Reduced control of SARS-CoV-2 infection associates with lower mucosal antibody responses in pregnancy.

Pregnant patients are at greater risk of hospitalization with severe COVID-19 than non-pregnant people. This was a retrospective observational cohort study of remnant clinical specimens from patients who visited acute care hospitals within the Johns Hopkins Health System in the Baltimore, MD-Washington DC, area between October 2020 and May 2022. Participants included confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected pregnant people and matched non-pregnant people (the matching criteria included age, race/ethnicity, area deprivation index, insurance status, and vaccination status to ensure matched demographics). The primary dependent measures were clinical COVID-19 outcomes, infectious virus recovery, viral RNA levels, and mucosal anti-spike (S) IgG titers from upper respiratory tract samples. A total of 452 individuals (117 pregnant and 335 non-pregnant) were included in the study, with both vaccinated and unvaccinated individuals represented. Pregnant patients were at increased risk of hospitalization (odds ratio [OR] = 4.2; confidence interval [CI] = 2.0-8.6), intensive care unit admittance (OR = 4.5; CI = 1.2-14.2), and being placed on supplemental oxygen therapy (OR = 3.1; CI = 1.3-6.9). Individuals infected during their third trimester had higher mucosal anti-S IgG titers and lower viral RNA levels (P < 0.05) than those infected during their first or second trimesters. Pregnant individuals experiencing breakthrough infections due to the Omicron variant had reduced anti-S IgG compared to non-pregnant patients (P < 0.05). The observed increased severity of COVID-19 and reduced mucosal antibody responses particularly among pregnant participants infected with the Omicron variant suggest that maintaining high levels of SARS-CoV-2 immunity through booster vaccines may be important for the protection of this at-risk population.IMPORTANCEIn this retrospective observational cohort study, we analyzed remnant clinical samples from non-pregnant and pregnant individuals with confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections who visited the Johns Hopkins Hospital System between October 2020 and May 2022. Disease severity, including intensive care unit admission, was greater among pregnant than non-pregnant patients. Vaccination reduced recovery of infectious virus and viral RNA levels in non-pregnant patients, but not in pregnant patients. In pregnant patients, increased nasopharyngeal viral RNA levels and recovery of infectious virus were associated with reduced mucosal IgG antibody responses, especially among women in their first trimester of pregnancy or experiencing breakthrough infections from Omicron variants. Taken together, this study provides insights into how pregnant patients are at greater risk of severe COVID-19. The novelty of this study is that it focuses on the relationship between the mucosal antibody response and its association with virus load and disease outcomes in pregnant people, whereas previous studies have focused on serological immunity. Vaccination status, gestational age, and SARS-CoV-2 omicron variant impact mucosal antibody responses and recovery of infectious virus from pregnant patients.

Outpatient COVID-19 convalescent plasma recipient antibody thresholds correlated to reduced hospitalizations within a randomized trial.

BACKGROUNDCOVID-19 convalescent plasma (CCP) virus-specific antibody levels that translate into recipient posttransfusion antibody levels sufficient to prevent disease progression are not defined.METHODSThis secondary analysis correlated donor and recipient antibody levels to hospitalization risk among unvaccinated, seronegative CCP recipients within the outpatient, double-blind, randomized clinical trial that compared CCP to control plasma. The majority of COVID-19 CCP arm hospitalizations (15/17, 88%) occurred in this unvaccinated, seronegative subgroup. A functional cutoff to delineate recipient high versus low posttransfusion antibody levels was established by 2 methods: (i) analyzing virus neutralization-equivalent anti-Spike receptor-binding domain immunoglobulin G (anti-S-RBD IgG) responses in donors or (ii) receiver operating characteristic (ROC) curve analysis.RESULTSSARS-CoV-2 anti-S-RBD IgG antibody was volume diluted 21.3-fold into posttransfusion seronegative recipients from matched donor units. Virus-specific antibody delivered was approximately 1.2 mg. The high-antibody recipients transfused early (symptom onset within 5 days) had no hospitalizations. A CCP-recipient analysis for antibody thresholds correlated to reduced hospitalizations found a statistical significant association between early transfusion and high antibodies versus all other CCP recipients (or control plasma), with antibody cutoffs established by both methods-donor-based virus neutralization cutoffs in posttransfusion recipients (0/85 [0%] versus 15/276 [5.6%]; P = 0.03) or ROC-based cutoff (0/94 [0%] versus 15/267 [5.4%]; P = 0.01).CONCLUSIONIn unvaccinated, seronegative CCP recipients, early transfusion of plasma units in the upper 30% of study donors' antibody levels reduced outpatient hospitalizations. High antibody level plasma units, given early, should be reserved for therapeutic use.TRIAL REGISTRATIONClinicalTrials.gov NCT04373460.FUNDINGDepartment of Defense (W911QY2090012); Defense Health Agency; Bloomberg Philanthropies; the State of Maryland; NIH (3R01AI152078-01S1, U24TR001609-S3, 1K23HL151826NIH); the Mental Wellness Foundation; the Moriah Fund; Octapharma; the Healthnetwork Foundation; the Shear Family Foundation; the NorthShore Research Institute; and the Rice Foundation.

