Variant mutation in SARS-CoV-2 nucleocapsid enhances viral infection via altered genomic encapsidation (preprint)

The evolution of SARS-CoV-2 variants and their respective phenotypes represents an important set of tools to understand basic coronavirus biology as well as the public health implications of individual mutations in variants of concern. While mutations outside of Spike are not well studied, the entire viral genome is undergoing evolutionary selection, particularly the central disordered linker region of the nucleocapsid (N) protein. Here, we identify a mutation (G215C), characteristic of the Delta variant, that introduces a novel cysteine into this linker domain, which results in the formation of a disulfide bond and a stable N-N dimer. Using reverse genetics, we determined that this cysteine residue is necessary and sufficient for stable dimer formation in a WA1 SARS-CoV-2 background, where it results in significantly increased viral growth both in vitro and in vivo. Finally, we demonstrate that the N:G215C virus packages more nucleocapsid per virion and that individual virions are larger, with elongated morphologies.

Cross-neutralizing antibody against emerging Omicron subvariants of SARS-CoV-2 in infection-naïve individuals with homologous BNT162b2 or BNT162b2(WT+ BA.4/5) bivalent booster vaccination (preprint)

Since the emergence of SARS-CoV-2, different variants and subvariants successively emerged to dominate global virus circulation as a result of immune evasion, replication fitness or both. COVID-19 vaccines continue to be updated in response to the emergence of antigenically divergent viruses, the first being the bivalent RNA vaccines that encodes for both the Wuhan-like and Omicron BA.5 subvariant spike proteins. Repeated infections and vaccine breakthrough infections have led to complex immune landscapes in populations making it increasingly difficult to assess the intrinsic neutralizing antibody responses elicited by the vaccines. Hong Kong’s intensive COVID-19 containment policy through 2020–2021 permitted us to identify sera from a small number of infection naïve individuals who received 3 doses RNA vaccine BNT162b2 of vaccines encoding the Wuhan-like spike who were boosted with a fourth dose monovalent Wuhan-like (WT) vaccine or the bivalent Wuhan-like and BA.4/5 spike (WT + BA.4/5) expressing vaccine. While neutralizing antibody to wild-type virus was comparable in both vaccine groups, BNT162b2 bivalent vaccine elicited significantly higher plaque neutralizing antibodies to Omicron subvariants BA.5, XBB.1.5, XBB.1.16, XBB.1.9.1, XBB.2.3.2, EG.5.1, HK.3, BA.2.86 and JN.1, compared to BNT162b2 monovalent vaccine. The single amino acid substitution that differentiates the spike of JN.1 from BA.2.86 resulted in a profound antigenic change.

Sex and gender differences in adverse events following receipt of influenza and COVID-19 vaccination among healthcare workers (preprint)

