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.

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.

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.

Presence of Human Hepegivirus-1 in a Cohort of People Who Inject Drugs.

BACKGROUND: Next-generation metagenomic sequencing (NGMS) has opened new frontiers in microbial discovery but has been clinically characterized in only a few settings. OBJECTIVE: To explore the plasma virome of persons who inject drugs and to characterize the sensitivity and accuracy of NGMS compared with quantitative clinical standards. DESIGN: Longitudinal and cross-sectional studies. SETTING: A clinical trial (ClinicalTrials.gov: NCT01285050) and a well-characterized cohort study of persons who have injected drugs. PARTICIPANTS: Persons co-infected with hepatitis C virus (HCV) and HIV. MEASUREMENTS: Viral nucleic acid in plasma by NGMS and quantitative polymerase chain reaction (PCR). RESULTS: Next-generation metagenomic sequencing generated a total of 600 million reads, which included the expected HIV and HCV RNA sequences. HIV and HCV reads were consistently identified only when samples contained more than 10 000 copies/mL or IU/mL, respectively, as determined by quantitative PCR. A novel RNA virus, human hepegivirus-1 (HHpgV-1), was also detected by NGMS in 4 samples from 2 persons in the clinical trial. Through use of a quantitative PCR assay for HHpgV-1, infection was also detected in 17 (10.9%) of 156 members of a cohort of persons who injected drugs. In these persons, HHpgV-1 viremia persisted for a median of at least 4538 days and was associated with detection of other bloodborne viruses, such as HCV RNA and SEN virus D. LIMITATION: The medical importance of HHpgV-1 infection is unknown. CONCLUSION: Although NGMS is insensitive for detection of viruses with relatively low plasma nucleic acid concentrations, it may have broad potential for discovery of new viral infections of possible medical importance, such as HHpgV-1. PRIMARY FUNDING SOURCE: National Institutes of Health.

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.

Respiratory virus disease and outcomes at a large academic medical center in the United States: a retrospective observational study of the early 2023/2024 respiratory viral season.

Respiratory disease, attributed to influenza, respiratory syncytial virus (RSV), and SARS-CoV-2, was reported nationally during the 2023/2024 respiratory viral season. The emergence of novel SARS-CoV-2 variants was considered a significant factor contributing to the rise in COVID-19 cases. Data from the Johns Hopkins Hospital System (JHHS) showed that enterovirus/rhinovirus had also been circulating at high rates. Analyzing clinical outcomes of the most prevalent respiratory viruses is crucial for understanding the role of circulating viral genotypes. A retrospective cohort of patients who tested positive for SARS-CoV-2, influenza, RSV, or enterovirus/rhinovirus between 1 June and 31 December 2023 was included in the study. Remnant clinical samples were utilized for targeted viral whole-genome sequencing and genotyping. Patients' metadata and outcomes following infection were studied, stratified by viral variants and genotypes. The increase of SARS-CoV-2 positivity in December was associated with the predominance of JN.1. Admissions for patients under 18 years old were primarily associated with enterovirus/rhinovirus and RSV, while older age groups were mainly linked to SARS-CoV-2 and influenza infections. SARS-CoV-2-related admissions increased with the predominance of the JN.1 variant in December. No significant difference in admissions for influenza subtypes, rhinovirus species, or SARS-CoV-2 variants was observed. RSV A was associated with slightly higher odds of admission compared with RSV B. Our data highlight the importance of systematically analyzing respiratory viral infections to inform public health strategies and clinical management, especially as SARS-CoV-2 becomes endemic. The findings highlight the value of expanded genomic surveillance in elucidating the clinical significance of viral evolution.IMPORTANCEThe analysis of the epidemiology and clinical outcomes of multiple co-circulating respiratory viruses in the early 2023/2024 respiratory virus season highlights the emergence of the SARS-CoV-2 JN.1 variant as well as underscores the importance of enterovirus/rhinovirus in respiratory infections. Understanding these dynamics is essential for refining public health strategies and clinical management, especially as SARS-CoV-2 transitions to an endemic status. This work emphasizes the need for ongoing surveillance, robust diagnostic algorithms, and detailed genomic analyses to anticipate and mitigate the burden of respiratory viral infections, ultimately contributing to more informed decision-making in healthcare settings and better patient outcomes.

A Third COVID-19 Vaccine Dose in Kidney Transplant Recipients Induces Antibody Response to Vaccine and Omicron Variants but Shows Limited Ig Subclass Switching.