Antigen-display exosomes provide adjuvant-free protection against SARS-CoV-2 disease at nanogram levels of spike protein.

As the only bionormal nanovesicle, exosomes have high potential as a nanovesicle for delivering vaccines and therapeutics. We show here that the loading of type-1 membrane proteins into the exosome membrane is induced by exosome membrane anchor domains, EMADs, that maximize protein delivery to the plasma membrane, minimize protein sorting to other compartments, and direct proteins into exosome membranes. Using SARS-CoV-2 spike as an example and EMAD13 as our most effective exosome membrane anchor, we show that cells expressing a spike-EMAD13 fusion protein produced exosomes that carry dense arrays of spike trimers on 50% of all exosomes. Moreover, we find that immunization with spike-EMAD13 exosomes induced strong neutralizing antibody responses and protected hamsters against SARS-CoV-2 disease at doses of just 0.5-5 ng of spike protein, without adjuvant, demonstrating that antigen-display exosomes are particularly immunogenic, with important implications for both structural and expression-dependent vaccines.

A SARS-CoV-2 RBD vaccine fused to the chemokine MIP-3α elicits sustained murine antibody responses over 12 months and enhanced lung T-cell responses.

Background: Previous studies have demonstrated enhanced efficacy of vaccine formulations that incorporate the chemokine macrophage inflammatory protein 3α (MIP-3α) to direct vaccine antigens to immature dendritic cells. To address the reduction in vaccine efficacy associated with a mutation in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutants, we have examined the ability of receptor-binding domain vaccines incorporating MIP-3α to sustain higher concentrations of antibody when administered intramuscularly (IM) and to more effectively elicit lung T-cell responses when administered intranasally (IN). Methods: BALB/c mice aged 6-8 weeks were immunized intramuscularly or intranasally with DNA vaccine constructs consisting of the SARS-CoV-2 receptor-binding domain alone or fused to the chemokine MIP-3α. In a small-scale (n = 3/group) experiment, mice immunized IM with electroporation were followed up for serum antibody concentrations over a period of 1 year and for bronchoalveolar antibody levels at the termination of the study. Following IN immunization with unencapsulated plasmid DNA (n = 6/group), mice were evaluated at 11 weeks for serum antibody concentrations, quantities of T cells in the lungs, and IFN-γ- and TNF-α-expressing antigen-specific T cells in the lungs and spleen. Results: At 12 months postprimary vaccination, recipients of the IM vaccine incorporating MIP-3α had significantly, approximately threefold, higher serum antibody concentrations than recipients of the vaccine not incorporating MIP-3α. The area-under-the-curve analyses of the 12-month observation interval demonstrated significantly greater antibody concentrations over time in recipients of the MIP-3α vaccine formulation. At 12 months postprimary immunization, only recipients of the fusion vaccine had concentrations of serum-neutralizing activity deemed to be effective. After intranasal immunization, only recipients of the MIP-3α vaccine formulations developed T-cell responses in the lungs significantly above those of PBS controls. Low levels of serum antibody responses were obtained following IN immunization. Conclusion: Although requiring separate IM and IN immunizations for optimal immunization, incorporating MIP-3α in a SARS-CoV-2 vaccine construct demonstrated the potential of a stable and easily produced vaccine formulation to provide the extended antibody and T-cell responses that may be required for protection in the setting of emerging SARS-CoV-2 variants. Without electroporation, simple, uncoated plasmid DNA incorporating MIP-3α administered intranasally elicited lung T-cell responses.

Three doses of COVID-19 mRNA vaccine induce class-switched antibody responses in inflammatory arthritis patients on immunomodulatory therapies.

Patients with inflammatory arthritis (IA) are at increased risk of severe COVID-19 due to medication-induced immunosuppression that impairs host defenses. The aim of this study was to assess antibody and B cell responses to COVID-19 mRNA vaccination in IA patients receiving immunomodulatory therapies. Adults with IA were enrolled through the Johns Hopkins Arthritis Center and compared with healthy controls (HC). Paired plasma and peripheral blood mononuclear cell (PBMC) samples were collected prior to and 30 days or 6 months following the first two doses of mRNA vaccines (D2; HC=77 and IA=31 patients), or 30 days following a third dose of mRNA vaccines (D3; HC=11 and IA=96 patients). Neutralizing antibody titers, total binding antibody titers, and B cell responses to vaccine and Omicron variants were analyzed. Anti-Spike (S) IgG and S-specific B cells developed appropriately in most IA patients following D3, with reduced responses to Omicron variants, and negligible effects of medication type or drug withholding. Neutralizing antibody responses were lower compared to healthy controls after both D2 and D3, with a small number of individuals demonstrating persistently undetectable neutralizing antibody levels. Most IA patients respond as well to mRNA COVID-19 vaccines as immunocompetent individuals by the third dose, with no evidence of improved responses following medication withholding. These data suggest that IA-associated immune impairment may not hinder immunity to COVID-19 mRNA vaccines in most individuals.

