Persistence of an infectious form of SARS-CoV-2 post protease inhibitor treatment of permissive cells in vitro (preprint)

Reports have described SARS-CoV-2 rebound in COVID-19 patients treated with nirmatrelvir, a 3CL protease inhibitor. The cause remains a mystery, although drug resistance, re-infection, and lack of adequate immune responses have been excluded. We now present virologic findings that provide a clue to the cause of viral rebound, which occurs in [~]20% of the treated cases. The persistence of an intermediary form of infectious SARS-CoV-2 was experimentally documented in vitro after treatment with nirmatrelvir or another 3CL protease inhibitor, but not with a polymerase inhibitor, remdesivir. This infectious intermediate decayed slowly with a half-life of [~]1 day, suggesting that its persistence could outlive the treatment course to re-ignited SARS-CoV-2 infection as the drug is eliminated. Additional studies are needed to define the nature of this viral intermediate, but our findings point to a particular direction for future investigation and offer a specific treatment recommendation that should be tested clinically.

Antibodies that neutralize all current SARS-CoV-2 variants of concern by conformational locking (preprint)

SARS-CoV-2 continues to evolve and evade most existing neutralizing antibodies, including all clinically authorized antibodies. We have isolated and characterized two human monoclonal antibodies, 12-16 and 12-19, which exhibited neutralizing activities against all SARS-CoV-2 variants tested, including BQ.1.1 and XBB.1.5. They also blocked infection in hamsters challenged with Omicron BA.1 intranasally. Structural analyses revealed both antibodies targeted a conserved quaternary epitope located at the interface between the N-terminal domain and subdomain 1, revealing a previously unrecognized site of vulnerability on SARS-CoV-2 spike. These antibodies prevent viral receptor engagement by locking the receptor-binding domain of spike in the down conformation, revealing a novel mechanism of virus neutralization for non-RBD antibodies. Deep mutational scanning showed that SARS-CoV-2 could mutate to escape 12-19, but the responsible mutations are rarely found in circulating viruses. Antibodies 12-16 and 12-19 hold promise as prophylactic agents for immunocompromised persons who do not respond robustly to COVID-19 vaccines.

Antibody avidity and multi-specificity combined to confer protection against SARS-CoV-2 and resilience against viral escape (preprint)

SARS-CoV-2, the causative agent of COVID-19, has been responsible for a global pandemic. Monoclonal antibodies have been used as antiviral therapeutics, but have been limited in efficacy by viral sequence variability in emerging variants of concern (VOCs), and in deployment by the need for high doses. In this study, we leverage the MULTI-specific, multi-Affinity antiBODY (Multabody, MB) platform, derived from the human apoferritin protomer, to drive the multimerization of antibody fragments and generate exceptionally potent and broad SARS-CoV-2 neutralizers. CryoEM revealed a high degree of homogeneity for the core of these engineered antibody-like molecules at 2.1 [A] resolution. We demonstrate that neutralization potency improvements of the MB over corresponding IgGs translates into superior in vivo protection: in the SARS-CoV-2 mouse challenge model, comparable in vivo protection was achieved for the MB delivered at 30x lower dose compared to the corresponding IgGs. Furthermore, we show how MBs potently neutralize SARS-CoV-2 VOCs by leveraging augmented avidity, even when corresponding IgGs lose their ability to neutralize potently. Multiple mAb specificities could also be combined into a single MB molecule to expand the neutralization breadth beyond SARS-CoV-2 to other sarbecoviruses. Our work demonstrates how avidity and multi-specificity combined can be leveraged to confer protection and resilience against viral diversity that exceeds that of traditional monoclonal antibody therapies.

Antigenic characterization of the SARS-CoV-2 Omicron subvariant BA.2.75 (preprint)

The SARS-CoV-2 Omicron subvariant BA.2.75 emerged recently and appears to be spreading rapidly. It has nine mutations in its spike compared to BA.2, raising concerns it may further evade vaccine-elicited and therapeutic antibodies. Here, we found BA.2.75 to be moderately more neutralization resistant to sera from vaccinated/boosted individuals than BA.2 (1.8-fold), similar to BA.2.12.1 (1.1-fold), but more neutralization sensitive than BA.4/5 (0.6-fold). Relative to BA.2, BA.2.75 showed heightened resistance to class 1 and class 3 monoclonal antibodies to the receptor-binding domain, while gaining sensitivity to class 2 antibodies. The resistance was largely conferred by the G446S and R460K mutations. Of note, BA.2.75 was slightly resistant (3.7-fold) to bebtelovimab, the only therapeutic antibody with potent activity against all Omicron subvariants. BA.2.75 also exhibited higher receptor binding affinity than other Omicron subvariants. BA.2.75 provides yet another example of the ongoing evolution of SARS-CoV-2 as it gains transmissibility while incrementally evading antibody neutralization.

