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

Syed, Abdullah Muhammad; Taha, Taha Y.; Tabata, Takako; Chen, Irene P.; Ciling, Alison; Khalid, Mir M.; Sreekumar, Bharath; Chen, Peiyi; Hayashi, Jennifer M.; Soczek, Katarzyna M; Ott, Mélanie; Doudna, Jennifer A.

doi:10.1126/science.abl6184

Cited by 231 publications

(135 citation statements)

References 35 publications

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“…The rapidly fixed mutation R203M in Delta variants, which is in the same location as the R203K mutation that occurs in Alpha variant, is likely not a coincidence. The improved mRNA packaging and luciferase induction associated with the R203M mutation based on the virus-like particle (VLP) assay also confirmed these results, although the mechanism of the N protein mutations is attributed to a previously unknown strategy 25 . The 203 mutation may influence the polymerization, phase separation or phosphorylation of the N protein and further impact the assembly of ribonucleocapsid (RNP).…”

Section: Discussionmentioning

confidence: 63%

“…In addition to these adaptive S protein mutations, there are adaptive mutations in other parts of the viral genome that contribute to the virulence, spread and immune evasion of the virus. Several researchers have highlighted the potential importance of mutations in the nucleocapsid (N) protein 12,23-25 . Our previous work has found that the cooccurring mutations R203K/G204R in the N protein increase fitness and infectivity of SARS-CoV-2 and facilitate the emergence of the Alpha variant 23 .…”

Section: Introductionmentioning

confidence: 99%

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Nucleocapsid 203 mutations enhance SARS-CoV-2 immune evasion

Lin

Xue

Zhang

et al. 2021

Preprint

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Previous work indicated that the nucleocapsid 203 mutation increase the virulence and transmission of the SARS-CoV-2 Alpha variant. However, Delta later outcompeted Alpha and other lineages, promoting a new wave of infections. Delta also possesses a nucleocapsid 203 mutation, R203M. Large-scale epidemiological analyses suggest a synergistic effect of the 203 mutation and the spike L452R mutation, associated with Delta expansion. Viral competition experiments demonstrate the synergistic effect in fitness and infectivity. More importantly, we found that the combination of R203M and L452R brings in a 3.2-fold decrease in neutralizing titers to the neutralizing serum relative to L452R-only virus. R203M/L452R show an increased fitness after the initiation of global vaccination programmes, possibly associated with the enhanced immune evasion. Another rapidly emerging variant Omicron also bears the 203 mutation. Thus, we proposed that nucleocapsid mutations play an essential role for the rise and predominance of variants in concern.

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Section: Discussionmentioning

confidence: 63%

Section: Introductionmentioning

confidence: 99%

Nucleocapsid 203 mutations enhance SARS-CoV-2 immune evasion

Lin

Xue

Zhang

et al. 2021

Preprint

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“…Another study found that four mutations in the nucleocapsid (N) protein, which is an important protein for viral replication, improve the viruses’ ability to form infectious particles. These four mutations in N, which are universally found in more transmissible SARS-CoV-2 variants, increased mRNA delivery and expression and two of the mutations produced more infectious virus (29). This is consistent with other studies that found that samples from people infected with the Delta variant have higher viral RNA levels and an increased duration of viral shedding compared to samples collected from people infected with other variants (14, 30-32).…”

Section: Discussionmentioning

confidence: 99%

Genomic and Virological Characterization of SARS-CoV-2 Variants in a Subset of Unvaccinated and Vaccinated U.S. Military Personnel

Smith

Singh

Green

et al. 2021

Preprint

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The emergence of SARS-CoV-2 variants complicates efforts to control the COVID-19 pandemic. Increasing genomic surveillance of SARS-CoV-2 is imperative for early detection of emerging variants, to trace the movement of variants, and to monitor effectiveness of countermeasures. Additionally, determining the amount of viable virus present in clinical samples is helpful to better understand the impact these variants have on viral shedding. In this study, we analyzed nasal swab samples collected between March 2020 and early November 2021 from a cohort of United States (U.S.) military personnel and healthcare system beneficiaries stationed worldwide as a part of the Defense Health Agency’s (DHA) Global Emerging Infections Surveillance (GEIS) program. SARS-CoV-2 quantitative real time reverse-transcription PCR (qRT-PCR) positive samples were characterized by next-generation sequencing and a subset was analyzed for isolation and quantification of viable virus. Not surprisingly, we found that the Delta variant is the predominant strain circulating among U.S. military personnel beginning in July 2021 and primarily represents cases of vaccine breakthrough infections (VBIs). Among VBIs, we found a 50-fold increase in viable virus in nasal swab samples from Delta variant cases when compared to cases involving other variants. Notably, we found a 40-fold increase in viable virus in nasal swab samples from VBIs involving Delta as compared to unvaccinated personnel infected with other variants prior to the availability of approved vaccines. This study provides important insight about the genomic and virological characterization of SARS-CoV-2 isolates from a unique study population with a global presence.ImpactThe COVID-19 pandemic is currently a leading cause of death globally and new SARS-CoV-2 variants continue to emerge. Genomic surveillance of variants is necessary to characterize mutations that could affect transmissibility and spread, antigenicity or virulence. Furthermore, wet lab studies are necessary to evaluate how genetic differences may affect viral fitness and transmissibility. The Delta variant is currently the predominant strain globally and determining factors that drive its ability to spread rapidly is important. In this study, we characterized SARS-CoV-2 positive samples from U.S. military personnel and their beneficiaries by next-generation sequencing and isolation of viable virus. Consistent with other studies, we found higher levels of infectious virus from Delta samples when compared to non-Delta infections. Strikingly, we found the difference in titer between Delta and other strains to be so profound as to be unaffected by vaccination status, suggesting that increased transmissibility of the Delta variant is in part due to higher amounts of virus shedding. This helps explain the rapid spread of the Delta variant and provides the impetus to increase control measures such as vaccination, boosters, masking and distancing requirements. It will be necessary to continue genomic and virological characterization of new variants, such as Omicron.

