Contribution of low population immunity to the severe Omicron BA.2 outbreak in Hong Kong (preprint)

Monitoring population protective immunity against SARS-CoV-2 variants is critical for risk assessment. In this serosurveillance study, older adults show much lower seropositive rates of neutralizing antibody (NAb) against ancestral virus than the younger population. The increase in NAb seopositive rate generally follows the population vaccination uptake rate, but older adults have a much lower NAb seropositive rate than vaccination uptake rate. For all age groups, the seropositive rates of NAb against Omicron variant are much lower than those against the ancestral virus. During the fifth wave of COVID-19 in Hong Kong which is dominated by Omicron sublineage BA.2, the case-fatality rate is exceptionally high in the ≥80 year-old age group (9.2%). Our study suggests that the severe BA.2 outbreak in Hong Kong can be attributed by the lack of protective immunity in the population, especially among the vulnerable older adults, and highlights the importance of continual surveillance of protective immunity against emerging variants of SARS-CoV-2.

Neutralization of SARS-CoV-2 Omicron variant by sera from BNT162b2 or Coronavac vaccine recipients (preprint)

ABSTRACT Background The SARS-CoV-2 Omicron variant, designated as a Variant of Concern(VOC) by the World Health Organization, carries numerous spike protein mutations which have been found to evade neutralizing antibodies elicited by COVID-19 vaccines. The susceptibility of Omicron variant by vaccine-induced neutralizing antibodies are urgently needed for risk assessment. Methods Omicron variant strains HKU691 and HKU344-R346K were isolated from patients using TMPRSS2-overexpressing VeroE6 cells. Whole genome sequence was determined using nanopore sequencing. Neutralization susceptibility of ancestral lineage A virus and the Omicron, Delta and Beta variants to sera from 25 BNT162b2 and 25 Coronavac vaccine recipients was determined using a live virus microneutralization assay. Results The Omicron variant strain HKU344-R346K has an additional spike R346K mutation, which is present in 8.5% of strains in GISAID database. Only 20% and 24% of BNT162b2 recipients had detectable neutralizing antibody against the Omicron variant HKU691 and HKU344-R346K, respectively, while none of the Coronavac recipients had detectable neutralizing antibody titer against either Omicron isolates. For BNT162b2 recipients, the geometric mean neutralization antibody titers(GMT) of the Omicron variant isolates(5.43 and 6.42) were 35.7-39.9-fold lower than that of the ancestral virus(229.4), and the GMT of both omicron isolates were significantly lower than those of the beta and delta variants. There was no significant difference in the GMT between HKU691 and HKU344-R346K. Conclusions Omicron variant escapes neutralizing antibodies elicited by BNT162b2 or CoronaVac. The additional R346K mutation did not affect the neutralization susceptibility. Our data suggest that the Omicron variant may be associated with lower COVID-19 vaccine effectiveness.

Emerging SARS-CoV-2 variants expand species tropism to rodents (preprint)

Mice are not susceptible to wildtype SARS-CoV-2 infection. Emerging SARS-CoV-2 variants including B.1.1.7, B.1.351, P.1, and P.3 contain mutations in spike, which have been suggested to associate with an increased recognition of mouse ACE2, raising the postulation that they may have evolved to expand species tropism to rodents. Here, we investigated the capacity of B.1.1.7 and other emerging SARS-CoV-2 variants in infecting mouse (Mus musculus) and rats (Rattus norvegicus) under in vitro and in vivo settings. Our results show that B.1.1.7 and P.3, but not B.1 or wildtype SARS-CoV-2, can utilize mouse and rat ACE2 for virus entry in vitro. High infectious virus titers, abundant viral antigen expression, and pathological changes are detected in the nasal turbinate and lung of B.1.1.7-inocluated mice and rats. Together, these results reveal that the current predominant circulating SARS-CoV-2 variant, B.1.1.7, has gained the capability to expand species tropism to rodents.

A molecularly engineered, broad-spectrum anti-coronavirus lectin inhibits SARS-CoV-2 and MERS-CoV infection in vivo (preprint)