SARS-CoV-2 omicron variants succumb in vitro to Artemisia annua hot water extracts (preprint)

The SARS-CoV-2 (COVID-19) global pandemic continuous to infect and kill millions while rapidly evolving new variants that are more transmissible and evading vaccine-elicited antibodies. Artemisia annua L. extracts have shown potency against all previously tested variants. Here we further queried extract efficacy against omicron and its recent subvariants. Using Vero E6 cells, we measured the in vitro efficacy (IC50) of stored (frozen) dried-leaf hot-water A. annua L. extracts of four cultivars (A3, BUR, MED, and SAM) against SARS-CoV-2 variants: original WA1 (WT), BA.1.1.529+R346K (omicron), BA.2, BA.2.12.1, and BA.4. IC50 values normalized to the extract artemisinin (ART) content ranged from 0.5-16.5 uM ART. When normalized to dry mass of the extracted A. annua leaves, values ranged from 20-106 ug. Although IC50 values for these new variants are slightly higher than those reported for previously tested variants, they were within limits of assay variation. There was no measurable loss of cell viability at leaf dry weights less than or equal to 50 ug of any cultivar extract. Results continue to indicate that oral consumption of A. annua hot-water extracts (tea infusions) could potentially provide a cost-effective approach to help stave off this pandemic virus and its rapidly evolving variants.

An Immunostimulatory Glycolipid That Blocks SARS-CoV-2, RSV, and Influenza Infections In Vivo (preprint)

The fear and devastation caused by the COVID-19 pandemic has been mitigated by the successful development and deployment of prophylactic vaccines that substantially lowered the incidences of symptomatic infection, hospitalization, and death. However, as the causative agent SARS-CoV-2 continues to spread and evolve worldwide, vaccine-breakthrough infections have become frequent, especially after the emergence of viral variants that are antigenically distant from the ancestral strain used in the current vaccines. Additional approaches are therefore needed in our prevention tool kit. Here, we report on a glycolipid termed 7DW8-5 that exploits the host innate immune system to enable rapid control of viral infections in vivo. This glycolipid binds to CD1d on antigen-presenting cells and thereby stimulates NKT cells to release a cascade of cytokines and chemokines. The intranasal administration of 7DW8-5 prior to virus exposure significantly blocked infection by three different authentic variants of SARS-CoV-2, as well as by respiratory syncytial virus and influenza virus, in mice or hamsters. We also found that this protective antiviral effect is both host-directed and mechanism-specific, requiring both the CD1d molecule and interferon- gamma. A chemical compound like 7DW8-5 that is easy to administer and cheap to manufacture may be useful not only in slowing the spread of COVID-19 but also in responding to future pandemics that include currently ongoing influenza, SARS-CoV-2 and respiratory syncytia virus- associated tripledemic long before vaccines or drugs are developed.

SARS-CoV-2 Omicron BA.2.12.1, BA.4, and BA.5 subvariants evolved to extend antibody evasion (preprint)

The Omicron subvariant BA.2 accounts for a large majority of the SARS-CoV-2 infection worldwide today. However, its recent descendants BA.2.12.1 and BA.4/5 have surged dramatically to become dominant in the United States and South Africa, respectively. That these novel Omicron subvariants carry additional mutations in their spike proteins raises concerns that they may further evade neutralizing antibodies, thereby further compromising the efficacy of our COVID-19 vaccines and therapeutic monoclonals. We now report findings from a systematic antigenic analysis of these surging Omicron subvariants. BA.2.12.1 is only modestly (1.8-fold) more resistant to sera from vaccinated and boosted individuals than BA.2. On the other hand, BA.4/5 is substantially (4.2-fold) more resistant and thus more likely to lead to vaccine breakthrough infections. Mutation at spike residue L452 found in both BA.2.12.1 and BA.4/5 facilitates escape from some antibodies directed to the so-called Class 2 and Class 3 regions of the receptor-binding domain (RBD). The F486V mutation found in BA.4/5 facilitates escape from certain Class 1 and Class 2 antibodies to the RBD but compromises the spike affinity for the cellular receptor ACE2. The R493Q reversion mutation, however, restores receptor affinity and consequently the fitness of BA.4/5. Among therapeutic antibodies authorized for clinical use, only bebtelovimab (LY-COV1404) retains full potency against both BA.2.12.1 and BA.4/5. The Omicron lineage of SARS-CoV-2 continues to evolve, successively yielding subvariants that are not only more transmissible but also more evasive to antibodies.

