key: cord-0748731-5t25ewrx authors: Mostafa, Heba H.; Luo, Chun Huai; Morris, C. Paul; Li, Maggie; Swanson, Nicholas J.; Amadi, Adannaya; Gallagher, Nicholas; Pekosz, Andrew title: SARS-CoV-2 infections in mRNA vaccinated individuals are biased for viruses encoding spike E484K and associated with reduced infectious virus loads that correlate with respiratory antiviral IgG levels date: 2022-04-04 journal: J Clin Virol DOI: 10.1016/j.jcv.2022.105151 sha: e2420195ebe3b9e3c860693e48381eb339962ce8 doc_id: 748731 cord_uid: 5t25ewrx INTRODUCTION: COVID-19 large scale immunization in the US has been associated with breakthrough positive molecular testing. In this study, we investigated whether a positive test is associated with a high anti-viral IgG, specific viral variant, recovery of infectious virus, or symptomatic infection during an early phase after vaccination rollout. METHODS: We identified 133 SARS-CoV-2 positive patients who had received two doses of either Pfizer-BioNTech (BNT162b2) or Moderna (mRNA-1273) vaccines, the 2nd of which was received between January and April of 2021. The positive samples were collected between January and May of 2021. Samples were sequenced to characterize the whole genome and Spike protein changes and cycle thresholds that reflect viral loads were determined using a single molecular assay. Respiratory SARS-CoV-2 IgG antibodies were examined using ELISA and specimens were grown on cell culture to assess the recovery of infectious virus as compared to a control unvaccinated cohort. RESULTS: Of 133 specimens, 24 failed sequencing and yielded a negative or very low viral load on the repeat PCR. Of 109 specimens that were used for further genome analysis, 68 (62.4%) were from symptomatic infections, 11 (10.1%) were admitted for COVID-19, and 2 (1.8%) required ICU admission with no associated mortality. The predominant virus variant was the Alpha (B.1.1.7), however a significant association between lineage B.1.526 and amino acid change S: E484K with positives after vaccination was noted. A significant reduction of the recovery of infectious virus on cell culture was accompanied by an increase in localized IgG levels in respiratory samples of vaccinated individuals. CONCLUSIONS: Vaccination reduces the recovery of infectious virus in breakthrough infections caused primarily by the Alpha variant accompanied by an increase in upper respiratory tract IgG levels. SARS-CoV-2 has caused a devastating pandemic. Millions of global deaths have been recorded with thousands of new cases diagnosed daily, a trend that significantly changed with the large-scale vaccination in certain countries including the US (https://coronavirus.jhu.edu/map.html). Even though vaccines have high efficacy (1, 2) and undoubtedly, have reduced COVID-19 mortality and severe disease in countries that accelerated mass immunization (3), breakthrough infections have been reported. With the appearance of SARS-CoV-2 variants that are more transmissible or capable of evading vaccine induced immune responses, surveillance has become of utmost importance and genome characterization of positives after vaccination is essential. Currently, data support that vaccines approved for use in the US are effective against most of the currently circulating variants (4, 5) . With the general increase in the circulation of variants of concern (VOC), it is expected to see a high percentage of breakthrough infections caused by these variants. In this manuscript, we provide a comprehensive analysis of the first 133 positives after vaccination diagnosed by Johns Hopkins Clinical Virology laboratory during the early phase after vaccination rollout. Samples were enrolled in our whole genome sequencing for surveillance pipeline and were retested by the PerkinElmer PCR assay to obtain comparable cycle threshold values (Cts). The recovery of infectious virus from positives after vaccination was determined as well as local SARS-CoV-2 IgG levels in the respiratory samples using ELISA and compared to a control unvaccinated cohort. The research was performed with a waiver of consent under IRB00221396. Whole genomes were made publicly available at GISAID. Remnant Nasopharyngeal (symptomatic patients) or lateral mid-turbinate nasal swabs (asymptomatic screening) after standard of care diagnostic or screening testing were collected and used for genome sequencing. At Johns Hopkins