Coronaviruses have repeatedly crossed species barriers to cause epidemics1. “Pan-coronavirus” antivirals targeting conserved viral components involved in coronavirus replication, such as the extensively glycosylated spike protein, can be designed. Here we show that the rationally engineered H84T-banana lectin (H84T-BanLec), which specifically recognizes high-mannose found on viral proteins but seldom on healthy human cells2, potently inhibits the highly virulent MERS-CoV, pandemic SARS-CoV-2 and its variants, and other human-pathogenic coronaviruses at nanomolar concentrations. MERS-CoV-infected human DPP4-transgenic mice treated by H84T-BanLec have significantly higher survival, lower viral burden, and reduced pulmonary damage. Similarly, prophylactic or therapeutic H84T-BanLec is effective against SARS-CoV-2 in hamsters. Importantly, intranasally and intraperitoneally administered H84T-BanLec are comparably effective. Time-of-drug-addition assay shows that H84T-BanLec targets virus entry. Real-time structural analysis with high-speed atomic force microscopy depicts multi-molecular associations of H84T-BanLec dimers with the SARS-CoV-2 spike trimer. Single-molecule force spectroscopy demonstrates binding of H84T-BanLec to multiple SARS-CoV-2 spike mannose sites with high affinity, and that H84T-BanLec competes with SARS-CoV-2 spike for binding to cellular ACE2. Modelling experiments identify distinct high-mannose glycans in spike recognized by H84T-BanLec. The multiple H84T-BanLec binding sites on spike likely account for the activity against SARS-CoV-2 variants and the lack of resistant mutants. The broad-spectrum H84T-BanLec should be clinically evaluated in respiratory viral infections including COVID-19.

Low dose inocula of SARS-CoV-2 B.1.1.7 variant initiate more robust infections in the upper respiratory tract of hamsters than earlier D614G variants (preprint)

There is a lack of experimental evidence to explain how the B.1.1.7 variant spreads more quickly than pre-existing variants in humans. We found that B.1.1.7 displays increased competitive fitness over earlier D614G lineages in an in-vitro system. Furthermore, we demonstrated that B.1.1.7 variant is able to replicate and shed more efficiently in the nasal cavity than other variants with lower dose and shorter duration of exposure.

Neurosensory Rehabilitation and Olfactory Network Recovery in COVID-19-related Olfactory Dysfunction (preprint)

Background: Non-conductive olfactory dysfunction (OD) is an important extra-pulmonary manifestation of coronavirus disease 2019 (COVID-19). Prolonged COVID-19-related OD is a serious neurosensory disability. Treatment for the restoration of smell is urgently needed. Case presentation Two patients presenting with prolonged COVID-19-related OD underwent structural and resting-state functional magnetic resonance imaging (rs-fMRI) brain scans. Two healthy controls were recruited for radiological comparison. One patient received olfactory treatment (OT) by the combination of oral vitamin A and smell training via the novel electronic portable aromatic rehabilitation (EPAR) diffusers. After four-weeks of OT, clinical recuperation of smell was correlated with interval increase of bilateral OB volumes [right: 22.5mm 3 to 49.5mm 3 (120%), left: 37.5mm 3 to 42mm 3 (12%)] and the enhancement of mean olfactory functional connectivity [0.09 to 0.15 (66.6%)]. Conclusions: Olfactory network functional defects and OB volume loss were identified in patients presenting with prolonged COVID-19-related OD. Preliminary evidence demonstrated that the combination of oral vitamin A and smell training may induce neurogenesis at the olfactory apparatus and achieve olfactory neurosensory rehabilitation. This observation should be validated in large scale randomized–controlled trials.

SARS-CoV-2 spike D614G variant exhibits highly efficient replication and transmission in hamsters (preprint)

SARS-CoV-2 causes disease varying in severity from asymptomatic infections to severe respiratory distress and death in humans. The viral factors which determine transmissibility and pathogenicity are not yet clearly characterized. We used the hamster infection model to compare the replication ability and pathogenicity of five SARS-CoV-2 strains isolated from early cases originating in Wuhan, China, in February, and infected individuals returning from Europe and elsewhere in March 2020. The HK-13 and HK-95 isolates showed distinct pathogenicity in hamsters, with higher virus titers and more severe pathological changes in the lungs observed compared to other isolates. HK-95 contains a D614G substitution in the spike protein and demonstrated higher viral gene expression and transmission efficiency in hamsters. Intra-host diversity analysis revealed that further quasi species were generated during hamster infections, indicating that strain-specific adaptive mutants with advantages in replication and transmission will continue to arise and dominate subsequent waves of SARS-CoV-2 dissemination.

Pathogenicity, immunogenicity, and protective ability of an attenuated SARS-CoV-2 variant with a deletion at the S1/S2 junction of the spike protein (preprint)

SARS-CoV-2 is zoonotic origin and contains a PRRA polybasic cleavage motif which is considered critical for efficient infection and transmission in humans. We previously reported on a panel of attenuated SARS-CoV-2 variants with deletion at the S1/S2 junction of spike protein. Here we characterize pathogenicity, immunogenicity, and protective ability of a further cell-adapted SARS-CoV-2 variant, Ca-DelMut, in in vitro and in vivo systems. Ca-DelMut replicates more efficiently than wild type or parental virus in cells, but causes no apparent disease in hamsters, despite replicating in respiratory tissues. Unlike wild type virus, Ca-DelMut causes no apparent pathological changes and does not induce elevated proinflammatory cytokines in hamster infections, but still triggers a strong neutralizing antibody response in hamsters. Ca-DelMut immunized hamsters challenged with wild type SARS-CoV-2 are fully protected with no sign of virus replication in the upper or lower respiratory tract of challenged animals, demonstrating sterilizing immunity.

