission 1-4 and significantly reduced efficacy of vaccines in preventing symptomatic disease particularly with the most immune evasive Variant of Concern (VOC) Beta . In 2021, SARS-CoV-2 accelerated its spread initially in India through the VOC Delta and this now has become the dominant variant globally, supplanting Alpha, Beta and Gamma (https://nextstrain.org/ncov/open/global). Biological mechanisms for the fitness dominance of Delta include its increased Angiotensin Converting Enzyme 2 (ACE2) affinity through 452 and 478 Spike polymorphisms , combined with the furin cleavage site polymorphism P681R that drives more efficient late-stage viral fusion in concert with the serine protease TMPRSS2 . Whilst these observations are in vitro, high viral loads sustained within the nasopharyngeal tract , a significantly higher secondary attack rate compared to the previous dominating VOC Alpha 9 and other viral variants, all suggest that these changes have significant implications on in vivo fitness. Whilst vaccines were highly efficacious against Delta, the rapid emergence of the Omicron variant in late 2021 has led to high transmission rates in both convalescent and vaccinated groups. During 2021 our aim was to develop a simple and rapid means of viral variant isolation and characterisation. To do so, we developed a series of platforms that combined firstly fast turnaround of whole genome sequencing , secondly rapid and sensitive viral isolation and finally rapid viral characterisation. The utility of our platforms was then tested stringently at several levels. Firstly, in rapid isolation of many globally relevant variants isolated from swabs with low viral loads (PCR Ct >30). Secondly, we characterised variants against a range of humoral responses and therapeutics and established relative viral fitness, quantification and characterisation to enable studies of neutralisation (or equivalent therapeutic screens) within one week of obtaining the original nasopharyngeal swab. Finally, with the recent expansion of Delta in the Australian community, we demonstrate the utility of our methods to rapidly and sensitively determine in vivo viral titres directly from nasopharyngeal swabs overnight. When the latter values are combined with diagnostic PCR, it enables real-time in vivo measurement of infectivity to particle ratio calculations (viral fitness). Following characterisation of our platform, Omicron was observed in vaccinated travellers returning to Australia in late November. In this latter setting, we rapidly isolated and tested Omicron for humoral neutralisation within one week of receiving the swab specimens and provided the first evidence of patient infectivity when Omicron was present initially in many breakthrough vaccine cases. The latter highlighted the solutions herein are valuable for the continued COVID-19 response as they enable the development of a scale of relative variant threat that combines measures of viral fitness with measurements of humoral/therapeutic evasion. Importantly, this platform can be performed at scale and in formats consistent with global accessibility and use.