Rapid assembly of SARS-CoV-2 genomes reveals attenuation of the Omicron BA.1 variant through NSP6.

Although the SARS-CoV-2 Omicron variant (BA.1) spread rapidly across the world and effectively evaded immune responses, its viral fitness in cell and animal models was reduced. The precise nature of this attenuation remains unknown as generating replication-competent viral genomes is challenging because of the length of the viral genome (~30 kb). Here, we present a plasmid-based viral genome assembly and rescue strategy (pGLUE) that constructs complete infectious viruses or noninfectious subgenomic replicons in a single ligation reaction with >80% efficiency. Fully sequenced replicons and infectious viral stocks can be generated in 1 and 3 weeks, respectively. By testing a series of naturally occurring viruses as well as Delta-Omicron chimeric replicons, we show that Omicron nonstructural protein 6 harbors critical attenuating mutations, which dampen viral RNA replication and reduce lipid droplet consumption. Thus, pGLUE overcomes remaining barriers to broadly study SARS-CoV-2 replication and reveals deficits in nonstructural protein function underlying Omicron attenuation.

SARS-CoV-2 infection of airway organoids reveals conserved use of Tetraspanin-8 by Ancestral, Delta, and Omicron variants.

Ancestral SARS coronavirus-2 (SARS-CoV-2) and variants of concern (VOC) caused a global pandemic with a spectrum of disease severity. The mechanistic explaining variations related to airway epithelium are relatively understudied. Here, we biobanked airway organoids (AO) by preserving stem cell function. We optimized viral infection with H1N1/PR8 and comprehensively characterized epithelial responses to SARS-CoV-2 infection in phenotypically stable AO from 20 different subjects. We discovered Tetraspanin-8 (TSPAN8) as a facilitator of SARS-CoV-2 infection. TSPAN8 facilitates SARS-CoV-2 infection rates independently of ACE2-Spike interaction. In head-to-head comparisons with Ancestral SARS-CoV-2, Delta and Omicron VOC displayed lower overall infection rates of AO but triggered changes in epithelial response. All variants shared highest tropism for ciliated and goblet cells. TSPAN8-blocking antibodies diminish SARS-CoV-2 infection and may spur novel avenues for COVID-19 therapy.

Omicron mutations enhance infectivity and reduce antibody neutralization of SARS-CoV-2 virus-like particles.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant contains extensive sequence changes relative to the earlier-arising B.1, B.1.1, and Delta SARS-CoV-2 variants that have unknown effects on viral infectivity and response to existing vaccines. Using SARS-CoV-2 virus-like particles (VLPs), we examined mutations in all four structural proteins and found that Omicron and Delta showed 4.6-fold higher luciferase delivery overall relative to the ancestral B.1 lineage, a property conferred mostly by enhancements in the S and N proteins, while mutations in M and E were mostly detrimental to assembly. Thirty-eight antisera samples from individuals vaccinated with Pfizer/BioNTech, Moderna, or Johnson & Johnson vaccines and convalescent sera from unvaccinated COVID-19 survivors had 15-fold lower efficacy to prevent cell transduction by VLPs containing the Omicron mutations relative to the ancestral B.1 spike protein. A third dose of Pfizer vaccine elicited substantially higher neutralization titers against Omicron, resulting in detectable neutralizing antibodies in eight out of eight subjects compared to one out of eight preboosting. Furthermore, the monoclonal antibody therapeutics casirivimab and imdevimab had robust neutralization activity against B.1 and Delta VLPs but no detectable neutralization of Omicron VLPs, while newly authorized bebtelovimab maintained robust neutralization across variants. Our results suggest that Omicron has similar assembly efficiency and cell entry compared to Delta and that its rapid spread is due mostly to reduced neutralization in sera from previously vaccinated subjects. In addition, most currently available monoclonal antibodies will not be useful in treating Omicron-infected patients with the exception of bebtelovimab.

Viral E protein neutralizes BET protein-mediated post-entry antagonism of SARS-CoV-2.

Inhibitors of bromodomain and extraterminal domain (BET) proteins are possible anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) prophylactics as they downregulate angiotensin-converting enzyme 2 (ACE2). Here we show that BET proteins should not be inactivated therapeutically because they are critical antiviral factors at the post-entry level. Depletion of BRD3 or BRD4 in cells overexpressing ACE2 exacerbates SARS-CoV-2 infection; the same is observed when cells with endogenous ACE2 expression are treated with BET inhibitors during infection and not before. Viral replication and mortality are also enhanced in BET inhibitor-treated mice overexpressing ACE2. BET inactivation suppresses interferon production induced by SARS-CoV-2, a process phenocopied by the envelope (E) protein previously identified as a possible "histone mimetic." E protein, in an acetylated form, directly binds the second bromodomain of BRD4. Our data support a model where SARS-CoV-2 E protein evolved to antagonize interferon responses via BET protein inhibition; this neutralization should not be further enhanced with BET inhibitor treatment.

Limited cross-variant immunity from SARS-CoV-2 Omicron without vaccination.

