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 be associated with a relatively high rate of 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.

Neutralizing activity and 3-month durability of tixagevimab and cilgavimab prophylaxis against Omicron sublineages in transplant recipients.

Neutralizing antibody (nAb) responses are attenuated in solid organ transplant recipients (SOTRs) despite severe acute respiratory syndrome-coronavirus-2 vaccination. Preexposure prophylaxis (PrEP) with the antibody combination tixagevimab and cilgavimab (T+C) might augment immunoprotection, yet in vitro activity and durability against Omicron sublineages BA.4/5 in fully vaccinated SOTRs have not been delineated. Vaccinated SOTRs, who received 300 + 300 mg T+C (ie, full dose), within a prospective observational cohort submitted pre and postinjection samples between January 31, 2022, and July 6, 2022. The peak live virus nAb was measured against Omicron sublineages (BA.1, BA.2, BA.2.12.1, and BA.4), and surrogate neutralization (percent inhibition of angiotensin-converting enzyme 2 receptor binding to full length spike, validated vs live virus) was measured out to 3 months against sublineages, including BA.4/5. With live virus testing, the proportion of SOTRs with any nAb increased against BA.2 (47%-100%; P < .01), BA.2.12.1 (27%-80%; P < .01), and BA.4 (27%-93%; P < .01), but not against BA.1 (40%-33%; P = .6). The proportion of SOTRs with surrogate neutralizing inhibition against BA.5, however, fell to 15% by 3 months. Two participants developed mild severe acute respiratory syndrome-coronavirus-2 infection during follow-up. The majority of fully vaccinated SOTRs receiving T+C PrEP achieved BA.4/5 neutralization, yet nAb activity commonly waned by 3 months postinjection. It is critical to assess the optimal dose and interval of T+C PrEP to maximize protection in a changing variant climate.

The D614G mutation redirects SARS-CoV-2 spike to lysosomes and suppresses deleterious traits of the furin cleavage site insertion mutation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) egress occurs by lysosomal exocytosis. We show that the Spike D614G mutation enhances Spike trafficking to lysosomes, drives Spike-mediated reprogramming of lysosomes, and reduces cell surface Spike expression by ~3-fold. D614G is not a human-specific adaptation. Rather, it is an adaptation to the earlier furin cleavage site insertion (FCSI) mutation that occurred at the genesis of SARS-CoV-2. While advantageous to the virus, furin cleavage of spike has deleterious effects on spike structure and function, inhibiting its trafficking to lysosomes and impairing its infectivity by the transmembrane serine protease 2(TMPRSS2)-independent, endolysosomal pathway. D614G restores spike trafficking to lysosomes and enhances the earliest events in SARS-CoV-2 infectivity, while spike mutations that restore SARS-CoV-2's TMPRSS2-independent infectivity restore spike's trafficking to lysosomes. Together, these and other results show that D614G is an intragenic suppressor of deleterious traits linked to the FCSI and lend additional support to the endolysosomal model of SARS-CoV-2 egress and entry.

Association of Frailty, Age, and Biological Sex With Severe Acute Respiratory Syndrome Coronavirus 2 Messenger RNA Vaccine-Induced Immunity in Older Adults.

BACKGROUND: Male sex and old age are risk factors for severe coronavirus disease 2019, but the intersection of sex and aging on antibody responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines has not been characterized. METHODS: Plasma samples were collected from older adults (aged 75-98 years) before and after 3 doses of SARS-CoV-2 mRNA vaccination, and from younger adults (aged 18-74 years) post-dose 2, for comparison. Antibody binding to SARS-CoV-2 antigens (spike protein [S], S receptor-binding domain, and nucleocapsid), functional activity against S, and live-virus neutralization were measured against the vaccine virus and the Alpha, Delta, and Omicron variants of concern (VOCs). RESULTS: Vaccination induced greater antibody titers in older females than in older males, with both age and frailty associated with reduced antibody responses in males but not females. Responses declined significantly in the 6 months after the second dose. The third dose restored functional antibody responses and eliminated disparities caused by sex, age, and frailty in older adults. Responses to the VOCs, particularly the Omicron variant, were significantly reduced relative to the vaccine virus, with older males having lower titers to the VOCs than older females. Older adults had lower responses to the vaccine and VOC viruses than younger adults, with greater disparities in males than in females. CONCLUSIONS: Older and frail males may be more vulnerable to breakthrough infections owing to low antibody responses before receipt of a third vaccine dose. Promoting third dose coverage in older adults, especially males, is crucial to protecting this vulnerable population.

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.

Convalescent plasma with a high level of virus-specific antibody effectively neutralizes SARS-CoV-2 variants of concern.

The ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants severely limits available effective monoclonal antibody therapies. Effective drugs are also supply limited. COVID-19 convalescent plasma (CCP) qualified for high antibody levels effectively reduces immunocompetent outpatient hospitalization. The Food and Drug Administration currently allows outpatient CCP for the immunosuppressed. Viral-specific antibody levels in CCP can range 10- to 100-fold between donors, unlike the uniform viral-specific monoclonal antibody dosing. Limited data are available on the efficacy of polyclonal CCP to neutralize variants. We examined 108 pre-δ/pre-ο donor units obtained before March 2021, 20 post-δ COVID-19/postvaccination units, and 1 pre-δ/pre-ο hyperimmunoglobulin preparation for variant-specific virus (vaccine-related isolate [WA-1], δ, and ο) neutralization correlated to Euroimmun S1 immunoglobulin G antibody levels. We observed a two- to fourfold and 20- to 40-fold drop in virus neutralization from SARS-CoV-2 WA-1 to δ or ο, respectively. CCP antibody levels in the upper 10% of the 108 donations as well as 100% of the post-δ COVID-19/postvaccination units and the hyperimmunoglobulin effectively neutralized all 3 variants. High-titer CCP neutralizes SARS-CoV-2 variants despite no previous donor exposure to the variants.