SARS-CoV-2 spike D614G variant exhibits highly efficient replication and transmission in hamsters (preprint)

SARS-CoV-2 causes disease varying in severity from asymptomatic infections to severe respiratory distress and death in humans. The viral factors which determine transmissibility and pathogenicity are not yet clearly characterized. We used the hamster infection model to compare the replication ability and pathogenicity of five SARS-CoV-2 strains isolated from early cases originating in Wuhan, China, in February, and infected individuals returning from Europe and elsewhere in March 2020. The HK-13 and HK-95 isolates showed distinct pathogenicity in hamsters, with higher virus titers and more severe pathological changes in the lungs observed compared to other isolates. HK-95 contains a D614G substitution in the spike protein and demonstrated higher viral gene expression and transmission efficiency in hamsters. Intra-host diversity analysis revealed that further quasi species were generated during hamster infections, indicating that strain-specific adaptive mutants with advantages in replication and transmission will continue to arise and dominate subsequent waves of SARS-CoV-2 dissemination.

A SARS-CoV-2 BioID-based virus-host membrane protein interactome and virus peptide compendium: new proteomics resources for COVID-19 research (preprint)

Key steps of viral replication take place at host cell membranes, but the detection of membrane-associated protein-protein interactions using standard affinity-based approaches (e.g. immunoprecipitation coupled with mass spectrometry, IP-MS) is challenging. To learn more about SARS-CoV-2 - host protein interactions that take place at membranes, we utilized a complementary technique, proximity-dependent biotin labeling (BioID). This approach uncovered a virus-host topology network comprising 3566 proximity interactions amongst 1010 host proteins, highlighting extensive virus protein crosstalk with: (i) host protein folding and modification machinery; (ii) membrane-bound vesicles and organelles, and; (iii) lipid trafficking pathways and ER-organelle membrane contact sites. The design and implementation of sensitive mass spectrometric approaches for the analysis of complex biological samples is also important for both clinical and basic research proteomics focused on the study of COVID-19. To this end, we conducted a mass spectrometry-based characterization of the SARS-CoV-2 virion and infected cell lysates, identifying 189 unique high-confidence virus tryptic peptides derived from 17 different virus proteins, to create a high quality resource for use in targeted proteomics approaches. Together, these datasets comprise a valuable resource for MS-based SARS-CoV-2 research, and identify novel virus-host protein interactions that could be targeted in COVID-19 therapeutics.

Elucidation of remdesivir cytotoxicity pathways through genome-wide CRISPR-Cas9 screening and transcriptomics (preprint)

The adenosine analogue remdesivir has emerged as a frontline antiviral treatment for SARS-CoV-2, with preliminary evidence that it reduces the duration and severity of illness1. Prior clinical studies have identified adverse events1,2, and remdesivir has been shown to inhibit mitochondrial RNA polymerase in biochemical experiments7, yet little is known about the specific genetic pathways involved in cellular remdesivir metabolism and cytotoxicity. Through genome-wide CRISPR-Cas9 screening and RNA sequencing, we show that remdesivir treatment leads to a repression of mitochondrial respiratory activity, and we identify five genes whose loss significantly reduces remdesivir cytotoxicity. In particular, we show that loss of the mitochondrial nucleoside transporter SLC29A3 mitigates remdesivir toxicity without a commensurate decrease in SARS-CoV-2 antiviral potency and that the mitochondrial adenylate kinase AK2 is a remdesivir kinase required for remdesivir efficacy and toxicity. This work elucidates the cellular mechanisms of remdesivir metabolism and provides a candidate gene target to reduce remdesivir cytotoxicity.

Pathogenicity, immunogenicity, and protective ability of an attenuated SARS-CoV-2 variant with a deletion at the S1/S2 junction of the spike protein (preprint)

SARS-CoV-2 contains a PRRA polybasic cleavage motif considered critical for efficient infection and transmission in humans. We previously reported that virus variants with spike protein S1/S2 junction deletions spanning this motif are attenuated. Here we characterize a further cell-adapted SARS-CoV-2 variant, Ca-DelMut. Ca-DelMut replicates more efficiently than wild type or parental virus in cells, but causes no apparent disease in hamsters, despite replicating in respiratory tissues. Unlike wild type virus, Ca-DelMut does not induce proinflammatory cytokines in hamster infections, but still triggers a strong neutralizing antibody response. Ca-DelMut-immunized hamsters challenged with wild type SARS-CoV-2 are fully protected, demonstrating sterilizing immunity.

