Quantifying Absolute Neutralization Titers against SARS-CoV-2 by a Standardized Virus Neutralization Assay Allows for CrossCohort Comparisons of COVID-19 Sera

The global coronavirus disease 2019 (COVID-19) pandemic has mobilized efforts to develop vaccines and antibody-based therapeutics, including convalescent-phase plasma therapy, that inhibit viral entry by inducing or transferring neutralizing antibodies (nAbs) against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (CoV2-S). However, rigorous efficacy testing requires extensive screening with live virus under onerous biosafety level 3 (BSL3) conditions, which limits high-throughput screening of patient and vaccine sera. Myriad BSL2-compatible surrogate virus neutralization assays (VNAs) have been developed to overcome this barrier. Yet, there is marked variability between VNAs and how their results are presented, making intergroup comparisons difficult. To address these limitations, we developed a standardized VNA using CoV2-S pseudotyped particles (CoV2pp) based on vesicular stomatitis virus bearing the Renilla luciferase gene in place of its G glyco-protein (VSVDG); this assay can be robustly produced at scale and generate accurate neutralizing titers within 18 h postinfection. Our standardized CoV2pp VNA showed a strong positive correlation with CoV2-S enzyme-linked immunosorbent assay (ELISA) results and live-virus neutralizations in confirmed convalescent-patient sera. Three independent groups subsequently validated our standardized CoV2pp VNA (n . 120). Our data (i) show that absolute 50% inhibitory concentration (absIC50), absIC80, and absIC90 values can be legitimately compared across diverse cohorts, (ii) highlight the substantial but consistent variability in neutralization potency across these cohorts, and (iii) support the use of the absIC80 as a more meaningful metric for assessing the neutralization potency of a vaccine or convalescent-phase sera. Lastly, we used our CoV2pp in a screen to identify ultrapermissive 293T clones that stably express ACE2 or ACE2 plus TMPRSS2. When these are used in combination with our CoV2pp, we can produce CoV2pp sufficient for 150,000 standardized VNAs/week. IMPORTANCE Vaccines and antibody-based therapeutics like convalescent-phase plasma therapy are premised upon inducing or transferring neutralizing antibodies that inhibit SARS-CoV-2 entry into cells. Virus neutralization assays (VNAs) for measuring neutralizing antibody titers (NATs) are an essential part of determining vaccine or therapeutic efficacy. However, such efficacy testing is limited by the inherent dangers of working with the live virus, which requires specialized high-level biocontainment facilities. We there-fore developed a standardized replication-defective pseudotyped particle system that mimics the entry of live SARS-CoV-2. This tool allows for the safe and efficient measurement of NATs, determination of other forms of entry inhibition, and thorough investigation of virus entry mechanisms. Four independent labs across the globe validated our standardized VNA using diverse cohorts. We argue that a standardized and scalable assay is necessary for meaningful comparisons of the myriad of vaccines and antibody-based therapeutics becoming available. Our data provide generalizable metrics for assessing their efficacy.Fil: Oguntuyo, Kasopefoluwa. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Stevens, Christian S.. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Hung, Chuan Tien. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Ikegame, Satoshi. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Acklin, Joshua A.. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Kowdle, Shreyas S.. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Carmichael, Jillian C.. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Chiu, Hsin Ping. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Azarm, Kristopher D.. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Haas, Griffin D.. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Amanat, Fatima. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Klingler, Jéromine. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Baine, Ian. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Arinsburg, Suzanne. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Bandres, Juan C.. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Siddiquey, Mohammed N. A.. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Schilke, Robert M.. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Woolard, Matthew D.. State University of Louisiana; Estados UnidosFil: Zhang, Hongbo. State University of Louisiana; Estados UnidosFil: Duty, Andrew J.. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Kraus, Thomas A.. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Moran, Thomas M.. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Tortorella, Domenico. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Lim, Jean K.. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Gamarnik, Andrea Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Hioe, Catarina E.. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Zolla Pazner, Susan. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Ivanov, Stanimir S.. State University of Louisiana; Estados UnidosFil: Kamil, Jeremy. State University of Louisiana; Estados UnidosFil: Krammer, Florian. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Lee, Benhur. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Ojeda, Diego Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; ArgentinaFil: González López Ledesma, María Mora. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Costa Navarro, Guadalupe Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Pallarés, H. M.. No especifíca;Fil: Sanchez, Lautaro Nicolas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Perez, P.. No especifíca;Fil: Ostrowsk, M.. No especifíca;Fil: Villordo, S. M.. No especifíca;Fil: Alvarez, D. E.. No especifíca;Fil: Caramelo, J. J.. No especifíca;Fil: Carradori, J.. No especifíca;Fil: Yanovsky, M. J.. No especifíca