SARS-CoV-2 Omicron BA.2.12.1, BA.4, and BA.5 subvariants evolved to extend antibody evasion (preprint)

The Omicron subvariant BA.2 accounts for a large majority of the SARS-CoV-2 infection worldwide today. However, its recent descendants BA.2.12.1 and BA.4/5 have surged dramatically to become dominant in the United States and South Africa, respectively. That these novel Omicron subvariants carry additional mutations in their spike proteins raises concerns that they may further evade neutralizing antibodies, thereby further compromising the efficacy of our COVID-19 vaccines and therapeutic monoclonals. We now report findings from a systematic antigenic analysis of these surging Omicron subvariants. BA.2.12.1 is only modestly (1.8-fold) more resistant to sera from vaccinated and boosted individuals than BA.2. On the other hand, BA.4/5 is substantially (4.2-fold) more resistant and thus more likely to lead to vaccine breakthrough infections. Mutation at spike residue L452 found in both BA.2.12.1 and BA.4/5 facilitates escape from some antibodies directed to the so-called Class 2 and Class 3 regions of the receptor-binding domain (RBD). The F486V mutation found in BA.4/5 facilitates escape from certain Class 1 and Class 2 antibodies to the RBD but compromises the spike affinity for the cellular receptor ACE2. The R493Q reversion mutation, however, restores receptor affinity and consequently the fitness of BA.4/5. Among therapeutic antibodies authorized for clinical use, only bebtelovimab (LY-COV1404) retains full potency against both BA.2.12.1 and BA.4/5. The Omicron lineage of SARS-CoV-2 continues to evolve, successively yielding subvariants that are not only more transmissible but also more evasive to antibodies.

Antibody Evasion Properties of SARS-CoV-2 Omicron Sublineages (preprint)

The identification of the Omicron variant (B.1.1.529.1 or BA.1) of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) in Botswana in November 20211 immediately raised alarms due to the sheer number of mutations in the spike glycoprotein that could lead to striking antibody evasion. We2 and others3-6 recently reported results in this Journal confirming such a concern. Continuing surveillance of Omicron evolution has since revealed the rise in prevalence of two sublineages, BA.1 with an R346K mutation (BA.1+R346K) and B.1.1.529.2 (BA.2), with the latter containing 8 unique spike mutations while lacking 13 spike mutations found in BA.1. We therefore extended our studies to include antigenic characterization of these new sublineages. Polyclonal sera from patients infected by wild-type SARS-CoV-2 or recipients of current mRNA vaccines showed a substantial loss in neutralizing activity against both BA.1+R346K and BA.2, with drops comparable to that already reported for BA.12,3,5,6. These findings indicate that these three sublineages of Omicron are antigenically equidistant from the wild-type SARS-CoV-2 and thus similarly threaten the efficacies of current vaccines. BA.2 also exhibited marked resistance to 17 of 19 neutralizing monoclonal antibodies tested, including S309 (sotrovimab)7, which had retained appreciable activity against BA.1 and BA.1+R346K2-4,6 . This new finding shows that no presently approved or authorized monoclonal antibody therapy could adequately cover all sublineages of the Omicron variant.

Striking Antibody Evasion Manifested by the Omicron Variant of SARS-CoV-2 (preprint)

The Omicron (B.1.1.529) variant of SARS-CoV-2 was only recently detected in southern Africa, but its subsequent spread has been extensive, both regionally and globally1. It is expected to become dominant in the coming weeks2, probably due to enhanced transmissibility. A striking feature of this variant is the large number of spike mutations3 that pose a threat to the efficacy of current COVID-19 vaccines and antibody therapies4. This concern is amplified by the findings from our study. We found B.1.1.529 to be markedly resistant to neutralization by serum not only from convalescent patients, but also from individuals vaccinated with one of the four widely used COVID-19 vaccines. Even serum from persons vaccinated and boosted with mRNA-based vaccines exhibited substantially diminished neutralizing activity against B.1.1.529. By evaluating a panel of monoclonal antibodies to all known epitope clusters on the spike protein, we noted that the activity of 18 of the 19 antibodies tested were either abolished or impaired, including ones currently authorized or approved for use in patients. In addition, we also identified four new spike mutations (S371L, N440K, G446S, and Q493R) that confer greater antibody resistance to B.1.1.529. The Omicron variant presents a serious threat to many existing COVID-19 vaccines and therapies, compelling the development of new interventions that anticipate the evolutionary trajectory of SARS-CoV-2.