Spike-protein proteolytic antibodies in COVID-19 convalescent plasma contribute to SARS-CoV-2 neutralization.

Understanding the mechanisms of antibody-mediated neutralization of SARS-CoV-2 is critical in combating the COVID-19 pandemic. Based on previous reports of antibody catalysis, we investigated the proteolysis of spike (S) by antibodies in COVID-19 convalescent plasma (CCP) and its contribution to viral neutralization. Quenched fluorescent peptides were designed based on S epitopes to sensitively detect antibody-mediated proteolysis. We observed epitope cleavage by CCP from different donors which persisted when plasma was heat-treated or when IgG was isolated from plasma. Further, purified CCP antibodies proteolyzed recombinant S domains, as well as authentic viral S. Cleavage of S variants suggests CCP antibody-mediated proteolysis is a durable phenomenon despite antigenic drift. We differentiated viral neutralization occurring via direct interference with receptor binding from that occurring by antibody-mediated proteolysis, demonstrating that antibody catalysis enhanced neutralization. These results suggest that antibody-catalyzed damage of S is an immunologically relevant function of neutralizing antibodies against SARS-CoV-2.

Outpatient COVID-19 convalescent plasma recipient antibody thresholds correlated to reduced hospitalizations within a randomized trial.

BACKGROUND: The COVID-19 convalescent plasma (CCP) viral specific antibody levels that translate into recipient post-transfusion antibody levels sufficient to prevent disease progression is not defined. METHODS: This secondary analysis correlated donor and recipient antibody levels to hospitalization risk among unvaccinated, seronegative CCP recipients within the outpatient, double blind, randomized clinical trial that compared CCP to control plasma. The majority of COVID-19 CCP arm hospitalizations (15/17, 88%) occurred in this unvaccinated, seronegative subgroup. A functional cutoff to delineate recipient high versus low post-transfusion antibody levels was established by two methods: 1) analyzing virus neutralization-equivalent anti-S-RBD IgG responses in donors or 2) receiver operating characteristic (ROC) analysis. RESULTS: SARS-CoV-2 anti-S-RBD IgG antibody was diluted by a factor of 21.3 into post-transfusion seronegative recipients from matched donor units. Viral specific antibody delivered approximated 1.2 mg. The high antibody recipients transfused early (symptom onset within 5 days) had no hospitalizations. A CCP recipient analysis for antibody thresholds correlated to reduced hospitalizations found a significant association with Fisher's exact test between early and high antibodies versus all other CCP recipients (or control plasma) with antibody cutoffs established by both methods-donor virus neutralization-based cutoff: (0/85; 0% versus 15/276; 5.6%) p=0.03 or ROC based cutoff: (0/94; 0% versus 15/267; 5.4%) p=0.01. CONCLUSION: In unvaccinated, seronegative CCP recipients, early transfusion of plasma units corresponding to the upper 30% of all study donors reduced outpatient hospitalizations. These high antibody level plasma units, given early, should be reserved for therapeutic use.Trial registration: NCT04373460. FUNDING: Defense Health Agency and others.

SARS-CoV-2 seroprevalence among blood donors in Uganda: 2019-2022.

BACKGROUND: The true burden of COVID-19 in low- and middle-income countries remains poorly characterized, especially in Africa. Even prior to the availability of SARS-CoV-2 vaccines, countries in Africa had lower numbers of reported COVID-19 related hospitalizations and deaths than other regions globally. METHODS: Ugandan blood donors were evaluated between October 2019 and April 2022 for IgG antibodies to SARS-CoV-2 nucleocapsid (N), spike (S), and five variants of the S protein using multiplexed electrochemiluminescence immunoassays (MesoScale Diagnostics, Rockville, MD). Seropositivity for N and S was assigned using manufacturer-provided cutoffs and trends in seroprevalence were estimated by quarter. Statistically significant associations between N and S antibody seropositivity and donor characteristics in November-December 2021 were assessed by chi-square tests. RESULTS: A total of 5393 blood unit samples from donors were evaluated. N and S seropositivity increased throughout the pandemic to 82.6% in January-April 2022. Among seropositive individuals, N and S antibody levels increased ≥9-fold over the study period. In November-December 2021, seropositivity to N and S antibody was higher among repeat donors (61.3%) compared with new donors (55.1%; p = .043) and among donors from Kampala (capital city of Uganda) compared with rural regions (p = .007). Seropositivity to S antibody was significantly lower among HIV-seropositive individuals (58.8% vs. 84.9%; p = .009). CONCLUSIONS: Despite previously reported low numbers of COVID-19 cases and related deaths in Uganda, high SARS-CoV-2 seroprevalence and increasing antibody levels among blood donors indicated that the country experienced high levels of infection over the course of the pandemic.

Reduced control of SARS-CoV-2 infection is associated with lower mucosal antibody responses in pregnant women.