Changes in symptomatology, reinfection, and transmissibility associated with the SARS-CoV-2 variant B.1.1.7: an ecological study

Background The SARS-CoV-2 variant B.1.1.7 was first identified in December, 2020, in England. We aimed to investigate whether increases in the proportion of infections with this variant are associated with differences in symptoms or disease course, reinfection rates, or transmissibility. Methods We did an ecological study to examine the association between the regional proportion of infections with the SARS-CoV-2 B.1.1.7 variant and reported symptoms, disease course, rates of reinfection, and transmissibility. Data on types and duration of symptoms were obtained from longitudinal reports from users of the COVID Symptom Study app who reported a positive test for COVID-19 between Sept 28 and Dec 27, 2020 (during which the prevalence of B.1.1.7 increased most notably in parts of the UK). From this dataset, we also estimated the frequency of possible reinfection, defined as the presence of two reported positive tests separated by more than 90 days with a period of reporting no symptoms for more than 7 days before the second positive test. The proportion of SARS-CoV-2 infections with the B.1.1.7 variant across the UK was estimated with use of genomic data from the COVID-19 Genomics UK Consortium and data from Public Health England on spike-gene target failure (a non-specific indicator of the B.1.1.7 variant) in community cases in England. We used linear regression to examine the association between reported symptoms and proportion of B.1.1.7. We assessed the Spearman correlation between the proportion of B.1.1.7 cases and number of reinfections over time, and between the number of positive tests and reinfections. We estimated incidence for B.1.1.7 and previous variants, and compared the effective reproduction number, Rt, for the two incidence estimates. Findings From Sept 28 to Dec 27, 2020, positive COVID-19 tests were reported by 36 920 COVID Symptom Study app users whose region was known and who reported as healthy on app sign-up. We found no changes in reported symptoms or disease duration associated with B.1.1.7. For the same period, possible reinfections were identified in 249 (0·7% [95% CI 0·6–0·8]) of 36 509 app users who reported a positive swab test before Oct 1, 2020, but there was no evidence that the frequency of reinfections was higher for the B.1.1.7 variant than for pre-existing variants. Reinfection occurrences were more positively correlated with the overall regional rise in cases (Spearman correlation 0·56–0·69 for South East, London, and East of England) than with the regional increase in the proportion of infections with the B.1.1.7 variant (Spearman correlation 0·38–0·56 in the same regions), suggesting B.1.1.7 does not substantially alter the risk of reinfection. We found a multiplicative increase in the Rt of B.1.1.7 by a factor of 1·35 (95% CI 1·02–1·69) relative to pre-existing variants. However, Rt fell below 1 during regional and national lockdowns, even in regions with high proportions of infections with the B.1.1.7 variant. Interpretation The lack of change in symptoms identified in this study indicates that existing testing and surveillance infrastructure do not need to change specifically for the B.1.1.7 variant. In addition, given that there was no apparent increase in the reinfection rate, vaccines are likely to remain effective against the B.1.1.7 variant. Funding Zoe Global, Department of Health (UK), Wellcome Trust, Engineering and Physical Sciences Research Council (UK), National Institute for Health Research (UK), Medical Research Council (UK), Alzheimer's Society