Isolation and comparative analysis of antibodies that broadly neutralize sarbecoviruses (preprint)

The devastation caused by SARS-CoV-2 has made clear the importance of pandemic preparedness. To address future zoonotic outbreaks due to related viruses in the sarbecovirus subgenus, we identified a human monoclonal antibody, 10-40, that neutralized or bound all sarbecoviruses tested in vitro and protected against SARS-CoV-2 and SARS-CoV in vivo. Comparative studies with other receptor-binding domain (RBD)-directed antibodies showed 10-40 to have the greatest breadth against sarbecoviruses and thus its promise as an agent for pandemic preparedness. Moreover, structural analyses on 10-40 and similar antibodies not only defined an epitope cluster in the inner face of the RBD that is well conserved among sarbecoviruses, but also uncovered a new antibody class with a common CDRH3 motif. Our analyses also suggested that elicitation of this class of antibodies may not be overly difficult, an observation that bodes well for the development of a pan-sarbecovirus vaccine.

A monoclonal antibody that neutralizes SARS-CoV-2 variants, SARS-CoV, and other sarbecoviruses (preprint)

The repeated emergence of highly pathogenic human coronaviruses as well as their evolving variants highlight the need to develop potent and broad-spectrum antiviral therapeutics and vaccines. By screening monoclonal antibodies (mAbs) isolated from COVID-19-convalescent patients, we found one mAb, 2-36, with cross-neutralizing activity against SARS-CoV. We solved the cryo-EM structure of 2-36 in complex with SARS-CoV-2 or SARS-CoV spike, revealing a highly conserved epitope in the receptor-binding domain (RBD). Antibody 2-36 neutralized not only all current circulating SARS-CoV-2 variants and SARS-COV, but also a panel of bat and pangolin sarbecoviruses that can use human angiotensin-converting enzyme 2 (ACE2) as a receptor. We selected 2-36-escape viruses in vitro and confirmed that K378T in SARS-CoV-2 RBD led to viral resistance. Taken together, 2-36 represents a strategic reserve drug candidate for the prevention and treatment of possible diseases caused by pre-emergent SARS-related coronaviruses. Its epitope defines a promising target for the development of a pan-sarbecovirus vaccine.

Artemisia annua hot-water extracts show potent activity in vitro against Covid-19 variants including delta (preprint)

Ethnopharmacological relevanceFor millennia in Southeast Asia, Artemisia annua L. was used to treat "fever". This medicinal plant is effective against numerous infectious microbial and viral diseases and is used by many global communities as a source of artemisinin derivatives that are first-line drugs to treat malaria. Aim of the StudyThe SARS-CoV-2 (Covid-19) global pandemic has killed millions and evolved numerous variants, with delta being the most transmissible to date and causing break-through infections of vaccinated individuals. We further queried the efficacy of A. annua cultivars against new variants. Materials and MethodsUsing Vero E6 cells, we measured anti-SARS-CoV-2 activity of dried-leaf hot-water A. annua extracts of four cultivars, A3, BUR, MED, and SAM, to determine their efficacy against five fully infectious variants of the virus: alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2), and kappa (B.1.617.1). ResultsIn addition to being effective against the original wild type WA1, A. annua cultivars A3, BUR, MED and SAM were also potent against all five variants. IC50 and IC90 values based on measured artemisinin content ranged from 0.3-8.4 M and 1.4-25.0 M, respectively. The IC50 and IC90 values based on dried leaf weight (DW) used to make the tea infusions ranged from 11.0-67.7 g DW and 59.5-160.6 g DW, respectively. Cell toxicity was insignificant at a leaf dry weight of [≤]50 g in the extract of any cultivar. ConclusionsResults suggest that oral consumption of A. annua hot-water extracts (tea infusions), could provide a cost-effective therapy to help stave off the rapid global spread of these variants, buying time for broader implementation of vaccines.