Solid organ transplant recipients (SOTRs) suffer more frequent and more severe infections due to their compromised immune responses resulting from immunosuppressive treatments designed to prevent organ rejection. Pharmacological immunosuppression can adversely affect immune responses to vaccination. A cohort of kidney transplant recipients (KTRs) received their third dose of ancestral, monovalent COVID-19 vaccine in the context of a clinical trial and antibody responses to the vaccine strain, as well as to Omicron variants BA.1 and BA.5 were investigated and compared with healthy controls. Total IgG and live virus neutralizing antibody titers were reduced in KTRs compared to controls for all variants. KTRs displayed altered IgG subclass switching, with significantly lower IgG3 antibodies. Responses in KTRs were also very heterogeneous, with some individuals showing strong responses but a significant number showing no Omicron-specific neutralizing antibodies. Taken together, immune responses after COVID-19 vaccination in KTRs were not only lower than healthy controls but highly variable, indicating that simply increasing the number of vaccine doses alone may not be sufficient to provide greater protection in this population. Importance: This study addresses the challenges faced by kidney transplant recipients (KTRs) in mounting effective immune responses against COVID-19. By evaluating the antibody responses to a third dose of monovalent mRNA COVID-19 vaccine and its effectiveness against Omicron subvariants (BA.1 and BA.5), this study reveals significant reductions in both binding and neutralizing antibodies in KTRs compared to healthy controls. The research highlights altered IgG subclass switching and heterogeneous responses within the KTR population. Reduced recognition of variants, coupled with differences in IgG subclasses, decreases both the quality and quantity of protective antibodies after vaccination in KTRs. These findings underscore the need for tailored vaccination strategies for immunosuppressed populations such as KTRs. Alternative formulations and doses of COVID-19 vaccines should be considered for people with severely compromised immune systems, as more frequent vaccinations may not significantly improve the response, especially regarding neutralizing antibodies.

COVID-19 Diarrhea is Inflammatory, Caused by Direct Viral Effects Plus Major Role of Virus-induced Cytokines.

BACKGROUND & AIMS: Diarrhea occurs in up to 50% of cases of COVID-19. Nonetheless, the pathophysiologic mechanism(s) have not been determined. METHODS: This was examined using normal human enteroid monolayers exposed apically to live SARS-CoV-2 or non-replicating virus-like particles (VLPs) bearing the 4 SARS-CoV-2 structural proteins or irradiated virus, all of which bound and entered enterocytes. RESULTS: Live virus and VLPs incrieased secretion of multiple cytokines and reduced mRNAs of ACE2, NHE3, and DRA. Interleukin (IL)-6 plus IL-8 alone reduced NHE3 mRNA and protein and DRA mRNA and protein. Neither VLPs nor IL-6 plus IL-8 alone altered Cl- secretion, but together they caused Cl- secretion, which was Ca2+-dependent, CFTR-independent, blocked partially by a specific TMEM16A inhibitor, and entirely by a general TMEM16 family inhibitor. VLPs and irradiated virus, but not IL-6 plus IL-8, produced Ca2+ waves that began within minutes of VLP exposure, lasted for at least 60 minutes, and were prevented by pretreatment with apyrase, a P2Y1 receptor antagonist, and general TMEM16 family inhibitor but not by the specific TMEM16A inhibitor. CONCLUSIONS: The pathophysiology of COVID-19 diarrhea appears to be a unique example of a calcium-dependent inflammatory diarrhea that is caused by direct viral effects plus the virus-induced intestinal epithelial cytokine secretion.

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.

Association of neurotropic viruses in HIV-infected individuals who died of secondary complications of tuberculosis, cryptococcosis, or toxoplasmosis in South India.

The frequencies of 10 opportunistic DNA viruses were determined by multiplex real-time PCR in paired cerebrospinal fluid (CSF) and brain tissue of HIV-infected individuals. In the CSF, viruses were detectable in 45/55 cases: JC virus (JCV) in 62%, Epstein-Barr virus (EBV) in 44%, cytomegalovirus (CMV) in 25%, varicella-zoster virus (VZV) in 3.6%, herpes simplex virus 1 (HSV-1) in 1.8%, and human herpesvirus 6 (HHV-6) in 1.8% of cases. A single virus was detectable in 20 cases, 19 cases had coinfection with two viruses, and 6 cases were positive for three viruses. JCV was detectable in the CSF of 62% of cases and in 42% of brain tissues, with higher loads in progressive multifocal leukoencephalopathy (PML) (P < 0.05).

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.

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.

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.

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.

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.

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.