IntroductionActive and passive surveillance studies have found that a greater proportion of females report adverse events (AE) following receipt of either the COVID-19 or seasonal influenza vaccine compared to males. We sought to determine the intersection of biological sex and sociocultural gender differences in prospective active reporting of vaccine outcomes, which remains poorly characterized. MethodsThis cohort study enrolled Johns Hopkins Health System healthcare workers (HCWs) who were recruited from the annual fall 2019-2022 influenza vaccine and the fall 2022 COVID-19 bivalent vaccine campaigns. Vaccine recipients were enrolled the day of vaccination and AE surveys were administered two days post-vaccination (DPV) for bivalent COVID-19 and Influenza vaccine recipients. Data were collected regarding the presence of a series of solicited local and systemic AEs. Open-ended answers about participants experiences with AEs also were collected for the COVID-19 vaccine recipients. ResultsFemales were more likely to report local AEs after influenza (OR=2.28, p=0.001) or COVID-19 (OR=2.57, p=0.008) vaccination compared to males, regardless of age or race. Males and females had comparable probabilities of reporting systemic AEs after influenza (OR=1.18, p=0.552) or COVID-19 (OR=0.96, p=0.907) vaccination. Exogenous hormones from birth control use did not impact the rates of reported AEs following COVID-19 vaccination among reproductive-aged female HCWs. Women reported more interruptions in their daily routine following COVID-19 vaccination than men and were more likely to seek out self-treatment. More women than men scheduled their COVID-19 vaccination before their days off in anticipation of AEs. ConclusionsOur findings highlight the need for sex- and gender-inclusive policies to inform more effective occupational health vaccination strategies. Further research is needed to evaluate the potential disruption of AEs on occupational responsibilities following mandated vaccination for healthcare workers and to more fully characterize the post-vaccination behavioral differences between men and women. KEY MESSAGEO_ST_ABSWhat is already known on this topicC_ST_ABS{Rightarrow} Among diversely aged adults 18-64 years, females report more AEs to vaccines, including the influenza and COVID-19 vaccines, than males. {Rightarrow}Vaccine AEs play a role in shaping vaccine hesitancy and uptake. {Rightarrow}Vaccine uptake related to influenza and COVID-19 are higher among men than women. {Rightarrow}Research that addresses both the sex and gender disparities of vaccine outcomes and behaviors is lacking. What this study adds{Rightarrow} This prospective active reporting study uses both quantitative and qualitative survey data to examine sex and gender differences in AEs following influenza or COVID-19 vaccination among a cohort of reproductive-aged healthcare workers. How this study might affect research, practice, or policy{Rightarrow} Sex and gender differences in AEs and perceptions relating to vaccination should drive the development of more equitable and effective vaccine strategies and policies in occupational health settings.

Innate Immune Activation and Mitochondrial ROS Invoke Persistent Cardiac Conduction System Dysfunction after COVID-19 (preprint)

BackgroundCardiac risk rises during acute SARS-CoV-2 infection and in long COVID syndrome in humans, but the mechanisms behind COVID-19-linked arrhythmias are unknown. This study explores the acute and long term effects of SARS-CoV-2 on the cardiac conduction system (CCS) in a hamster model of COVID-19. MethodsRadiotelemetry in conscious animals was used to non-invasively record electrocardiograms and subpleural pressures after intranasal SARS-CoV-2 infection. Cardiac cytokines, interferon-stimulated gene expression, and macrophage infiltration of the CCS, were assessed at 4 days and 4 weeks post-infection. A double-stranded RNA mimetic, polyinosinic:polycytidylic acid (PIC), was used in vivo and in vitro to activate viral pattern recognition receptors in the absence of SARS-CoV-2 infection. ResultsCOVID-19 induced pronounced tachypnea and severe cardiac conduction system (CCS) dysfunction, spanning from bradycardia to persistent atrioventricular block, although no viral protein expression was detected in the heart. Arrhythmias developed rapidly, partially reversed, and then redeveloped after the pulmonary infection was resolved, indicating persistent CCS injury. Increased cardiac cytokines, interferon-stimulated gene expression, and macrophage remodeling in the CCS accompanied the electrophysiological abnormalities. Interestingly, the arrhythmia phenotype was reproduced by cardiac injection of PIC in the absence of virus, indicating that innate immune activation was sufficient to drive the response. PIC also strongly induced cytokine secretion and robust interferon signaling in hearts, human iPSC-derived cardiomyocytes (hiPSC-CMs), and engineered heart tissues, accompanied by alterations in electrical and Ca2+ handling properties. Importantly, the pulmonary and cardiac effects of COVID-19 were blunted by in vivo inhibition of JAK/STAT signaling or by a mitochondrially-targeted antioxidant. ConclusionsThe findings indicate that long term dysfunction and immune cell remodeling of the CCS is induced by COVID-19, arising indirectly from oxidative stress and excessive activation of cardiac innate immune responses during infection, with implications for long COVID Syndrome.

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

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.