Improved detection of antibody against SARS-CoV-2 by microsphere-based antibody assay (preprint)

Objective: Currently available COVID-19 antibody tests using enzyme immunoassay (EIA) or immunochromatographic assay have variable sensitivity and specificity. Here, we developed and evaluated a novel microsphere-based antibody assay (MBA) for the detection of immunoglobulin G (IgG) against SARS-CoV-2 nucleoprotein (NP) and spike protein receptor binding domain (RBD). Method: We developed a microsphere-based assay (MBA) to determine the levels of IgG against SARS-CoV-2 NP and spike RBD. The seropositive cut-off mean fluorescent intensity (MFI) was set using a cohort of 294 anonymous serum specimens collected in 2018. The specificity was assessed using serum specimens collected from organ donors or influenza patients before 2020. Seropositive rate was determined among patients with COVID-19. Time-to-seropositivity and signal-to-cutoff (S/CO) ratio were compared between MBA and EIA. Results: MBA had a specificity of 100% (93/93; 95% confidence interval [CI], 96-100%) for anti-NP IgG and 98.9% (92/93; 95% CI 94.2-100%) for anti-RBD IgG. The MBA seropositive rate for convalescent serum specimens of COVID-19 patients were 89.8% (35/39) for anti-NP IgG and 79.5% (31/39) for anti-RBD IgG. The time-to-seropositivity was shorter with MBA than that of EIA. When compared with EIA, MBA could better differentiate between COVID-19 patients and negative controls with significantly higher S/CO ratio for COVID-19 patients and lower S/CO ratio with negative controls. MBA also had fewer specimens in the equivocal range (S/CO 0.9-1.1) than EIA. Conclusion: MBA is robust and simple, and is suitable for clinical microbiology laboratory for the accurate determination of anti-SARS-CoV-2 antibody for retrospective diagnosis, serosurveillance, and vaccine trials.

Mapping the Immunodominance Landscape of SARS-CoV-2 Spike Protein for the Design of Vaccines against COVID-19 (preprint)

The ongoing coronavirus disease 2019 (COVID-19) pandemic is a serious threat to global public health, and imposes severe burdens on the entire human society. The severe acute respiratory syndrome (SARS) coronavirus-2 (SARS-CoV-2) can cause severe respiratory illness and death. Currently, there are no specific antiviral drugs that can treat COVID-19. Several vaccines against SARS-CoV-2 are being actively developed by research groups around the world. The surface S (spike) protein and the highly expressed internal N (nucleocapsid) protein of SARS-CoV-2 are widely considered as promising candidates for vaccines. In order to guide the design of an effective vaccine, we need experimental data on these potential epitope candidates. In this study, we mapped the immunodominant (ID) sites of S protein using sera samples collected from recently discharged COVID-19 patients. The SARS-CoV-2 S protein-specific antibody levels in the sera of recovered COVID-19 patients were strongly correlated with the neutralising antibody titres. We used epitope mapping to determine the landscape of ID sites of S protein, which identified nine linearized B cell ID sites. Four out of the nine ID sites were found in the receptor-binding domain (RBD). Further analysis showed that these ID sites are potential high-affinity SARS-CoV-2 antibody binding sites. Peptides containing two out of the nine sites were tested as vaccine candidates against SARS-CoV-2 in a mouse model. We detected epitope-specific antibodies and SARS-CoV-2-neutralising activity in the immunised mice. This study for the first time provides human serological data for the design of vaccines against COVID-19.

Respiratory Virus Shedding in Exhaled Breath and Efficacy of Face Masks (preprint)

There are few studies describing the presence of respiratory viruses in respiratory droplets and aerosols in the exhaled breath of infected persons, and the efficacy of facemasks as a source control to prevent respiratory virus transmission. Here, we recruited children and adults with acute respiratory illness and collected respiratory droplets and aerosols, with and without surgical facemasks. We identified human coronaviruses, influenza virus and rhinovirus from both respiratory droplets and aerosols. Surgical face masks reduced detection of coronavirus RNA in both respiratory droplets and aerosols, but only respiratory droplets and not aerosols for influenza virus RNA. Our results provide mechanistic evidence that surgical facemasks could prevent transmission of human coronavirus and influenza virus infections if worn by symptomatic individuals.Authors Donald K Milton and Benjamin J Cowling are joint senior authors.