Search

Drug resistance poses a significant challenge in the development of effective therapies against SARS-CoV-2. Here, we identified two double mutations, M49K/M165V and M49K/S301P, in the 3C-like protease (3CLpro) that confer resistance to a novel non-covalent inhibitor, WU-04, which is currently in phase III clinical trials (NCT06197217). Crystallographic analysis indicates that the M49K mutation destabilizes the WU-04-binding pocket, impacting the binding of WU-04 more significantly than the binding of 3CLpro substrates. The M165V mutation directly interferes with WU-04 binding. The S301P mutation, which is far from the WU-04-binding pocket, indirectly affects WU-04 binding by restricting the rotation of 3CLpro's C-terminal tail and impeding 3CLpro dimerization. We further explored 3CLpro mutations that confer resistance to two clinically used inhibitors: ensitrelvir and nirmatrelvir, and revealed a trade-off between the catalytic activity, thermostability, and drug resistance of 3CLpro. We found that mutations at the same residue (M49) can have distinct effects on the 3CLpro inhibitors, highlighting the importance of developing multiple antiviral agents with different skeletons for fighting SARS-CoV-2. These findings enhance our understanding of SARS-CoV-2 resistance mechanisms and inform the development of effective therapeutics. [ABSTRACT FROM AUTHOR]

An attenuated SARS-CoV-2 virus with modified viral transcriptional regulatory sequences and deletion of open-reading frames 3, 6, 7 and 8 (∆3678) was previously reported to protect hamsters from SARS-CoV-2 infection and transmission. Here we report that a single-dose intranasal vaccination of ∆3678 protects K18-hACE2 mice from wild-type or variant SARS-CoV-2 challenge. Compared with wild-type virus infection, the ∆3678 vaccination induces equivalent or higher levels of lung and systemic T cell, B cell, IgA, and IgG responses. The results suggest ∆3678 as an attractive mucosal vaccine candidate to boost pulmonary immunity against SARS-CoV-2. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the COVID-19 global pandemic. SARS-CoV-2 is an enveloped RNA virus that relies on its trimeric surface glycoprotein spike for entry into host cells. Here we describe the COVID-19 vaccine candidate MV-014-212, a live, attenuated, recombinant human respiratory syncytial virus expressing a chimeric SARS-CoV-2 spike as the only viral envelope protein. MV-014-212 was attenuated and immunogenic in African green monkeys (AGMs). One mucosal administration of MV-014-212 in AGMs protected against SARS-CoV-2 challenge, reducing by more than 200-fold the peak shedding of SARS-CoV-2 in the nose. MV-014-212 elicited mucosal immunoglobulin A in the nose and neutralizing antibodies in serum that exhibited cross-neutralization against virus variants of concern Alpha, Beta, and Delta. Intranasally delivered, live attenuated vaccines such as MV-014-212 entail low-cost manufacturing suitable for global deployment. MV-014-212 is currently in Phase 1 clinical trials as an intranasal COVID-19 vaccine. [ABSTRACT FROM AUTHOR]

The newly emerged Omicron SARS-CoV-2 has several distinct sublineages including BA.1, BA.2, and BA.3. BA.1 accounts for the initial surge and is being replaced by BA.2, whereas BA.3 is at a low prevalence at this time. Here we report the neutralization of BNT162b2-vaccinated sera (collected 1 month after dose 3) against the three Omicron sublineages. To facilitate the neutralization testing, we have engineered the complete BA.1, BA.2, or BA.3 spike into an mNeonGreen USA-WA1/2020 SRAS-CoV-2. All BNT162b2-vaccinated sera neutralize USA-WA1/2020, BA.1-, BA.2-, and BA.3-spike SARS-CoV-2s with titers of >20; the neutralization geometric mean titers (GMTs) against the four viruses are 1211, 336, 300, and 190, respectively. Thus, the BA.1-, BA.2-, and BA.3-spike SARS-CoV-2s are 3.6-, 4.0-, and 6.4-fold less efficiently neutralized than the USA-WA1/2020, respectively. Our data have implications in vaccine strategy and understanding the biology of Omicron sublineages. It is essential to test the neutralization of approved vaccines against SARSCoV-2 Omicron sublineages. Kurhade et al. find that sera from people with three doses of BNT162b2 neutralize Omicron BA.1, BA.2, and BA.3 to a lesser extent than the original strain USAWA1/2020. [ABSTRACT FROM AUTHOR]