Genomic assessment of quarantine measures to prevent SARS-CoV-2 importation and transmission

Mitigation of SARS-CoV-2 transmission from international travel is a priority. We evaluated the effectiveness of travellers being required to quarantine for 14-days on return to England in Summer 2020. We identified 4,207 travel-related SARS-CoV-2 cases and their contacts, and identified 827 associated SARS-CoV-2 genomes. Overall, quarantine was associated with a lower rate of contacts, and the impact of quarantine was greatest in the 16–20 age-group. 186 SARS-CoV-2 genomes were sufficiently unique to identify travel-related clusters. Fewer genomically-linked cases were observed for index cases who returned from countries with quarantine requirement compared to countries with no quarantine requirement. This difference was explained by fewer importation events per identified genome for these cases, as opposed to fewer onward contacts per case. Overall, our study demonstrates that a 14-day quarantine period reduces, but does not completely eliminate, the onward transmission of imported cases, mainly by dissuading travel to countries with a quarantine requirement

Genomic epidemiology of SARS-CoV-2 in a UK university identifies dynamics of transmission

AbstractUnderstanding SARS-CoV-2 transmission in higher education settings is important to limit spread between students, and into at-risk populations. In this study, we sequenced 482 SARS-CoV-2 isolates from the University of Cambridge from 5 October to 6 December 2020. We perform a detailed phylogenetic comparison with 972 isolates from the surrounding community, complemented with epidemiological and contact tracing data, to determine transmission dynamics. We observe limited viral introductions into the university; the majority of student cases were linked to a single genetic cluster, likely following social gatherings at a venue outside the university. We identify considerable onward transmission associated with student accommodation and courses; this was effectively contained using local infection control measures and following a national lockdown. Transmission clusters were largely segregated within the university or the community. Our study highlights key determinants of SARS-CoV-2 transmission and effective interventions in a higher education setting that will inform public health policy during pandemics.</jats:p

The HSV-1 mechanisms of cell-to-cell spread and fusion are critically dependent on host PTP1B.

All herpesviruses have mechanisms for passing through cell junctions, which exclude neutralizing antibodies and offer a clear path to neighboring, uninfected cells. In the case of herpes simplex virus type 1 (HSV-1), direct cell-to-cell transmission takes place between epithelial cells and sensory neurons, where latency is established. The spreading mechanism is poorly understood, but mutations in four different HSV-1 genes can dysregulate it, causing neighboring cells to fuse to produce syncytia. Because the host proteins involved are largely unknown (other than the virus entry receptor), we were intrigued by an earlier discovery that cells infected with wild-type HSV-1 will form syncytia when treated with salubrinal. A biotinylated derivative of this drug was used to pull down cellular complexes, which were analyzed by mass spectrometry. One candidate was a protein tyrosine phosphatase (PTP1B), and although it ultimately proved not to be the target of salubrinal, it was found to be critical for the mechanism of cell-to-cell spread. In particular, a highly specific inhibitor of PTP1B (CAS 765317-72-4) blocked salubrinal-induced fusion, and by itself resulted in a dramatic reduction in the ability of HSV-1 to spread in the presence of neutralizing antibodies. The importance of this phosphatase was confirmed in the absence of drugs by using PTP1B-/- cells. Importantly, replication assays showed that virus titers were unaffected when PTP1B was inhibited or absent. Only cell-to-cell spread was altered. We also examined the effects of salubrinal and the PTP1B inhibitor on the four Syn mutants of HSV-1, and strikingly different responses were found. That is, both drugs individually enhanced fusion for some mutants and reduced fusion for others. PTP1B is the first host factor identified to be specifically required for cell-to-cell spread, and it may be a therapeutic target for preventing HSV-1 reactivation disease

Parameters influencing salubrinal-induced fusion.