Medical System, SARS-CoV-2 clinical testing is performed for inpatients and outpatients as well as standard of care asymptomatic presurgical screening and the laboratory serves a large geographic area in the National Capital Region (Baltimore, Virginia, and D.C.) (6) . Molecular methods used for screening and diagnosis include the NeuMoDx SARS-CoV-2 (Qiagen), Cobas SARS-CoV-2 (Roche), Xpert Xpress SARS-CoV-2/Flu/RSV (Cepheid), in addition to the RealStar® SARS-CoV-2 RT-PCR (Altona Diagnostics), ePlex Respiratory Pathogen Panel 2 (Roche), Aptima SARS-CoV-2 (Hologic), and Accula SARS-CoV-2 assays (ThermoFisher Scientific) (7-10). Breakthrough infections selected for the study were the first 133 positive samples from vaccinated patients who: 1) received two doses of either fizer/BioNTech BNT162b2 or Moderna mRNA-1273, and 2) were identified by our group through our SARS-CoV-2 genomic surveillance. Positive samples from vaccinated patients had collection times that ranged from 2 to 99 days from the second dose (Table S1) . Tables S1 and S2 and Table 1 summarizes the clinical and metadata of the vaccinated and control cohorts. The control group used for whole genome comparison were randomly selected from samples sequenced by our group (Table S3 includes GISAID submission information), using MatchIt in R (method= 'optimal', ratio=5) based on collection date (to control for differences that might be related to the frequency of the circulating variants at a given time frame). This control group (N = 335) was independent of the control samples used for cell culture, ELISA, and cycle thresholds comparisons (Table S3 and metadata shown in Table 1 ). Patient data was extracted by manual chart reviews of electronic health records. Symptomatic versus asymptomatic status was determined by the ordering clinician and included as a questionnaire in the patients' charts. Vaccination status was determined through documented vaccinations given and selfreports documented in the system. Notably, Johns Hopkins has an agreement with the State of Maryland and Chesapeake Regional Information System for our Patients (CRISP) to provide vaccination status for all Johns Hopkins Patients. Automated nucleic acid extraction was performed using the chemagic 360 (PerkinElmer) following the manufacturer's protocol, with an RNA elution volume of 60µL. Real-time reverse transcriptase PCR (rRT-PCR) was performed using the PerkinElmer SARS-CoV-2 Real-time RT-PCR Assay following the package insert (https://www.fda.gov/media/136410/download). Whole genome sequencing and analysis were performed using the ARTIC protocol as previously described (6, 11) . Thresholds were set to a minimum of 90% coverage and 100 mean depth. Lineages were assigned by Pangolin (COG-UK; coguk.io) and ambiguous mutations were visually confirmed with Integrated Genomics viewer (IGV) (Version 2.8.10). The EUROIMMUN Anti-SARS-CoV-2 ELISA (IgG) was run using undiluted respiratory samples and following the package insert (https://www.fda.gov/media/137609/download). This assay detects antibodies to the S1 domain of the spike protein of SARS-CoV-2. The assay has a cut-off < 0.8 for negative results and ≥ 0.8 to < 1.1 as borderline. The value 1.1 was used as a cut off for positivity for nasopharyngeal/ NMT swab specimen types even though these sources were not tested by the manufacturer. Vero-TMPRSS2 cells were cultured and infected with aliquots of swab specimens as previously described for VeroE6 cells (12) . The presence of SARS-CoV-2 was confirmed by reverse transcriptase PCR (qPCR). Statistics were performed using GraphPad Prism. Chi-square and Fisher Exact tests were used for categorical variable comparisons and t-test was used for comparing continuous independent variables. For Lineage and spike analyses ( Figure 1C had failed sequencing with 0% coverage which we believe was related to very low viral loads (Table S1 ). Twenty-one sequences had coverage of less than 50% and average depth of less than 50 and a lineage was not called. Of the genomes that had more than 50% coverage and more than an average depth of 50 (a total of 88), the majority (61%) belonged to the B.1.1.7 lineage (Alpha variant) (20I/501Y.V1 clade), consistent with its predominance in this time frame (11, 13) , followed by the 20C lineages (Iota variants) B.1.526 (9%) and B.1.526.1 (4.5%) ( Figure 1A and B) . When a cohort of our