Importance: Pregnant women are at increased risk of severe COVID-19, but the contribution of viral RNA load, the presence of infectious virus, and mucosal antibody responses remain understudied. Objective: To evaluate the association of COVID-19 outcomes following confirmed infection with vaccination status, mucosal antibody responses, infectious virus recovery and viral RNA levels in pregnant compared with non-pregnant women. Design: A retrospective observational cohort study of remnant clinical specimens from SARS-CoV-2 infected patients between October 2020-May 2022. Setting: Five acute care hospitals within the Johns Hopkins Health System (JHHS) in the Baltimore, MD-Washington, DC area. Participants: Participants included confirmed SARS-CoV-2 infected pregnant women and matched non-pregnant women (matching criteria included age, race/ethnicity, and vaccination status). Exposure: SARS-CoV-2 infection, with documentation of SARS-CoV-2 mRNA vaccination. Main Outcomes: The primary dependent measures were clinical COVID-19 outcomes, infectious virus recovery, viral RNA levels, and mucosal anti-spike (S) IgG titers from upper respiratory tract samples. Clinical outcomes were compared using odds ratios (OR), and measures of virus and antibody were compared using either Fisher's exact test, two-way ANOVA, or regression analyses. Results were stratified according to pregnancy, vaccination status, maternal age, trimester of pregnancy, and infecting SARS-CoV-2 variant. Resultss: A total of 452 individuals (117 pregnant and 335 non-pregnant) were included in the study, with both vaccinated and unvaccinated individuals represented. Pregnant women were at increased risk of hospitalization (OR = 4.2; CI = 2.0-8.6), ICU admittance, (OR = 4.5; CI = 1.2-14.2), and of being placed on supplemental oxygen therapy (OR = 3.1; CI =13-6.9). An age-associated decrease in anti-S IgG titer and corresponding increase in viral RNA levels (P< 0.001) was observed in vaccinated pregnant, but not non-pregnant, women. Individuals in their 3rd trimester had higher anti-S IgG titers and lower viral RNA levels (P< 0.05) than those in their 1st or 2nd trimesters. Pregnant individuals experiencing breakthrough infections due to the omicron variant had reduced anti-S IgG compared to non-pregnant women (P< 0.05). Conclusions and Relevance: In this cohort study, vaccination status, maternal age, trimester of pregnancy, and infecting SARS-CoV-2 variant were each identified as drivers of differences in mucosal anti-S IgG responses in pregnant compared with non-pregnant women. Observed increased severity of COVID-19 and reduced mucosal antibody responses particularly among pregnant participants infected with the Omicron variant suggest that maintaining high levels of SARS-CoV-2 immunity may be important for protection of this at-risk population.

Second-Phase Hepatitis C Plasma Viral Kinetics Directly Reflects Reduced Intrahepatic Burden of Hepatitis C Virus.

BACKGROUND: Mathematical models explain how antivirals control viral infections. Hepatitis C virus (HCV) treatment results in at least 2 phases of decline in viremia. The first phase reflects clearance of rapidly produced virions. The second phase is hypothesized to derive from loss of infected cells but has been challenging to prove. METHODS: Using single-cell methods, we quantified the number of hepatitis C virus (HCV)-infected hepatocytes in liver biopsies taken before and within 7 days of initiating direct-acting antivirals (DAAs) in a double-blinded randomized controlled trial testing 2 (sofosbuvir-velpatasvir) versus 3 (sofosbuvir-velpatasvir-voxilaprevir) DAAs. RESULTS: We employed thousands of intrahepatic measurements in 10 persons with chronic genotype 1a HCV infection: median proportion of infected hepatocytes declined from 11.3% (range, 1.3%-59%) to 0.6% (range, <0.3%-5.8%), a loss of 75%-95% infected hepatocytes. Plasma viremia correlated with numbers of HCV-infected hepatocytes (r = 0.77; P < .0001). Second-phase plasma dynamics and changes in infected hepatocytes were indistinct (P = .16), demonstrating that second-phase viral dynamics derive from loss of infected cells. DAAs led to a decline in intracellular HCV RNA and interferon-stimulated gene expression (P < .05 for both). CONCLUSIONS: We proved that second-phase viral dynamics reflect decay of intrahepatic burden of HCV, partly due to clearance of HCV RNA from hepatocytes. CLINICAL TRIALS REGISTRATION: NCT02938013.

Omicron Subvariants: Clinical, Laboratory, and Cell Culture Characterization.

