Distinct phenotype of SARS-CoV-2 Omicron BA.1 in human primary cells but no increased host range in cell lines of putative mammalian reservoir species.

SARS-CoV-2's genetic plasticity has led to several variants of concern (VOCs). Here we studied replicative capacity for seven SARS-CoV-2 isolates (B.1, Alpha, Beta, Gamma, Delta, Zeta, and Omicron BA.1) in primary reconstituted airway epithelia (HAE) and lung-derived cell lines. Furthermore, to investigate the host range of Delta and Omicron compared to ancestral SARS-CoV-2, we assessed replication in 17 cell lines from 11 non-primate mammalian species, including bats, rodents, insectivores and carnivores. Only Omicron's phenotype differed in vitro, with rapid but short replication and efficient production of infectious virus in nasal HAEs, in contrast to other VOCs, but not in lung cell lines. No increased infection efficiency for other species was observed, but Delta and Omicron infection efficiency was increased in A549 cells. Notably replication in A549 and Calu3 cells was lower than in nasal HAE. Our results suggest better adaptation of VOCs towards humans, without an extended host range, and may be relevant to the search for the putative intermediate host and reservoirs prior to the pandemic.

SARS-CoV-2 convalescence and hybrid immunity elicits mucosal immune responses.

BACKGROUND: Mucosal antibodies play a key role in the protection against SARS-CoV-2 infection in the upper respiratory tract, and potentially in limiting virus replication and therefore onward transmission. While systemic immunity to SARS-CoV-2 is well understood, we have a limited understanding about the antibodies present on the nasal mucosal surfaces. METHODS: In this study, we evaluated SARS-CoV-2 mucosal antibodies following previous infection, vaccination, or a combination of both. Paired nasal fluid and serum samples were collected from 143 individuals, which include convalescent, vaccinated, or breakthrough infections. FINDINGS: We detected a high correlation between IgG responses in serum and nasal fluids, which were higher in both compartments in vaccinated compared to convalescent participants. Contrary, nasal and systemic SARS-CoV-2 IgA responses were weakly correlated, indicating a compartmentalization between the local and systemic IgA responses. SARS-CoV-2 secretory component IgA (s-IgA) antibodies, present exclusively on mucosal surfaces, were detected in the nasal fluid only in a minority of vaccinated subjects and were significantly higher in previously infected individuals. Depletion of IgA antibodies in nasal fluids resulted in a tremendous reduction of neutralization activity against SARS-CoV-2, indicating that IgA is the crucial contributor to neutralization in the nasal mucosa. Neutralization against SARS-CoV-2 was higher in the mucosa of subjects with previous SARS-CoV-2 infections compared to vaccinated participants. INTERPRETATION: In summary, we demonstrate that currently available vaccines elicit strong systemic antibody responses, but SARS-CoV-2 infection generates higher titers of binding and neutralizing mucosal antibodies. Our results support the importance to develop SARS-CoV-2 vaccines that elicit mucosal antibodies. FUNDING: The work was funded by the COVID-19 National Research Program 78 (grant number 198412) of the Swiss National Science Foundation.

Analytical Sensitivity of Eight Different SARS-CoV-2 Antigen-Detecting Rapid Tests for Omicron-BA.1 Variant.

The emergence of each novel SARS-CoV-2 variant of concern (VOC) requires investigation of its potential impact on the performance of diagnostic tests in use, including antigen-detecting rapid diagnostic tests (Ag-RDTs). Although anecdotal reports have been circulating that the newly emerged Omicron-BA.1 variant is in principle detectable by Ag-RDTs, few data on sensitivity are available. We have performed (i) analytical sensitivity testing with cultured virus in eight Ag-RDTs and (ii) retrospective testing in duplicates with clinical samples from vaccinated individuals with Omicron-BA.1 (n = 59) or Delta (n = 54) breakthrough infection on seven Ag-RDTs. Overall, in our analytical study we have found heterogenicity between Ag-RDTs for detecting Omicron-BA.1. When using cultured virus, we observed a trend toward lower endpoint sensitivity for Omicron-BA.1 detection than for earlier circulating SARS-CoV-2 and the other VOCs. In our retrospective study, the detection of Delta and Omicron-BA.1 was assessed in a comparable set of stored clinical samples using seven Ag-RDTs. Four hundred ninety-seven of all 826 tests (60.17%) performed on Omicron-BA.1 samples were positive, compared to 489/756 (64.68%) for Delta samples. In the analytical study, the sensitivity for both Omicron-BA.1 and Delta between the Ag-RDTs was variable. All seven Ag-RDTs showed comparable sensitivities to detect Omicron-BA.1 and Delta in the retrospective study. IMPORTANCE Sensitivity for detecting Omicron-BA.1 shows high heterogenicity between Ag-RDTs, necessitating a careful consideration when using these tests to guide infection prevention measures. Analytical and retrospective testing is a proxy and timely solution to generate rapid performance data, but it is not a replacement for clinical evaluations, which are urgently needed. Biological and technical reasons for detection failure by some Ag-RDTs need to be further investigated.

Neutralization capacity of antibodies elicited through homologous or heterologous infection or vaccination against SARS-CoV-2 VOCs.