SARS-CoV-2 Delta and Omicron are globally relevant variants of concern. Although individuals infected with Delta are at risk of developing severe lung disease, infection with Omicron often causes milder symptoms, especially in vaccinated individuals1,2. The question arises of whether widespread Omicron infections could lead to future cross-variant protection, accelerating the end of the pandemic. Here we show that without vaccination, infection with Omicron induces a limited humoral immune response in mice and humans. Sera from mice overexpressing the human ACE2 receptor and infected with Omicron neutralize only Omicron, but not other variants of concern, whereas broader cross-variant neutralization was observed after WA1 and Delta infections. Unlike WA1 and Delta, Omicron replicates to low levels in the lungs and brains of infected animals, leading to mild disease with reduced expression of pro-inflammatory cytokines and diminished activation of lung-resident T cells. Sera from individuals who were unvaccinated and infected with Omicron show the same limited neutralization of only Omicron itself. By contrast, Omicron breakthrough infections induce overall higher neutralization titres against all variants of concern. Our results demonstrate that Omicron infection enhances pre-existing immunity elicited by vaccines but, on its own, may not confer broad protection against non-Omicron variants in unvaccinated individuals.

Neutralizing antibody activity against SARS-CoV-2 variants in gestational age-matched mother-infant dyads after infection or vaccination.

Pregnancy confers unique immune responses to infection and vaccination across gestation. To date, there are limited data comparing vaccine- and infection-induced neutralizing Abs (nAbs) against COVID-19 variants in mothers during pregnancy. We analyzed paired maternal and cord plasma samples from 60 pregnant individuals. Thirty women vaccinated with mRNA vaccines (from December 2020 through August 2021) were matched with 30 naturally infected women (from March 2020 through January 2021) by gestational age of exposure. Neutralization activity against the 5 SARS-CoV-2 spike sequences was measured by a SARS-CoV-2-pseudotyped spike virion assay. Effective nAbs against SARS-CoV-2 were present in maternal and cord plasma after both infection and vaccination. Compared with WT spike protein, these nAbs were less effective against the Delta and Mu spike variants. Vaccination during the third trimester induced higher cord-nAb levels at delivery than did infection during the third trimester. In contrast, vaccine-induced nAb levels were lower at the time of delivery compared with infection during the first trimester. The transfer ratio (cord nAb level divided by maternal nAb level) was greatest in mothers vaccinated in the second trimester. SARS-CoV-2 vaccination or infection in pregnancy elicits effective nAbs with differing neutralization kinetics that are influenced by gestational time of exposure.

Neutralizing immunity in vaccine breakthrough infections from the SARS-CoV-2 Omicron and Delta variants.

Virus-like particle (VLP) and live virus assays were used to investigate neutralizing immunity against Delta and Omicron SARS-CoV-2 variants in 259 samples from 128 vaccinated individuals. Following Delta breakthrough infection, titers against WT rose 57-fold and 3.1-fold compared with uninfected boosted and unboosted individuals, respectively, versus only a 5.8-fold increase and 3.1-fold decrease for Omicron breakthrough infection. Among immunocompetent, unboosted patients, Delta breakthrough infections induced 10.8-fold higher titers against WT compared with Omicron (p = 0.037). Decreased antibody responses in Omicron breakthrough infections relative to Delta were potentially related to a higher proportion of asymptomatic or mild breakthrough infections (55.0% versus 28.6%, respectively), which exhibited 12.3-fold lower titers against WT compared with moderate to severe infections (p = 0.020). Following either Delta or Omicron breakthrough infection, limited variant-specific cross-neutralizing immunity was observed. These results suggest that Omicron breakthrough infections are less immunogenic than Delta, thus providing reduced protection against reinfection or infection from future variants.

Rapid assessment of SARS-CoV-2-evolved variants using virus-like particles.

Efforts to determine why new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants demonstrate improved fitness have been limited to analyzing mutations in the spike (S) protein with the use of S-pseudotyped particles. In this study, we show that SARS-CoV-2 virus-like particles (SC2-VLPs) can package and deliver exogenous transcripts, enabling analysis of mutations within all structural proteins and at multiple steps in the viral life cycle. In SC2-VLPs, four nucleocapsid (N) mutations found universally in more-transmissible variants independently increased messenger RNA delivery and expression ~10-fold, and in a reverse genetics model, the serine-202→arginine (S202R) and arginine-203→methionine (R203M) mutations each produced >50 times as much virus. SC2-VLPs provide a platform for rapid testing of viral variants outside of a biosafety level 3 setting and demonstrate N mutations and particle assembly to be mechanisms that could explain the increased spread of variants, including B.1.617.2 (Delta, which contains the R203M mutation).

Transmission, infectivity, and neutralization of a spike L452R SARS-CoV-2 variant.

We identified an emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant by viral whole-genome sequencing of 2,172 nasal/nasopharyngeal swab samples from 44 counties in California, a state in the western United States. Named B.1.427/B.1.429 to denote its two lineages, the variant emerged in May 2020 and increased from 0% to >50% of sequenced cases from September 2020 to January 2021, showing 18.6%-24% increased transmissibility relative to wild-type circulating strains. The variant carries three mutations in the spike protein, including an L452R substitution. We found 2-fold increased B.1.427/B.1.429 viral shedding in vivo and increased L452R pseudovirus infection of cell cultures and lung organoids, albeit decreased relative to pseudoviruses carrying the N501Y mutation common to variants B.1.1.7, B.1.351, and P.1. Antibody neutralization assays revealed 4.0- to 6.7-fold and 2.0-fold decreases in neutralizing titers from convalescent patients and vaccine recipients, respectively. The increased prevalence of a more transmissible variant in California exhibiting decreased antibody neutralization warrants further investigation.