BACKGROUND: The variant of concern Omicron has become the sole circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant for the past several months. Omicron subvariants BA.1, BA.2, BA.3, BA.4, and BA.5 evolved over the time, with BA.1 causing the largest wave of infections globally in December 2021-January 2022. This study compared the clinical outcomes in patients infected with different Omicron subvariants and the relative viral loads and recovery of infectious virus from upper respiratory specimens. METHODS: SARS-CoV-2-positive remnant clinical specimens, diagnosed at the Johns Hopkins Microbiology Laboratory between December 2021 and July 2022, were used for whole-genome sequencing. The clinical outcomes of infections with Omicron subvariants were compared with infections with BA.1. Cycle threshold (Ct) values and the recovery of infectious virus on the VeroTMPRSS2 cell line from clinical specimens were compared. RESULTS: BA.1 was associated with the largest increase in SARS-CoV-2 positivity rate and coronavirus disease 2019 (COVID-19)-related hospitalizations at the Johns Hopkins system. After a peak in January, cases decreased in the spring, but the emergence of BA.2.12.1 followed by BA.5 in May 2022 led to an increase in case positivity and admissions. BA.1 infections had a lower mean Ct value when compared with other Omicron subvariants. BA.5 samples had a greater likelihood of having infectious virus at Ct values <20. CONCLUSIONS: Omicron subvariants continue to be associated with a relatively high rate of polymerase chain reaction (PCR) positivity and hospital admissions. The BA.5 infections are more while BA.2 infections are less likely to have infectious virus, suggesting potential differences in infectibility during the Omicron waves.

Omicron Subvariants: Clinical, Laboratory, and Cell Culture Characterization.

Background: The variant of concern, Omicron, has become the sole circulating SARS-CoV-2 variant for the past several months. Omicron subvariants BA.1, BA.2, BA.3, BA.4, and BA.5 evolved over the time, with BA.1 causing the largest wave of infections globally in December 2021- January 2022. In this study, we compare the clinical outcomes in patients infected with different Omicron subvariants and compare the relative viral loads, and recovery of infectious virus from upper respiratory specimens. Methods: SARS-CoV-2 positive remnant clinical specimens, diagnosed at the Johns Hopkins Microbiology Laboratory between December 2021 and July 2022, were used for whole genome sequencing. The clinical outcomes of infections with Omicron subvariants were compared to infections with BA.1. Cycle threshold values (Ct) and the recovery of infectious virus on VeroTMPRSS2 cell line from clinical specimens were compared. Results: The BA.1 was associated with the largest increase in SARS-CoV-2 positivity rate and COVID-19 related hospitalizations at the Johns Hopkins system. After a peak in January cases fell in the spring, but the emergence of BA.2.12.1 followed by BA.5 in May 2022 led to an increase in case positivity and admissions. BA.1 infections had a lower mean Ct when compared to other Omicron subvariants. BA.5 samples had a greater likelihood of having infectious virus at Ct values less than 20. Conclusions: Omicron subvariants continue to associate with a relatively high positivity and admissions. The BA.5 infections are more while BA.2 infections are less likely to have infectious virus, suggesting potential differences in infectibility during the Omicron waves. Funding: Centers for Disease Control and Prevention contract 75D30121C11061, NIH/NIAID Center of Excellence in Influenza Research and Surveillance contract HHS N2772201400007C, Johns Hopkins University, Maryland department of health, and The Modeling Infectious Diseases in Healthcare Network (MInD) under awards U01CK000589.

Evaluation of Four Point of Care (POC) Antigen Assays for the Detection of the SARS-CoV-2 Variant Omicron.

Ensuring SARS-CoV-2 diagnostics that can reliably detect emerging variants has been an ongoing challenge. Due to the rapid spread of the Omicron variant, point-of-care (POC) antigen tests have become more widely used. This study aimed at (i) comparing the analytical sensitivity (LOD) of 4 POC antigen assays, BD Veritor, Abbott BinaxNow, Orasure InteliSwab and Quidel QuickVue, for the Omicron versus the Delta variant and (ii) verifying the reproducible detection of Omicron by the 4 antigen assays. The LOD for all four assays were evaluated using Omicron and Delta virus stocks quantified for infectivity and genome copies. The four assays detected all replicates of Omicron and Delta dilutions at 104 and 105 TCID50/mL, respectively. We quantified both viral stocks using droplet digital PCR (ddPCR), which revealed that the Omicron stock had equivalent copies of the N gene to Delta at a one log lower infectious virus. The Abbott BinaxNow and Orasure InteliSwab had the highest analytical sensitivity for Omicron while the Orasure InteliSwab and the Quidel QuickVue had the highest analytical sensitivity for Delta. When 14 SARS-CoV-2 real-time PCR positive nasal/nasopharyngeal swab samples (12 Omicron and 2 Delta, mean Ct = 19.1), were tested by the four assays, only the QuickVue detected all samples. Antigen test positivity correlated with recovery of infectious virus on cell culture in 9 out of 13 tested specimens from symptomatic, asymptomatic, unvaccinated, and vaccinated individuals. Although our study confirms the reduced analytical sensitivity of antigen testing compared to molecular methods, the Omicron variant was detectable by the four evaluated rapid antigen tests. IMPORTANCE In the manuscript, we report an evaluation of the capability of 4 point of care (POC) antigen assays, the BD Veritor, Abbott BinaxNow, Orasure InteliSwab and Quidel QuickVue to detect the Omicron variant of SARS-CoV-2, and we compared their analytical sensitivity for Omicron versus Delta. In this analysis we found that all four assays detected Omicron and Delta at 104 and 105 TCID50/mL, respectively. We further quantified the viral stocks used by droplet digital (ddPCR) and found that the Omicron stock had equivalent copies of the N gene to Delta at a one log lower infectious virus titer and that an increased RNA to infectious virus ratio may be contributing to discrepancies in limit of detection in Omicron compared to Delta. We evaluated 14 SARS-CoV-2 real-time PCR positive nasal/nasopharyngeal swab samples (12 Omicron and 2 Delta), with an average cycle threshold value of 19.1, and only the QuickVue showed 100% agreement.