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“…The artificial, replication deficient retro-or lentiviral vectors can be problematic because viral glycoproteins, which mediate attachment, uptake and fusion with cellular membranes might perform differently in the context of viral vectors compared to their authentic viral host (Khoury et al, 2020; Syed et al, 2021). S-pseudotyped retro-or lentiviral vectors mostly bud from the plasma membrane where S is incorporated into the envelope of the vector with uncertain stoichiometry (Giroglou et al, 2004; Syed et al, 2021). Very much in contrast, the envelopes of coronaviruses carry a discrete number of S trimers per particle, which assemble in the ER-Golgi intermediate compartment together with M, E and N in a given, optimal stoichiometry (Ujike et al, 2016).…”

Section: Discussionmentioning

confidence: 99%

“…For SARS-CoV (Hsieh et al, 2005), and later SARS-CoV-2, VLPs were shown to mediate transport and delivery of reporter transcripts that encompass cis-acting packaging signal sequences mimicking the generation of authentic SARS-CoV-2 and their infectivity (Syed et al, 2021). This is a major achievement, but the readout still depends on de novo expression of the reporter transcript encoding luciferase in this report.…”

Section: Discussionmentioning

confidence: 99%

Quantitation of SARS-CoV-2 neutralizing antibodies with a virus-free, authentic test

Roessler

Pich

Albanese

et al. 2021

Preprint

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Neutralizing antibodies (NAbs), and their concentration in sera of convalescents and vaccinees are a solid correlate of protection from COVID-19. The antibody concentrations in clinical samples that neutralize SARS-CoV-2 are difficult and very cumbersome to assess with conventional virus neutralization tests (cVNTs), which require work with the infectious virus and biosafety level 3 containment precautions. Alternative virus neutralization tests currently in use are mostly surrogate tests based on direct or competitive ELISA formats or use viral vectors with the spike protein as the single structural component of SARS-CoV-2. To overcome these obstacles, we developed a virus-free, safe and very fast (4.5 h) in vitro diagnostic test based on engineered yet authentic SARS-CoV-2 virus-like-particles (VLPs). They share all features of the original SARS-CoV-2 but lack the viral RNA genome and thus are non-infectious. NAbs induced by infection or vaccination, but also potentially neutralizing monoclonal antibodies can be reliably quantified and assessed with ease and within hours with our test, because they interfere and block the ACE2-mediated uptake of VLPs by recipient cells. Results from the VLP neutralization test (VLPNT) show excellent correlation to a cVNT with fully infectious SARS-CoV-2 and allow to estimate the reduced neutralization capacity of COVID-19 vaccinee sera with variants of concern of SARS-CoV-2.Author summaryThe current pandemic caused by SARS-CoV-2 is a major challenge not only for COVID-19 patients, medical staff, healthcare systems and the general public, but also virologists and clinical laboratories. A particular challenge are safety issues which require biological safety level 3 to work with and study the pathogen. An alternative are virus-like particles (VLPs) of SARS-CoV-2, which are authentic in terms of viral structure and function but are harmless bioproducts in nature. We engineered VLPs which are close-to-perfect mimics of SARS-CoV-2 by all structural, biochemical, physical and functional criteria tested. SARS-CoV-2 VLPs were used in virus neutralization tests (VNTs). Because high concentrations of neutralizing antibodies correlate with protection from COVID-19 practical VNTs are urgently needed. We developed an authentic, virus-free, thus safe yet very fast in vitro diagnostic test with SARS-CoV-2 VLPs. Virus neutralizing antibodies induced by natural infection or vaccination but also certain monoclonal antibodies inhibit VLP fusion with recipient cells carrying ACE2. Quantitative results from a conventional neutralization test with fully infectious SARS-CoV-2 and results from the VLP-based neutralization test correlate perfectly. The setup of the test is very flexible and allows to analyze sera for their neutralizing capacity against different variants of concern and in a standardized assay format.

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

Cited by 231 publications

References 35 publications

Nucleocapsid 203 mutations enhance SARS-CoV-2 immune evasion

Nucleocapsid 203 mutations enhance SARS-CoV-2 immune evasion

Genomic and Virological Characterization of SARS-CoV-2 Variants in a Subset of Unvaccinated and Vaccinated U.S. Military Personnel

Quantitation of SARS-CoV-2 neutralizing antibodies with a virus-free, authentic test

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