The Omicron SARS-CoV-2 has several distinct sublineages, among which sublineage BA.1 is responsible for the initial Omicron surge and is now being replaced by BA.2 worldwide, whereas BA.3 is currently at a low frequency. The ongoing BA.1-to-BA.2 replacement underscores the importance to understand the cross-neutralization among the three Omicron sublineages. Here we test the neutralization of BA.1-infected human sera against BA.2, BA.3, and USA/WA1-2020 (a strain isolated in late January 2020). The BA.1-infected sera neutralize BA.1, BA.2, BA.3, and USA/WA1-2020 SARS-CoV-2s with geometric mean titers (GMTs) of 445, 107, 102, and 16, respectively. Thus, the neutralizing GMTs against heterologous BA.2, BA.3, and USA/WA1-2020 are 4.2-, 4.4-, and 28.4-fold lower than the GMT against homologous BA.1, respectively. These findings have implications in COVID-19 vaccine strategy. It is important to understand the cross-neutralization among distinct SARS-CoV-2 Omicron sublineages. Zou et al. show that sera from Omicron BA.1-infected people are significantly weaker in neutralizing Omicron BA.2, BA.3, and USA-WA1/2020 than neutralizing BA.1. [ABSTRACT FROM AUTHOR]

BNT162b2-elicited human sera neutralize the currently dominant Delta SARS-CoV-2 variant. Here, we report the ability of 20 human sera, drawn 2 or 4 weeks after two doses of BNT162b2, to neutralize USA-WA1/2020 SARS-CoV-2 bearing variant spikes from Delta plus (Delta-AY.1, Delta-AY.2), Delta-∆144 (Delta with the Y144 deletion of the Alpha variant), Lambda, B.1.1.519, Theta, and Mu lineage viruses. Geometric mean plaque reduction neutralization titers against Delta-AY.1, Delta-AY.2, and Mu viruses are slightly lower than against USA-WA1/2020, but all sera neutralize the variant viruses to titers of ≥80, and neutralization titers against the Delta-∆144, Lambda, B.1.1.519 and Theta variants not significantly reduced relative to those against USA-WA1/2020. The susceptibility of Delta plus, Lambda, B.1.1.519, Theta, Mu, and other variants to neutralization by the sera indicates that antigenic change has not led to virus escape from vaccine-elicited neutralizing antibodies and supports ongoing mass immunization with BNT162b2 to control the variants and to minimize the emergence of new variants. [ABSTRACT FROM AUTHOR]

The spread of the Omicron SARS-CoV-2 variant underscores the importance of analyzing the cross-protection from previous non-Omicron infection. We have developed a high-throughput neutralization assay for Omicron SARS-CoV-2 by engineering the Omicron spike gene into an mNeonGreen USA-WA1/2020 SARS-CoV-2 (isolated in January 2020). Using this assay, we determine the neutralization titers (defined as the maximal serum dilution that inhibited 50% of infectious virus) of patient sera collected at 1- or 6-months after infection with non-Omicron SARS-CoV-2. From 1- to 6-month post-infection, the neutralization titers against USA-WA1/2020 decrease from 601 to 142 (a 4.2-fold reduction), while the neutralization titers against Omicron-spike SARS-CoV-2 remain low at 38 and 32, respectively. Thus, at 1- and 6-months after non-Omicron SARS-CoV-2 infection, the neutralization titers against Omicron are 15.8- and 4.4-fold lower than those against USA-WA1/2020, respectively. The low cross-neutralization against Omicron from previous non-Omicron infection supports vaccination of formerly infected individuals to mitigate the health impact of the ongoing Omicron surge. The SARS-CoV-2 variant of concern Omicron has quickly spread. Here, Zou et al. develop a high-throughput neutralization test for Omicron SARS-CoV-2 and show that patients with previous non-Omicron infections do not develop robust neutralization against Omicron. [ABSTRACT FROM AUTHOR]