Growth media affects susceptibility of air-lifted human nasal epithelial cell cultures to SARS-CoV2, but not Influenza A, virus infection. (preprint)

Primary differentiated human epithelial cell cultures have been widely used by researchers to study viral fitness and virus-host interactions, especially during the COVID19 pandemic. These cultures recapitulate important characteristics of the respiratory epithelium such as diverse cell type composition, polarization, and innate immune responses. However, standardization and validation of these cultures remains an open issue. In this study, two different expansion medias were evaluated and the impact on the resulting differentiated culture was determined. Use of both Airway and Ex Plus media types resulted in high quality, consistent cultures that were able to be used for these studies. Upon histological evaluation, Airway-grown cultures were more organized and had a higher proportion of basal progenitor cells while Ex Plus- grown cultures had a higher proportion terminally differentiated cell types. In addition to having different cell type proportions and organization, the two different growth medias led to cultures with altered susceptibility to infection with SARS-CoV-2 but not Influenza A virus. RNAseq comparing cultures grown in different growth medias prior to differentiation uncovered a high degree of differentially expressed genes in cultures from the same donor. RNAseq on differentiated cultures showed less variation between growth medias but alterations in pathways that control the expression of human transmembrane proteases including TMPRSS11 and TMPRSS2 were documented. Enhanced susceptibility to SARS-CoV-2 cannot be explained by altered cell type proportions alone, rather serine protease cofactor expression also contributes to the enhanced replication of SARS-CoV-2 as inhibition with camostat affected replication of an early SARS-CoV-2 variant and a Delta, but not Omicron, variant showed difference in replication efficiency between culture types. Therefore, it is important for the research community to standardize cell culture protocols particularly when characterizing novel viruses.

Accuracy of Expired BinaxNOW Rapid Antigen Tests (preprint)

The widespread existence of expired antigen testing kits in households and potential coronavirus outbreaks necessitate evaluating the reliability of these expired kits. Our study examined BinaxNOW COVID-19 rapid antigen tests 27 months post-manufacture and 5 months past their FDA extended expiration dates, using SARS-CoV-2 variant XBB.1.5 viral stock. We conducted testing at two concentrations: the Limit of Detection (LoD) and 10 times the LoD. 100 expired and unexpired kits were tested at each concentration for a total of 400 antigen tests. At the LoD (2.32x10^2 TCID50/mL), both expired and unexpired tests displayed 100% sensitivity (95% CI 96.38% to 100%), with no statistical difference (95% CI -3.92% to 3.92%). Similarly, at 10 times the LoD, unexpired tests retained 100% sensitivity (95% CI 96.38% to 100%), while expired tests exhibited 99% sensitivity (95% CI 94.61% to 99.99%), demonstrating a statistically insignificant 1% difference (95% CI -2.49% to 4.49%, p=0.56). Expired rapid antigen tests had fainter lines than the unexpired tests at each viral concentration. The expired rapid antigens tests at LoD were only just visible. These findings carry significant implications for waste management, cost efficiency, and supply chain resilience in pandemic readiness efforts. They also provide critical insights for formulating clinical guidelines for interpreting results from expired kits. In light of expert warnings of a potential outbreak of a severity rivaling the Omicron variant, our study underscores the importance of maximizing the utility of expired antigen testing kits in managing future health emergencies.

Antibody Correlates of Protection for COVID-19 Convalescent Plasma Associated with Reduced Outpatient Hospitalizations (preprint)

SARS-CoV-2 antibody levels associated with reduced hospitalization risk remain undefined. Our outpatient COVID-19 convalescent plasma (CCP), placebo-controlled trial observed SARS-CoV-2 antibody levels decreasing 22-fold from matched donor units into post-transfusion seronegative recipients. Unvaccinated recipients were jointly stratified by a) early or late transfusion (< 5 or >5 days from symptom onset) and b) high or low post-transfusion SARS-CoV-2 antibody levels (< or > geometric mean). Early treatment with high post-transfusion antibody levels reduced hospitalization risk-0/102 (0%) compared to all other CCP recipients-17/370 (4.6%; Fisher exact-p-0.03) and to all control plasma recipients-35/461 (7.6%; Fisher exact p-0.001). A similar donor upper/lower half antibody level and early late transfusion stratified analyses indicated significant hospital risk reduction. Pre-transfusion nasal viral loads were similar in CCP and control recipients regardless of hospitalization outcome. Therapeutic CCP should comprise the upper 30% of donor antibody levels to provide effective outpatient use for immunocompromised and immunocompetent outpatients.