<p><b>(A)</b> Vero cells were infected with strains KOS, F, or 17 at MOIs of 0.5, 1, or 3 and were incubated in medium containing 50 μM salubrinal. At 12 hpi, the fusion ratio (% free nuclei) was determined by flow cytometry. The mean values (±SD) from 3 independent experiments are plotted. <b>(B)</b> After infection with strain 17 (MOI = 3), cells were incubated in medium containing DMSO, and 50 μM salubrinal was added for the indicated time intervals. After a total of 12 hpi, the fusion ratios were measured by flow cytometry. Data are from 2 independent experiments. A student T-test was used to determine statistical significance for samples compared to the DMSO control. <b>(C)</b> After infection with strain 17 (MOI = 3), cells were incubated for the indicated time periods in medium containing 50 μM salubrinal, which was then replaced with control medium. At 12 hpi, the fusion ratio was determined by flow cytometry, and the data were analyzed as in (3B).</p

PTP1B inhibitor XXII blocks cell-to-cell spread.

<p><b>(A)</b> Vero cells were infected with the KOS strain (100 PFU/well in 6-well plates) and incubated in infection medium or medium containing 5 mg/ml of pooled human IgG to neutralize extracellular virions. At 18, 24, 30, and 42 hpi, the cells were immunostained for the major capsid protein, VP5, and nuclei were stained with DAPI. <b>(B)</b> To quantify virus spreading, the areas of 10–15 VP5-positive plaques were measured per sample and these were plotted. <b>(C and D)</b> Vero cells were infected (100 PFU/well) with strains (C) KOS or (D) 17 and then treated with increasing amounts of inhibitor XXII, all in the presence of 5 mg/ml of pooled IgG. At 42 hpi, the cells were immunostained for VP5, and representative plaques are shown. The areas of 10–15 plaques were measured for each drug concentration and compared to those from the DMSO control. Data from three independent experiments were combined and are represented as the mean ±SD. A student T-test was used to determine statistical significance for samples compared to the DMSO control. <b>(E and F)</b> Virus replication assays were performed in Vero cells infected (MOI = 5) with strains (E) KOS or (F) 17. The cells were incubated in medium containing DMSO or 30 μM inhibitor XXII, and at 6-hour time points, duplicate samples were collected to measure the virus titers (cell lysate + medium), which were averaged and plotted. <b>(G)</b> Representative images of plaques produced by the KOS strain on HaCaT cells treated with DMSO or 30 μM inhibitor XXII in the presence of 5 mg/ml pooled IgG. <b>(H)</b> At 42 hpi, 20 plaques from each sample in (6G) were measured, and their areas were plotted relative to the average obtained for the DMSO control.</p

Salubrinal-induced fusion of HSV-infected cells is dependent on accessory proteins.

<p><b>(A)</b> Diagram of relevant HSV-1 proteins involved in cell-to-cell spread and syncytia formation. The core fusion proteins are blue (gB, gH/gL, and gD), and proteins that can be altered to create syncytial variants are purple (gK, UL20, UL24, and gB). Two of the accessory glycoproteins are green (gE and gI), and three of the accessory tegument proteins are yellow (UL11, UL16, and UL21). <b>(B)</b> Vero cells were infected (MOI = 1) with HSV-1 strains KOS or 17 and incubated in medium containing DMSO or 50 μM salubrinal. At 12 hpi, the cells were immunostained for ZO-1 (red), and nuclei were stained with DAPI. Examples of syncytia are indicated (arrows). <b>(C)</b> Cells were infected with the KOS strain (MOI = 0.5) and incubated in the presence of salubrinal, as indicated. At 18 hpi, the cells were immunostained for ZO-1, and DAPI-stained nuclei were scored as being inside syncytia or within single cells. 1000 nuclei were scored per image for 3 replicates. Data are represented as mean ±SD, and statistical significance was determined by a student T-test. <b>(D)</b> Cells were infected (MOI = 0.5) with WT, gEΔCT, or ΔUL16 viruses and incubated in the presence of 50 μM salubrinal for 18 hours. DAPI-stained nuclei were scored as in (1C).</p

HSV-1 cell-to-cell spread is limited in PTP1B^-/- MEFs.