A candidate multigenic SARS-CoV-2 vaccine based on an MVA vector expressing both viral N and S proteins (MVA-S + N) was immunogenic, and induced T-cell responses and binding antibodies to both antigens but in the absence of detectable neutralizing antibodies. Intranasal immunization with the vaccine diminished viral loads and lung inflammation in mice after SARS-CoV-2 challenge, which correlated with the T-cell response induced by the vaccine in the lung, indicating that T-cell immunity is also likely critical for protection against SARS-CoV-2 infection in addition to neutralizing antibodies. [ABSTRACT FROM AUTHOR]

There is a great need for the development of vaccines that induce potent and long-lasting protective immunity against SARS-CoV-2. Multimeric display of the antigen combined with potent adjuvant can enhance the potency and longevity of the antibody response. The receptor binding domain (RBD) of the spike protein is a primary target of neutralizing antibodies. Here, we developed a trimeric form of the RBD and show that it induces a potent neutralizing antibody response against live virus with diverse effector functions and provides protection against SARS-CoV-2 challenge in mice and rhesus macaques. The trimeric form induces higher neutralizing antibody titer compared to monomer with as low as 1μg antigen dose. In mice, adjuvanting the protein with a TLR7/8 agonist formulation alum-3M-052 induces 100-fold higher neutralizing antibody titer and superior protection from infection compared to alum. SARS-CoV-2 infection causes significant loss of innate cells and pathology in the lung, and vaccination protects from changes in innate cells and lung pathology. These results demonstrate RBD trimer protein as a suitable candidate for vaccine against SARS-CoV-2. Efficient vaccines for SARS-CoV-2 are needed. Here, the authors show that a trimeric form of the receptor-binding domain of SARS-CoV-2 spike adjuvanted with alum-3M-052 protects non-human primates from disease and inhibits infection. [ABSTRACT FROM AUTHOR]

The development of specific antiviral compounds to SARS-CoV-2 is an urgent task. One of the obstacles for the antiviral development is the requirement of biocontainment because infectious SARS-CoV-2 must be handled in a biosafety level-3 laboratory. Replicon, a non-infectious self-replicative viral RNA, could be a safe and effective tool for antiviral evaluation. Herein, we generated a PCR-based SARS-CoV-2 replicon. Eight fragments covering the entire SARS-CoV-2 genome except S, E, and M genes were amplified with HiBiT-tag sequence by PCR. The amplicons were ligated and in vitro transcribed to RNA. The cells electroporated with the replicon RNA showed more than 3000 times higher luminescence than MOCK control cells at 24 h post-electroporation, indicating robust translation and RNA replication of the replicon. The replication was drastically inhibited by remdesivir, an RNA polymerase inhibitor for SARS-CoV-2. The IC50 of remdesivir in this study was 0.29 μM, generally consistent to the IC50 obtained using infectious SARS-CoV-2 in a previous study (0.77 μM). Taken together, this system could be applied to the safe and effective antiviral evaluation without using infectious SARS-CoV-2. Because this is a PCR-based and transient replicon system, further improvement including the establishment of stable cell line must be achieved. [ABSTRACT FROM AUTHOR]