The displacement of the SARS-CoV-2 variant Delta with Omicron: An investigation of hospital admissions and upper respiratory viral loads.

BACKGROUND: The increase in SARS-CoV-2 infections in December 2021 was driven primarily by the Omicron variant, which largely displaced the Delta over a three-week span. Outcomes from infection with Omicron remain uncertain. We evaluated whether clinical outcomes and viral loads differed between Delta and Omicron infections during the period when both variants were co-circulating. METHODS: In this retrospective observational cohort study, remnant clinical specimens, positive for SARS-CoV-2 after standard of care testing at the Johns Hopkins Microbiology Laboratory, between the last week of November and the end of December 2021, were used for whole viral genome sequencing. Cycle threshold values (Ct) for viral RNA, the presence of infectious virus, and levels of respiratory IgG were measured, and clinical outcomes were obtained. Differences in each measure were compared between variants stratified by vaccination status. FINDINGS: The Omicron variant displaced Delta during the study period and constituted 95% of the circulating lineages by the end of December 2021. Patients with Omicron infections (N = 1,119) were more likely to be vaccinated compared to patients with Delta (N = 908), but were less likely to be admitted (0.33 CI 0.21-0.52), require ICU level care (0.38 CI 0.17-0.87), or succumb to infection (0.26 CI 0.06-1.02) regardless of vaccination status. There was no statistically significant difference in Ct values based on the lineage regardless of the vaccination status. Recovery of infectious virus in cell culture was reduced in boosted patients compared to fully vaccinated without a booster and unvaccinated when infected with the Delta lineage. However, in patients with Omicron infections, recovery of infectious virus was not affected by vaccination. INTERPRETATION: Compared to Delta, Omicron was more likely to cause breakthrough infections of vaccinated individuals, yet admissions were less frequent. Admitted patients might develop severe disease comparable to Delta. Efforts for reducing Omicron transmission are required as, though the admission risk might be lower, the increased numbers of infections cause large numbers of hospitalizations. FUNDING: NIH/NIAID Center of Excellence in Influenza Research and Surveillance contract HHS N2772201400007C, Johns Hopkins University, Maryland department of health, Centers for Disease Control and Prevention contract 75D30121C11061, and The Modeling Infectious Diseases in Healthcare Network (MInD) under awards U01CK000589.

Large Scale SARS-CoV-2 Molecular Testing and Genomic Surveillance Reveal Prolonged Infections, Protracted RNA shedding, and Viral Reinfections.

Large-scale SARS-CoV-2 molecular testing coupled with whole genome sequencing in the diagnostic laboratories is instrumental for real-time genomic surveillance. The extensive genomic, laboratory, and clinical data provide a valuable resource for understanding cases of reinfection versus prolonged RNA shedding and protracted infections. In this study, data from a total of 22,292 clinical specimens, positive by SARS-CoV-2 molecular diagnosis at Johns Hopkins clinical virology laboratory between March 11th 2020 to September 23rd 2021, were used to identify patients with two or more positive results. A total of 3,650 samples collected from 1,529 patients who had between 2 and 20 positive results were identified in a time frame that extended up to 403 days from the first positive. Cycle threshold values (Ct) were available for 1,622 samples, the median of which was over 30 by 11 days after the first positive. Extended recovery of infectious virus on cell culture was notable for up to 70 days after the first positive in immunocompromised patients. Whole genome sequencing data generated as a part of our SARS-CoV-2 genomic surveillance was available for 1,027 samples from patients that had multiple positive tests. Positive samples collected more than 10 days after initial positive with high quality sequences (coverage >90% and mean depth >100), were more likely to be from unvaccinated, or immunosuppressed patients. Reinfections with viral variants of concern were found in 3 patients more than 130 days from prior infections with a different viral clade. In 75 patients that had 2 or more high quality sequences, the acquisition of more substitutions or deletions was associated with lack of vaccination and longer time between the recovered viruses. Our study highlights the value of integrating genomic, laboratory, and clinical data for understanding the biology of SARS-CoV-2 as well as for setting a precedent for future epidemics and pandemics.

Adaptive immune responses in vaccinated patients with symptomatic SARS-CoV-2 Alpha infection.