characterized genomes for surveillance were randomly selected as a control group based on the sample collection dates, with only genomes that have ≥ 90% coverage used for the analysis (vaccinated: N = 67, and control: N = 335), a significantly higher proportion of individuals infected with lineage B.1.526 were vaccinated (Chi-square with Bonferroni correction p = 0.022, Figure 1C ). Spike substitution analysis showed that the S: E484K was associated with genomes of the vaccinated group (p = 0.0032, Figure 1D and Tables S1 and S3). (Table S2 ). The cohort was selected randomly to include the Alpha variant lineage as well as prior predominant lineages. To compare the recovery of infectious virus between positives from fully vaccinated (N = 114 with sufficient volume) and control (N = 124) groups, samples were cultured on Vero-TMPRSS2 cells. As expected, samples that had failed sequencing and were thought of as very low viral load did not yield infectious virus and were excluded from further analysis (Table S1 ). Of the fully vaccinated group, 17 of 92 samples (18.5%) showed CPE on cell culture compared to 80 out of 124 (64.5%) of the control group (Fisher Exact test, p < 0.00001). Notably, the control group recovery on cell culture was faster than the vaccinated group with 44 out of 80 samples (55%) positive 2 days after culture compared to no samples showing CPE for the vaccinated group in the same day ( Figure 2A and B) . To study the localized SARS-CoV-2 antibodies in the respiratory samples and their correlation to the observed CPE phenotype, respiratory samples were tested by ELISA for SARS-CoV-2 IgG. A significant increase in SARS-CoV-2 respiratory IgG levels were noted in the respiratory samples from vaccinated individuals when compared to the control group ( Figure 2C , t test, P < 0.0001) with 66.7% positive samples from the vaccinated group versus 5.9% from the control group ( Figure 2C ). To examine if the discrepant virus recovery on cell culture is secondary to differences in the viral loads in vaccinated versus unvaccinated groups (12) Consistent with data from the whole cohort, higher nasal/ nasopharyngeal IgG levels ( Figure 3C , P < 0.0001) was noted for the fully vaccinated group. Figure 4C , t test, P = 0.004). The absence of symptoms did not correlate with higher IgG levels when we compared samples from symptomatic vaccinated to asymptomatic vaccinated individuals ( Figure 5A ). Notably, a significant increase in the mean Ct value for the asymptomatic group was noted (27.6 versus 23.2, t test, P = 0.0048, Figure 5B ) as well as a reduction in the mean genome coverage (70.8% versus 84.9%, t test, P = 0.0284, Figure 5C ). Infectious virus was recovered from only 2 samples from asymptomatic patients (6.5%) in contrast to 15 from symptomatic patients (24.6%) (Table S1 ). In this study, we provide an analysis of a cohort of 133 SARS-CoV-2 positive specimens collected after the completion of COVID-19 vaccination during an early phase after vaccination rollout. Genomic Data from the CDC showed that the widely used mRNA vaccines in the US reduce the infection risk by 91% and data from different groups confirm that breakthrough infections after full vaccination were scarce prior to the surge of the Delta variant (14) (15) (16) . Data also show that vaccines reduce symptomatic and asymptomatic infections and RNA loads (17-20) and reduce viral replication in the respiratory tracts of animals (21) (22) (23) . The emergence of SARS-CoV-2 variants of concern and interest were associated with changes in the spike protein within regions that could affect the receptor binding domain or impact the neutralization of the virus by natural or vaccine induced immune responses (24) (25) (26) (27) . Those variants were associated with an increase in transmissibility and in particular the S: E484K substitution was associated with a compromise in the neutralization by monoclonal antibodies rendering this change "of therapeutic concern". The S: E484K independently emerged in multiple lineages in distant geographical locations including the Gamma and the Beta and those lineages showed some reduction in neutralization by sera collected from immunized individuals as well as decreased susceptibility to certain therapeutic monoclonal antibodies. Additionally, the Gamma and Beta were associated with reductions in the vaccine efficacy data in locations of