Adverse outcomes in SARS-CoV-2 infected pregnant mice are gestational age-dependent and resolve with antiviral treatment (preprint)

SARS-CoV-2 infection during pregnancy is associated with severe COVID-19 and adverse fetal outcomes, but the underlying mechanisms remain poorly understood. Moreover, clinical studies assessing therapeutics against SARS-CoV-2 in pregnancy are limited. To address these gaps, we developed a mouse model of SARS-CoV-2 infection during pregnancy. Outbred CD1 mice were infected at embryonic day (E) 6, E10, or E16 with a mouse adapted SARS-CoV-2 (maSCV2) virus. Outcomes were gestational age-dependent with greater morbidity, reduced pulmonary function, reduced anti-viral immunity, greater viral titers, and more adverse fetal outcomes occurring with infection at E16 (3rd trimester-equivalent) than with infection at either E6 (1st trimester-equivalent) or E10 (2nd trimester-equivalent). To assess the efficacy of ritonavir-boosted nirmatrelvir (recommended for pregnant individuals with COVID-19), we treated E16-infected dams with mouse equivalent doses of nirmatrelvir and ritonavir. Treatment reduced pulmonary viral titers, decreased maternal morbidity, and prevented adverse offspring outcomes. Our results highlight that severe COVID-19 during pregnancy and adverse fetal outcomes are associated with heightened virus replication in maternal lungs. Ritonavir-boosted nirmatrelvir mitigated adverse maternal and fetal outcomes of SARS-CoV-2 infection. These findings prompt the need for further consideration of pregnancy in preclinical and clinical studies of therapeutics against viral infections.

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

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 Outcome(s): 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. Results(s): 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 =1.3-6.9). 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.

Early transcriptional responses of human nasal epithelial cells to infection with Influenza A and SARS-CoV-2 virus differ and are influenced by physiological temperature (preprint)

Influenza A (IAV) and SARS-CoV-2 (SCV2) viruses represent an ongoing threat to public health. Both viruses target the respiratory tract, which consists of a gradient of cell types, receptor expression, and temperature. Environmental temperature has been an understudied contributor to infection susceptibility and understanding its impact on host responses to infection could help uncover new insights into severe disease risk factors. As the nasal passageways are the initial site of respiratory virus infection, in this study we investigated the effect of temperature on host responses in human nasal epithelial cells (hNECs) utilizing IAV and SCV2 in vitro infection models. We demonstrate that temperature affects SCV2, but not IAV, viral replicative fitness and that SCV2 infected cultures are slower to mount an infection-induced response, likely due to suppression by the virus. Additionally, we show that that temperature not only changes the basal transcriptomic landscape of epithelial cells, but that it also impacts the response to infection. The induction of interferon and other innate immune responses were not drastically affected by temperature, suggesting that while the baseline antiviral response at different temperatures remains consistent, there may be metabolic or signaling changes that affect how well the cultures are able to adapt to new pressures such as infection. Finally, we show that hNECs respond differently to IAV and SCV2 infection in ways that give insight into how the virus is able to manipulate the cell to allow for replication and release. Taken together, these data give new insight into the innate immune response to respiratory infections and can assist in identifying new treatment strategies for respiratory infections.