Benchmarks for protective immunity from infection or severe disease after SARS-CoV-2 vaccination are still being defined. Here, we characterized virus neutralizing and ELISA antibody levels, cellular immune responses, and viral variants in 4 separate groups: healthy controls (HCs) weeks (early) or months (late) following vaccination in comparison with symptomatic patients with SARS-CoV-2 after partial or full mRNA vaccination. During the period of the study, most symptomatic breakthrough infections were caused by the SARS-CoV-2 Alpha variant. Neutralizing antibody levels in the HCs were sustained over time against the vaccine parent virus but decreased against the Alpha variant, whereas IgG titers and T cell responses against the parent virus and Alpha variant declined over time. Both partially and fully vaccinated patients with symptomatic infections had lower virus neutralizing antibody levels against the parent virus than the HCs, similar IgG antibody titers, and similar virus-specific T cell responses measured by IFN-γ. Compared with HCs, neutralization activity against the Alpha variant was lower in the partially vaccinated infected patients and tended to be lower in the fully vaccinated infected patients. In this cohort of breakthrough infections, parent virus neutralization was the superior predictor of breakthrough infections with the Alpha variant of SARS-CoV-2.

A Quick Displacement of the SARS-CoV-2 variant Delta with Omicron: Unprecedented Spike in COVID-19 Cases Associated with Fewer Admissions and Comparable Upper Respiratory Viral Loads.

BACKGROUND: The increase in SARS-CoV-2 infections in December 2021 in the United States was driven primarily by the Omicron variant which largely displaced the Delta over a three week span. Outcomes from infection with the Omicron remain uncertain. We evaluate whether clinical outcomes and viral loads differ between Delta and Omicron infections during the period when both variants were co-circulating. METHODS: Remnant clinical specimens from patients that tested positive for SARS-CoV-2 after standard of care testing between the last week of November and the end of December 2021were used for whole viral genome sequencing. Cycle threshold values (Ct) for viral RNA, the presence of infectious virus, and levels of respiratory IgG were measured, and clinical outcomes were obtained. Differences in each measure were compared between variants stratified by vaccination status. RESULTS: The Omicron variant displaced the Delta during the study period and constituted 95% of the circulating lineages by the end of December 2021. Patients with Omicron infections (N= 1121) were more likely to be vaccinated compared to patients with Delta (N = 910), but were less likely to be admitted, require ICU level care, or succumb to infection regardless of vaccination status. There was no significant difference in Ct values based on the lineage regardless of the vaccination status. Recovery of infectious virus in cell culture was reduced in boosted patients compared to fully vaccinated without a booster and unvaccinated when infected with the Delta lineage. However, in patients with Omicron infections, recovery of infectious virus was not affected by vaccination. CONCLUSIONS: Omicron infections of vaccinated individuals are expected, yet admissions are less frequent. Admitted patients might develop severe disease comparable to Delta. Efforts for reducing the Omicron transmission are required as even though the admission risk is lower, the numbers of infections continue to be high. RESEARCH IN CONTEXT EVIDENCE BEFORE THIS STUDY: The unprecedented increase in COVID-19 cases in the month of December 2021, associated with the displacement of the Delta variant with the Omicron, triggered a lot of concerns. An understanding of the disease severity associated with infections with Omicron is essential as well as the virological determinants that contributed to its widespread predominance. We searched PubMed for articles published up to January 23, 2022, using the search terms ("Omicron") AND ("Disease severity") as well as ("Omicron") AND ("Viral load") And/ or ("Cell culture"). Our search yielded 3 main studies that directly assessed the omicron's clinical severity in South Africa, its infectious viral load compared to Delta, and the dynamics of viral RNA shedding. In South Africa, compared to Delta, Omicron infected patients showed a significant reduction in severe disease. In this study, Omicron and non-Omicron variants were characterized based on S gene target failure using the TaqPath COVID-19 PCR (Thermo Fisher Scientific). In the study from Switzerland that assessed the infectious viral load in Omicron versus Delta, the authors analyzed only 18 Omicron samples that were all from vaccinated individuals to show that compared to Delta, Omicron had equivalent infectious viral titers. The third study that assessed the Omicron viral dynamics showed that the peak viral RNA in Omicron infections is lower than Delta. No published studies assessed the clinical discrepancies of Omicron and Delta infected patients from the US, nor comprehensively assessed, by viral load and cell culture studies, the characteristics of both variants stratified by vaccination status. ADDED VALUE OF THIS STUDY: To the best of our knowledge, this is the only study to date to compare the clinical characteristics and outcomes after infection with the Omicron variant compared to Delta in the US using variants characterized by whole genome sequencing and a selective time frame when both variant co-circulated. It is also the first study to stratify the analysis based on the vaccination status and to compare fully vaccinated patients who didn't receive a booster vaccination to patients who received a booster vaccination. In addition, we provide a unique viral RNA and infectious virus load analyses to compare Delta and Omicron samples from unvaccinated, fully vaccinated, and patients with booster vaccination. IMPLICATIONS OF ALL THE AVAILABLE EVIDENCE: Omicron associated with a significant increase in infections in fully and booster vaccinated individuals but with less admissions and ICU level care. Admitted patients showed similar requirements for supplemental oxygen and ICU level care when compared to Delta admitted patients. Viral loads were similar in samples from Omicron and Delta infected patients regardless of the vaccination status. The recovery of infectious virus on cell culture was reduced in samples from patients infected with Delta who received a booster dose, but this was not the case with Omicron. The recovery of infectious virus was equivalent in Omicron infected unvaccinated, fully vaccinated, and samples from patients who received booster vaccination. FUNDING: NIH/NIAID Center of Excellence in Influenza Research and Surveillance contract HHS N2772201400007C, Johns Hopkins University, Maryland department of health, Centers for Disease Control and Prevention contract 75D30121C11061.