their predominance (28, 29) . The S: E484K is also present in some strains of lineage B.1.526, a lineage which was significantly associated with positives after vaccination in our cohort, even though in a previous study, it was not reported to associate with positives after vaccination (30) . Our study shows that the S: E484K is significantly associated with breakthrough cases after vaccination in a well-controlled analysis that used a large cohort of controls from a matched time frame of sample collection. In conclusion, our study combined genomic analysis, cell culture, and serology to correlate reduced recovery of infectious virus from positives after vaccination with increased localized IgG levels during the first few months after the COVID-19 vaccination rollout. Our data showed a significant association of S: E484K with positives after full vaccination using a well-controlled analysis and a relatively large sample size in a time when the Alpha variant predominated. We declare no relevant competing interests Whole genome data were made available publicly and raw genomic data requests could be directed to HHM. ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Table S1 . Detailed Breakthrough cohort sample data. not done; no left-over samples were available for serology or PerkinElmers PCR runs, ND; not detected, CPE; cytopathic effect Table S2 . Detailed data of the control group used for cell culture, Cycle thresholds, and ELISA comparisons. Group CCT. 2021. Safety, Immunogenicity, and Efficacy of the BNT162b2 Covid-19 Vaccine in Adolescents Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine BNT162b2 mRNA Covid-19 Vaccine in a Nationwide Mass Vaccination Setting National Study Group for C-V. 2021. Effectiveness of the BNT162b2 Covid-19 Vaccine against the B.1.1.7 and B.1.351 Variants SARS-CoV-2 mRNA vaccines induce broad CD4+ T cell responses that recognize SARS-CoV-2 variants and HCoV-NL63 Genomic diversity of SARS-CoV-2 during early introduction into the Baltimore-Washington metropolitan area Clinical performance of the GenMark Dx ePlex respiratory pathogen panels for upper and lower respiratory tract infections Multi-center Evaluation of the Cepheid Xpert(R) Xpress SARS-CoV-2/Flu/RSV Test Comparison of the analytical sensitivity of seven commonly used commercial SARS-CoV-2 automated molecular assays Comparing the analytical performance of three SARS-CoV-2 molecular diagnostic assays An Update on SARS-CoV-2 Diversity in the United States National Capital Region: Evolution of Novel and Variants of Concern Repeat COVID-19 Molecular Testing: Correlation of SARS-CoV-2 Culture with Molecular Assays and Cycle Thresholds Infection with the SARS-CoV-2 Delta Variant is Associated with Higher Recovery of Infectious Virus Compared to the Alpha Variant in both Unvaccinated and Vaccinated Individuals Post-vaccination SARS-CoV-2 infections and incidence of the B.1.427/B.1.429 variant among healthcare personnel at a northern California academic medical center Impact and effectiveness of mRNA BNT162b2 vaccine against SARS-CoV-2 infections and COVID-19 cases, hospitalisations, and deaths following a nationwide vaccination campaign in Israel: an observational study using national surveillance data Association Between Vaccination With BNT162b2 and Incidence of Symptomatic and Asymptomatic SARS-CoV-2 Infections Among Health Care Workers Interim Estimates of Vaccine Effectiveness of BNT162b2 and mRNA-1273 COVID-19 Vaccines in Preventing SARS-CoV-2 Infection Among Health Care Personnel, First Responders, and Other Essential and Frontline Workers -Eight Asymptomatic and Symptomatic SARS-CoV-2 Infections After BNT162b2 Vaccination in a Routinely Screened Workforce Initial report of decreased SARS-CoV-2 viral load after inoculation with the BNT162b2 vaccine Evaluation of the mRNA-1273 Vaccine against SARS-CoV-2 in Nonhuman Primates Single-shot Ad26 vaccine protects against SARS-CoV-2 in rhesus macaques BNT162b vaccines protect rhesus macaques from SARS-CoV-2 Emergence of SARS-CoV-2 B.1.1.7 Lineage -United States Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7 Increased resistance of SARS-CoV-2 variant P.1 to antibody neutralization Safety and Efficacy of Single-Dose Ad26.COV2.S Vaccine against Covid-19 Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil Rapid Emergence and Epidemiologic Characteristics of the SARS-CoV-2 B.1.526 Variant