Emerging SARS-CoV-2 variants raise questions about escape from previous immunity. As the population immunity to SARS-CoV-2 has become more complex due to prior infections with different variants, vaccinations or the combination of both, understanding the antigenic relationship between variants is needed. Here, we have assessed neutralizing capacity of 120 blood specimens from convalescent individuals infected with ancestral SARS-CoV-2, Alpha, Beta, Gamma or Delta, double vaccinated individuals and patients after breakthrough infections with Delta or Omicron-BA.1. Neutralization against seven authentic SARS-CoV-2 isolates (B.1, Alpha, Beta, Gamma, Delta, Zeta and Omicron-BA.1) determined by plaque-reduction neutralization assay allowed us to map the antigenic relationship of SARS-CoV-2 variants. Highest neutralization titers were observed against the homologous variant. Antigenic cartography identified Zeta and Omicron-BA.1 as separate antigenic clusters. Substantial immune escape in vaccinated individuals was detected for Omicron-BA.1 but not Zeta. Combined infection/vaccination derived immunity results in less Omicron-BA.1 immune escape. Last, breakthrough infections with Omicron-BA.1 lead to broadly neutralizing sera.

Infectious viral load in unvaccinated and vaccinated individuals infected with ancestral, Delta or Omicron SARS-CoV-2.

Infectious viral load (VL) expelled as droplets and aerosols by infected individuals partly determines transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). RNA VL measured by qRT-PCR is only a weak proxy for infectiousness. Studies on the kinetics of infectious VL are important to understand the mechanisms behind the different transmissibility of SARS-CoV-2 variants and the effect of vaccination on transmission, which allows guidance of public health measures. In this study, we quantified infectious VL in individuals infected with SARS-CoV-2 during the first five symptomatic days by in vitro culturability assay in unvaccinated or vaccinated individuals infected with pre-variant of concern (pre-VOC) SARS-CoV-2, Delta or Omicron BA.1. Unvaccinated individuals infected with pre-VOC SARS-CoV-2 had lower infectious VL than Delta-infected unvaccinated individuals. Full vaccination (defined as >2 weeks after receipt of the second dose during the primary vaccination series) significantly reduced infectious VL for Delta breakthrough cases compared to unvaccinated individuals. For Omicron BA.1 breakthrough cases, reduced infectious VL was observed only in boosted but not in fully vaccinated individuals compared to unvaccinated individuals. In addition, infectious VL was lower in fully vaccinated Omicron BA.1-infected individuals compared to fully vaccinated Delta-infected individuals, suggesting that mechanisms other than increased infectious VL contribute to the high infectiousness of SARS-CoV-2 Omicron BA.1. Our findings indicate that vaccines may lower transmission risk and, therefore, have a public health benefit beyond the individual protection from severe disease.

Enhanced fitness of SARS-CoV-2 variant of concern Alpha but not Beta.

Emerging variants of concern (VOCs) are driving the COVID-19 pandemic1,2. Experimental assessments of replication and transmission of major VOCs and progenitors are needed to understand the mechanisms of replication and transmission of VOCs3. Here we show that the spike protein (S) from Alpha (also known as B.1.1.7) and Beta (B.1.351) VOCs had a greater affinity towards the human angiotensin-converting enzyme 2 (ACE2) receptor than that of the progenitor variant S(D614G) in vitro. Progenitor variant virus expressing S(D614G) (wt-S614G) and the Alpha variant showed similar replication kinetics in human nasal airway epithelial cultures, whereas the Beta variant was outcompeted by both. In vivo, competition experiments showed a clear fitness advantage of Alpha over wt-S614G in ferrets and two mouse models-the substitutions in S were major drivers of the fitness advantage. In hamsters, which support high viral replication levels, Alpha and wt-S614G showed similar fitness. By contrast, Beta was outcompeted by Alpha and wt-S614G in hamsters and in mice expressing human ACE2. Our study highlights the importance of using multiple models to characterize fitness of VOCs and demonstrates that Alpha is adapted for replication in the upper respiratory tract and shows enhanced transmission in vivo in restrictive models, whereas Beta does not overcome Alpha or wt-S614G in naive animals.

Dual display: phage selection driven by co-engagement of two targets by two different antibody fragments.

Antibody phage display technology has supported the emergence of numerous therapeutic antibodies. The development of bispecific antibodies, a promising new frontier in antibody therapy, could be facilitated by new phage display approaches that enable pairs of antibodies to be co-selected based on co-engagement of their respective targets. We describe such an approach, making use of two complementary leucine zipper domains that heterodimerize with high affinity. Phagemids encoding a first antibody fragment (scFv) fused to phage coat protein via the first leucine zipper are rescued in bacteria expressing a second scFv fused to the second leucine zipper as a soluble periplasmic protein, so that it is acquired by phage during assembly. Using a soluble scFv specific for a human CD3-derived peptide, we show that its acquisition by phage displaying an irrelevant antibody is sufficiently robust to drive selection of rare phage (1 in 10(5)) over three rounds of panning. We then set up a model selection experiment using a cell line expressing the chemokine receptor CCR5 fused to the CD3 peptide together with a panel of phage clones capable displaying either an anti-CCR5 scFv or an irrelevant antibody, with or without the capacity to acquire the soluble anti-CD3 scFv. In this experiment we showed that rare phage (1 in 10(5)) capable of displaying the two different scFvs can be specifically enriched over four rounds of panning. This approach has the potential to be applied to the identification of pairs of ligands capable of co-engaging two different user-defined targets, which would facilitate the discovery of novel bispecific antibodies.