Antibody cocktails represent a promising approach to prevent SARS-CoV-2 escape. The determinants for selecting antibody combinations and the mechanism that antibody cocktails prevent viral escape remain unclear. We compared the critical residues in the receptor-binding domain (RBD) used by multiple neutralizing antibodies and cocktails and identified a combination of two antibodies CoV2-06 and CoV2-14 for preventing viral escape. The two antibodies simultaneously bind to non-overlapping epitopes and independently compete for receptor binding. SARS-CoV-2 rapidly escapes from individual antibodies by generating resistant mutations in vitro, but it doesn't escape from the cocktail due to stronger mutational constraints on RBD-ACE2 interaction and RBD protein folding requirements. We also identified a conserved neutralizing epitope shared between SARS-CoV-2 and SARS-CoV for antibody CoV2-12. Treatments with CoV2-06 and CoV2-14 individually and in combination confer protection in mice. These findings provide insights for rational selection and mechanistic understanding of antibody cocktails as candidates for treating COVID-19. Antibody cocktails represent a promising approach to prevent SARS-CoV-2 escape. Here, Ku et al., identify SARS-CoV-2 neutralizing antibodies from a phage library and identify an antibody combination that prevents viral escape and protects mice from viral challenge. [ABSTRACT FROM AUTHOR]

Virus neutralization remains the gold standard for determining antibody efficacy. Therefore, a high-throughput assay to measure SARS-CoV-2 neutralizing antibodies is urgently needed for COVID-19 serodiagnosis, convalescent plasma therapy, and vaccine development. Here, we report on a fluorescence-based SARS-CoV-2 neutralization assay that detects SARS-CoV-2 neutralizing antibodies in COVID-19 patient specimens and yields comparable results to plaque reduction neutralizing assay, the gold standard of serological testing. The fluorescence-based neutralization assay is specific to measure COVID-19 neutralizing antibodies without cross reacting with patient specimens with other viral, bacterial, or parasitic infections. Collectively, our approach offers a rapid platform that can be scaled to screen people for antibody protection from COVID-19, a key parameter necessary to safely reopen local communities. Neutralizing antibody titers in SARS-CoV-2 infected or vaccinated people are an important measure for vaccine development and public health decision-making. Here, the authors develop a fluorescence based SARS-CoV-2 assay to determine neutralizing antibody titers in COVID-19 patient sera in a high throughput set-up. [ABSTRACT FROM AUTHOR]

The original version of this Article contained an error in the Acknowledgements, which incorrectly listed the NIH grant "AI142759". This has been corrected in both the PDF and HTML versions of the Article. [Extracted from the article]

Correction to: I Nature Communications i https://doi.org/10.1038/s41467-020-19055-7, published online 15 October 2020 The original version of this Article contained an error in the Acknowledgements, which incorrectly listed the NIH grant "AI142759". This has been corrected in both the PDF and HTML versions of the Article. [Extracted from the article]

A high-throughput platform would greatly facilitate coronavirus disease 2019 (COVID-19) serological testing and antiviral screening. Here we present a high-throughput nanoluciferase severe respiratory syndrome coronavirus 2 (SARS-CoV-2-Nluc) that is genetically stable and replicates similarly to the wild-type virus in cell culture. SARS-CoV-2-Nluc can be used to measure neutralizing antibody activity in patient sera within 5 hours, and it produces results in concordance with a plaque reduction neutralization test (PRNT). Additionally, using SARS-CoV-2-Nluc infection of A549 cells expressing human ACE2 receptor (A549-hACE2), we show that the assay can be used for antiviral screening. Using the optimized SARS-CoV-2-Nluc assay, we evaluate a panel of antivirals and other anti-infective drugs, and we identify nelfinavir, rupintrivir, and cobicistat as the most selective inhibitors of SARS-CoV-2-Nluc (EC50 0.77 to 2.74 µM). In contrast, most of the clinically approved antivirals, including tenofovir alafenamide, emtricitabine, sofosbuvir, ledipasvir, and velpatasvir were inactive at concentrations up to 10 µM. Collectively, this high-throughput platform represents a reliable tool for rapid neutralization testing and antiviral screening for SARS-CoV-2. A high-throughput platform would greatly facilitate coronavirus disease 2019 (COVID-19) serological testing and antiviral screening. To address this, Shi and colleagues present a high-throughput nanoluciferase severe respiratory syndrome coronavirus 2 (SARS-CoV2-Nluc), and show that it has potential for large-scale vaccine evaluation and neutralizing antibody testing. [ABSTRACT FROM AUTHOR]