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.

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.

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.

Understanding the Effects of Music Care on the Lived Experience of Isolation and Loneliness in Long-Term Care: A Qualitative Study.

This qualitative study aims to understand the lived experience of residents and other stakeholders during the implementation of a comprehensive music program in long-term care. It was conducted using a subset of 15 long-term care homes from the Room 217 Foundation Music Care Partners (MCP) "Grow" study in Ontario, Canada. The MCP program's approach to music delivery uses therapeutic music practices such as "music care" to improve the care experience for caregivers and residents in long-term care homes. Thirty-two participants were interviewed, including staff, volunteers, and residents. Data were transcribed and analyzed using a modified grounded theory approach based on emergent themes. In total, seven themes arose from the data: limited resources, distinct experiences, life enrichment, dynamic relationships, program flexibility, potential continuity, and enhanced socialization. This study provides insight on barriers, enablers, and outcomes of the MCP program and on key considerations for implementing a novel interdisciplinary music program in a healthcare setting.

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.

Pharmacokinetics of high-titer anti-SARS-CoV-2 human convalescent plasma in high-risk children.

BACKGROUNDWhile most children who contract COVID-19 experience mild disease, high-risk children with underlying conditions may develop severe disease, requiring interventions. Kinetics of antibodies transferred via COVID-19 convalescent plasma early in disease have not been characterized.METHODSIn this study, high-risk children were prospectively enrolled to receive high-titer COVID-19 convalescent plasma (>1:320 anti-spike IgG; Euroimmun). Passive transfer of antibodies and endogenous antibody production were serially evaluated for up to 2 months after transfusion. Commercial and research ELISA assays, virus neutralization assays, high-throughput phage-display assay utilizing a coronavirus epitope library, and pharmacokinetic analyses were performed.RESULTSFourteen high-risk children (median age, 7.5 years) received high-titer COVID-19 convalescent plasma, 9 children within 5 days (range, 2-7 days) of symptom onset and 5 children within 4 days (range, 3-5 days) after exposure to SARS-CoV-2. There were no serious adverse events related to transfusion. Antibodies against SARS-CoV-2 were transferred from the donor to the recipient, but antibody titers declined by 14-21 days, with a 15.1-day half-life for spike protein IgG. Donor plasma had significant neutralization capacity, which was transferred to the recipient. However, as early as 30 minutes after transfusion, recipient plasma neutralization titers were 6.2% (range, 5.9%-6.7%) of donor titers.CONCLUSIONConvalescent plasma transfused to high-risk children appears to be safe, with expected antibody kinetics, regardless of weight or age. However, current use of convalescent plasma in high-risk children achieves neutralizing capacity, which may protect against severe disease but is unlikely to provide lasting protection.Trial registrationClinicalTrials.gov NCT04377672.FundingThe state of Maryland, Bloomberg Philanthropies, and the NIH (grants R01-AI153349, R01-AI145435-A1, K08-AI139371-A1, and T32-AI052071).

Increased viral variants in children and young adults with impaired humoral immunity and persistent SARS-CoV-2 infection: A consecutive case series.

BACKGROUND: There is increasing concern that persistent infection of SARS-CoV-2 within immunocompromised hosts could serve as a reservoir for mutation accumulation and subsequent emergence of novel strains with the potential to evade immune responses. METHODS: We describe three patients with acute lymphoblastic leukemia who were persistently positive for SARS-CoV-2 by real-time polymerase chain reaction. Viral viability from longitudinally-collected specimens was assessed. Whole-genome sequencing and serological studies were performed to measure viral evolution and evidence of immune escape. FINDINGS: We found compelling evidence of ongoing replication and infectivity for up to 162 days from initial positive by subgenomic RNA, single-stranded RNA, and viral culture analysis. Our results reveal a broad spectrum of infectivity, host immune responses, and accumulation of mutations, some with the potential for immune escape. INTERPRETATION: Our results highlight the potential need to reassess infection control precautions in the management and care of immunocompromised patients. Routine surveillance of mutations and evaluation of their potential impact on viral transmission and immune escape should be considered.

Antigen-Based Testing but Not Real-Time Polymerase Chain Reaction Correlates With Severe Acute Respiratory Syndrome Coronavirus 2 Viral Culture.

BACKGROUND: Individuals can test positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by molecular assays following the resolution of their clinical disease. Recent studies indicate that SARS-CoV-2 antigen-based tests are likely to be positive early in the disease course, when there is an increased likelihood of high levels of infectious virus. METHODS: Upper respiratory specimens from 251 participants with coronavirus disease 2019 symptoms (≤7 days from symptom onset) were prospectively collected and tested with a lateral flow antigen test and a real-time polymerase chain reaction (rt-PCR) assay for detection of SARS-CoV-2. Specimens from a subset of the study specimens were utilized to determine the presence of infectious virus in the VeroE6TMPRSS2 cell culture model. RESULTS: The antigen test demonstrated a higher positive predictive value (90%) than rt-PCR (70%) when compared to culture-positive results. The positive percentage agreement for detection of infectious virus for the antigen test was similar to rt-PCR when compared to culture results. CONCLUSIONS: The correlation between SARS-CoV-2 antigen and SARS-CoV-2 culture positivity represents a significant advancement in determining the risk for potential transmissibility beyond that which can be achieved by detection of SARS-CoV-2 genomic RNA. SARS-CoV-2 antigen testing can facilitate low-cost, scalable, and rapid time-to-result, while providing good risk determination of those who are likely harboring infectious virus, compared to rt-PCR.