<p><b>(A)</b> Virus replication assays were performed in PTP1B+ and PTP1B^-/- MEFs infected with strain 17 (MOI = 5). At 6-hour time points, duplicate samples were collected for measurements of the virus titers (cell lysate + medium), which were averaged and plotted. <b>(B)</b> To measure syncytia formation, PTP1B+ MEFs or PTP1B^-/- MEFs were infected (MOI = 3) with strain 17 and treated with DMSO, 50 μM salubrinal, or 50 μM salubrinal + 30 μM inhibitor XXII. At 24 hpi, cells were immunostained for ZO-1 and DAPI-stained nuclei in syncytia were manually counted. 1000 nuclei were scored per image, and 2 replicates were averaged. <b>(C)</b> To assay for cell-to-cell spread, PTP1B+ MEFs or PTP1B^-/- MEFs were infected (100 PFU/well) with strains KOS or 17, and the cells were incubated in medium containing 5 mg/ml pooled IgG. At 42 hpi, the cells were immunostained for VP5, and the areas of 10–15 plaques per sample were measured. Data are represented as mean ±SD from 3 independent experiments. A student T-test was used to determine statistical significance for the PTP1B^-/- samples compared to the PTP1B+ control. <b>(D and E)</b> PTP1B+ MEFs or PTP1B^-/- MEFs were infected with strain 17 (100 PFU/well) and incubated in medium containing 5 mg/ml of pooled human IgG along with either DMSO or 30 μM inhibitor XXII. At 42 hpi, cells were immunostained for VP5. (D) Representative plaques are shown. (E) To quantify the results, 10–15 plaques per sample were measured, and the mean plaque area was plotted from 2 independent experiments. A student T-test was used to determine statistical significance for samples compared to the PTP1B+ DMSO control. <b>(F)</b> To ascertain their ability to respond to salubrinal, uninfected PTP1B+ MEFs or PTP1B^-/- MEFs were incubated in medium containing DMSO, 50 μM salubrinal, or 1 μM thapsigargin for 2 hours. Cell lysates were harvested in the presence of phosphatase inhibitors and probed via western blotting for total eIF2α and phosphorylated eIF2α. Duplicate samples were analyzed in the same western blot and band intensities were quantified.</p

Host protein tyrosine phosphatase 1B is important for salubrinal-induced fusion.

<p><b>(A)</b> Vero cells were infected with strain 17 (MOI = 3) and incubated in DMSO, 50 μM salubrinal, or 50 μM guanabenz (GBZ) for 12 hours. Cell lysates were prepared in the presence of phosphatase inhibitors and probed via western blotting for total eIF2α and for phosphorylated eIF2α. <b>(B)</b> Cells were infected with KOS (MOI = 3) and incubated in DMSO, salubrinal, or guanabenz. At 24 hpi, cell lysates and media were harvested, and viral titers (cell lysates + media) were measured. Data are the averages of 2 replicates with statistical significance determined by a student T-test. <b>(C)</b> Cells were infected with strain 17 (MOI = 3) and treated with 50 μM salubrinal and increasing amounts of PTP1B inhibitors TCS-401 or XXII. At 12 hpi, the fusion ratio was determined by flow cytometry. Data are represented as the mean ±SD from 3 independent experiments, and a student T-test was used to determine statistical significance for samples compared to the salubrinal-only control. <b>(D)</b> C10 cells were transfected with plasmids encoding gB, gD, gH, and gL in a 3:1:1:1 ratio and treated with DMSO, 50 μM salubrinal, or 50 μM inhibitor XXII for 16 hours. The 10 largest syncytia per sample were measured by counting the number of nuclei per syncytium. A student T-test was used to compare samples to the DMSO-only control.</p