A third COVID-19 vaccine shot markedly boosts neutralizing antibody potency and breadth (preprint)

COVID-19 (coronavirus disease 2019) vaccines have been rapidly developed and deployed globally as a measure to combat the disease. These vaccines have been demonstrated to confer significant protection, but there have been reports of temporal decay in antibody titer. Furthermore, several variants have been identified with variable degrees of antibody resistance. These two factors suggest that a booster vaccination may be worthy of consideration. While such a booster dose has been studied as a series of three homologous vaccines in healthy individuals, to our knowledge, information on a heterologous regimen remains unreported, despite the practical benefits of such a scheme. Here, in this observational study, we investigated the serological profile of four healthy individuals who received two doses of the BNT162b2 vaccine, followed by a third booster dose with the Ad26.COV2.S vaccine. We found that while all individuals had spike-binding antibodies at each of the timepoints tested, there was an appreciable drop in titer by four months following the second vaccination. The third vaccine dose robustly increased titers beyond that of two vaccinations, and these elicited antibodies had neutralizing capability against all SARS-CoV-2 strains tested in both a recombinant vesicular stomatitis virus-based pseudovirus assay and an authentic SARS-CoV-2 assay, except for one individual against B.1.351 in the latter assay. Thus, a third COVID-19 vaccine dose in healthy individuals promoted not just neutralizing antibody potency, but also induced breadth against dominant SARS-CoV-2 variants. SignificanceCOVID-19 vaccines confer protection from symptomatic disease, but the elicited antibody titer has been found to decrease with time. Furthermore, SARS-CoV-2 variants with relative resistance against antibody neutralization have been identified. To overcome such issues, a third vaccine dose applied as a booster vaccine may be necessary. We studied four healthy individuals who received a heterologous booster dose as a third vaccine. All of these individuals had heightened neutralizing antibody titer following the booster vaccination, and could neutralize nearly all variants tested. Thus, a heterologous third COVID-19 vaccine dose may be a mechanism to both heighten and broaden antibody titers, and could be an additional strategy for controlling the SARS-CoV-2 pandemic.

Neutralizing antibody 5-7 defines a distinct site of vulnerability in SARS-CoV-2 spike N-terminal domain (preprint)

Antibodies that potently neutralize SARS-CoV-2 target mainly the receptor-binding domain or the N-terminal domain (NTD). Over a dozen potently neutralizing NTD- directed antibodies have been studied structurally, and all target a single antigenic supersite in NTD (site 1). Here we report the 3.7 [A] resolution cryo-EM structure of a potent NTD-directed neutralizing antibody 5-7, which recognizes a site distinct from other potently neutralizing antibodies, inserting a binding loop into an exposed hydrophobic pocket between the two sheets of the NTD {beta}-sandwich. Interestingly, this pocket has been previously identified as the binding site for hydrophobic molecules including heme metabolites, but we observe their presence to not substantially impede 5-7 recognition. Mirroring its distinctive binding, antibody 5-7 retains a distinctive neutralization potency with variants of concern (VOC). Overall, we reveal a hydrophobic pocket in NTD proposed for immune evasion can actually be used by the immune system for recognition.

SARS-CoV-2 Infection Causes Dopaminergic Neuron Senescence (preprint)

COVID-19 patients commonly present with neurological signs of central nervous system (CNS) and/or peripheral nervous system dysfunction. However, which neural cells are permissive to infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been controversial. Here, we show that midbrain dopamine (DA) neurons derived from human pluripotent stem cells (hPSCs) are selectively permissive to SARS-CoV-2 infection both in vitro and upon transplantation in vivo, and that SARS-CoV-2 infection triggers a DA neuron inflammatory and cellular senescence response. A high-throughput screen in hPSC-derived DA neurons identified several FDA approved drugs, including riluzole, metformin, and imatinib, that can rescue the cellular senescence phenotype and prevent SARS-CoV-2 infection. RNA-seq analysis of human ventral midbrain tissue from COVID-19 patients, using formalin-fixed paraffin-embedded autopsy samples, confirmed the induction of an inflammatory and cellular senescence signature and identified low levels of SARS-CoV-2 transcripts. Our findings demonstrate that hPSC-derived DA neurons can serve as a disease model to study neuronal susceptibility to SARS-CoV-2 and to identify candidate neuroprotective drugs for COVID-19 patients. The susceptibility of hPSC-derived DA neurons to SARS-CoV-2 and the observed inflammatory and senescence transcriptional responses suggest the need for careful, long-term monitoring of neurological problems in COVID-19 patients.