Convergent Evolution of A-Lineage (Clade 19B) SARS-CoV-2 Spike Sequences with B-Lineage Variants of Concern Affects Virus Replication in a Temperature-Dependent Manner on Human Nasal Epithelial Cell Cultures (preprint)

The first three months of the COVID-19 pandemic was dominated by two SARS-CoV-2 lineages: A-lineages (Clade 19B) and B-lineages (Clade 19A). However, with the emergence of the Spike D614G substitution in B.1 lineages (Clade 20A), both early lineages were outcompeted and remained near-extinction from mid-2020 onwards. In early-2021, there was a re-emergence and persistence of novel A-lineage variants with substitutions in the Spike gene resembling those found in Variants of Concern (VOCs). An early A.3 variant (MD-HP00076/2020) and three A.2.5 variants (MD-HP02153/2021, MD-HP05922/2021 and CA-VRLC091/2021) were isolated and characterized for their genomic sequences, antibody neutralization, and in vitro replication. All A.2.5 isolates had five Spike mutations relative to the A.3 variant sequence: D614G, L452R, {Delta}141-143, D215A, and ins215AGY. Plaque reduction neutralization assays demonstrated that A.2.5 isolates had a 2.5 to 5-fold reduction in neutralization using contemporaneous COVID-19 convalescent plasma when compared to A.3. In vitro viral characterization in VeroE6 cell lines revealed that the A.3 isolate grew faster and spread more than A.2.5. On VeroE6-TMPRSS2 cells, significant syncytia formation was also observed with the A.2.5 isolates, however Spike cleavage efficiency did not explain these differences. In human nasal epithelial cell (hNEC) cultures, the A.2.5 isolates grew significantly faster and to higher total infectious virus titers than A.3. All A.2.5 lineage isolates grew significantly faster at 37{degrees}C than at 33{degrees}C irrespective of cell type, and to higher peak titers except compared to A.3. This suggests A.2.5's adapted to improve replication using similar mutations found in the B-lineage SARS-CoV-2 variants.

Early Treatment, Inflammation and Post-COVID Conditions (preprint)

ABSTRACT Background: Post-COVID conditions (PCC) are common and have significant morbidity. Risk factors for PCC include advancing age, female sex, obesity, and diabetes mellitus. Little is known about early treatment, inflammation, and PCC. Methods: Among 883 individuals with confirmed SARS-CoV-2 infection participating in a randomized trial of CCP vs. control plasma with available biospecimens and symptom data, the association between early COVID treatment, cytokine levels and PCC was evaluated. Cytokine and chemokine levels were assessed at baseline, day 14 and day 90 using a multiplexed sandwich immuosassay (Mesoscale Discovery). Presence of any self-reported PCC symptoms was assessed at day 90. Associations between COVID treatment, cytokine levels and PCC were examined using multivariate logistic regression models. Results: One-third of the 882 participants had day 90 PCC symptoms, with fatigue (14.5%) and loss of smell (14.5%) being most common. Cytokine levels decreased from baseline to day 90. In a multivariable analysis including diabetes, body mass index, race, and vaccine status, female sex (adjusted odds ratio[AOR]=2.70[1.93-3.81]), older age (AOR=1.32[1.17-1.50]), and elevated baseline levels of IL-6 (AOR=1.59[1.02-2.47]) were associated with development of PCC. There was a trend for decreased PCC in those with early CCP treatment (<5 days after symptom onset) compared to late CCP treatment. Conclusion: Increased IL-6 levels were associated with the development of PCC and there was a trend for decreased PCC with early CCP treatment in this predominately unvaccinated population. Future treatment studies should evaluate the effect of early treatment and anti-IL-6 therapies on PCC development.

Pathogenesis of Breakthrough Infections with SARS-CoV-2 Variants in Syrian Hamsters (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, has evolved into multiple variants. Animal models are important to understand variant pathogenesis, particularly for those with mutations that have significant phenotypic or epidemiological effects. Here, cohorts of naive or previously infected Syrian hamsters (Mesocricetus auratus) were infected with variants to investigate viral pathogenesis and disease protection. Naive hamsters infected with SARS-CoV-2 variants had consistent clinical outcomes, tissue viral titers, and pathology, while hamsters that recovered from initial infection and were reinfected demonstrated less severe clinical disease and lung pathology than their naive counterparts. Males had more frequent clinical signs than females in most variant groups, but few sex variations in tissue viral titers and lung pathology were observed. These findings support the use of Syrian hamsters as a SARS-CoV-2 model and highlight the importance of considering sex differences when using this species.