Infection With the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Delta Variant Is Associated With Higher Recovery of Infectious Virus Compared to the Alpha Variant in Both Unvaccinated and Vaccinated Individuals.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant of concern (VOC) B.1.617.2 (Delta) displaced B.1.1.7 (Alpha) and is associated with increases in coronavirus disease 2019 (COVID-19) cases, greater transmissibility, and higher viral RNA loads, but data are lacking regarding the infectious virus load and antiviral antibody levels in the nasal tract. METHODS: Whole genome sequencing, cycle threshold (Ct) values, infectious virus, anti-SARS-CoV-2 immunoglobulin G (IgG) levels, and clinical chart reviews were combined to characterize SARS-CoV-2 lineages circulating in the National Capital Region between January and September 2021 and differentiate infections in vaccinated and unvaccinated individuals by the Delta, Alpha, and B.1.2 (the predominant lineage prior to Alpha) variants. RESULTS: The Delta variant displaced the Alpha variant to constitute 99% of the circulating lineages in the National Capital Region by August 2021. In Delta infections, 28.5% were breakthrough cases in fully vaccinated individuals compared to 4% in the Alpha infected cohort. Breakthrough infections in both cohorts were associated with comorbidities, but only Delta infections were associated with a significant increase in the median days after vaccination. More than 74% of Delta samples had infectious virus compared to <30% from the Alpha cohort. The recovery of infectious virus with both variants was associated with low levels of local SARS-CoV-2 IgG. CONCLUSIONS: Infection with the Delta variant was associated with more frequent recovery of infectious virus in vaccinated and unvaccinated individuals compared to the Alpha variant but was not associated with an increase in disease severity in fully vaccinated individuals. Infectious virus was correlated with the presence of low amounts of antiviral IgG in the nasal specimens.

Infection with the SARS-CoV-2 Delta Variant is Associated with Higher Infectious Virus Loads Compared to the Alpha Variant in both Unvaccinated and Vaccinated Individuals.

BACKGROUND: The emerging SARS-CoV-2 variant of concern (VOC) B.1.6.17.2 (Delta) quickly displaced the B.1.1.7 (Alpha) and is associated with increases in COVID-19 cases nationally. The Delta variant has been associated with greater transmissibility and higher viral RNA loads in both unvaccinated and fully vaccinated individuals. Data is lacking regarding the infectious virus load in Delta infected individuals and how that compares to individuals infected with other SARS-CoV-2 lineages. METHODS: Whole genome sequencing of 2,785 clinical isolates was used to characterize the prevalence of SARS-CoV-2 lineages circulating in the National Capital Region between January and July 2021. Clinical chart reviews were performed for the Delta, Alpha, and B.1.2 (a control predominant lineage prior to both VOCs) variants to evaluate disease severity and outcome and Cycle threshold values (Cts) were compared. The presence of infectious virus was determined using Vero-TMPRSS2 cells and anti-SARS-CoV-2 IgG levels were determined from upper respiratory specimen. An analysis of infection in unvaccinated and fully vaccinated populations was performed. RESULTS: The Delta variant displaced the Alpha variant to constitute 88.2% of the circulating lineages in the National Capital Region by July, 2021. The Delta variant associated with increased breakthrough infections in fully vaccinated individuals that were mostly symptomatic when compared to the Alpha breakthrough infections, though it is important to note there was a significantly longer period of time between vaccination and infection with Delta infections. The recovery of infectious virus on cell culture was significantly higher with the Delta variant compared to Alpha in both vaccinated and unvaccinated groups. The impact of vaccination on reducing the recovery of infectious virus from clinical samples was only observed with Alpha variant infections but was strongly associated with low localized SARS-CoV-2 IgG for both variants. A comparison of Ct values showed a significant decrease in the Delta compared to Alpha with no significant differences between unvaccinated and vaccinated groups. CONCLUSIONS: Our data indicate that the Delta variant is associated with increased infectious virus loads when compared to the Alpha variant and decreased upper respiratory antiviral IgG levels. Measures to reduce transmission in addition to increasing vaccinations rates have to be implemented to reduce Delta variant spread. FUNDING: NIH/NIAID Center of Excellence in Influenza Research and Surveillance contract HHS N2772201400007C, Johns Hopkins University, Maryland department of health, Centers for Disease Control and Prevention contract 75D30121C11061.