Viral and host small RNA transcriptome analysis of SARS-CoV-1 and SARS-CoV-2-infected human cells reveals novel viral short RNAs. (preprint)

RNA viruses have been shown to express various short RNAs, some of which have regulatory roles during replication, transcription, and translation of viral genomes. However, short viral RNAs (svRNAs) generated by SARS-CoV-1 and SARS-CoV-2 remained largely unexplored, mainly due limitations of the widely used library preparation methods for small RNA deep sequencing and corresponding data processing. By analyzing publicly available small RNA-seq datasets, we observed that human cells infected by SARS-CoV-1 or SARS-CoV-2 produce multiple short viral RNAs (svRNAs), ranging in size from 15 to 26 nt and deriving predominantly from (+) RNA strands. In addition, we verified the presence of the five most abundant SARS-CoV-2 svRNAs in SARS-CoV-2-infected human lung adenocarcinoma cells by qPCR. Interestingly, the copy number of the observed SARS-CoV-2 svRNAs dramatically exceeded the expression of previously reported viral miRNAs in the same cells. We hypothesize that the reported SARS-CoV-2 svRNAs could serve as biomarkers for early infection stages due to their high abundance. Finally, we found that both SARS-CoV-1 and SARS-CoV-2 infection induced up- and down-regulation of multiple endogenous human short RNAs that align predominantly to protein-coding and lncRNA transcripts. Interestingly, a significant proportion of short RNAs derived from full-length viral genomes also aligned to various hg38 sequences, suggesting opportunities to investigate regulatory roles of svRNAs during infection. Further characterization of the small RNA landscape of both viral and host genomes is clearly warranted to improve our understanding of molecular events related to infection and to design more efficient strategies for therapeutic interventions as well as early diagnosis.

Methodological approaches to optimize multiplex oral fluid SARS-CoV-2 IgG assay performance and correlation with serologic and neutralizing antibody responses (preprint)

Background. Oral fluid (hereafter, saliva) is a non-invasive and attractive alternative to blood for SARS-CoV-2 IgG testing; however, the heterogeneity of saliva as a matrix poses challenges for immunoassay performance. Objectives. To optimize performance of a magnetic microparticle-based multiplex immunoassay (MIA) for SARS-CoV-2 IgG measurement in saliva, with consideration of: i) threshold setting and validation across different MIA bead batches; ii) sample qualification based on salivary total IgG concentration; iii) calibration to U.S. SARS-CoV-2 serological standard binding antibody units (BAU); and iv) correlations with blood-based SARS-CoV-2 serological and neutralizing antibody (nAb) assays. Methods. The salivary SARS-CoV-2 IgG MIA included 2 nucleocapsid (N), 3 receptor-binding domain (RBD), and 2 spike protein (S) antigens. Gingival crevicular fluid (GCF) swab saliva samples were collected before December, 2019 (n=555) and after molecular test-confirmed SARS-CoV-2 infection from 113 individuals (providing up to 5 repeated-measures; n=398) and used to optimize and validate MIA performance (total n=953). Combinations of IgG responses to N, RBD and S and total salivary IgG concentration (g/mL) as a qualifier of nonreactive samples were optimized and validated, calibrated to the U.S. SARS-CoV-2 serological standard, and correlated with blood-based SARS-CoV-2 IgG ELISA and nAb assays. Results. The sum of signal to cutoff (S/Co) to all seven MIA SARS-CoV-2 antigens and disqualification of nonreactive saliva samples with >15 g/mL total IgG led to correct classification of 62/62 positives (sensitivity [Se]=100.0%; 95% confidence interval [CI]=94.8%, 100.0%) and 108/109 negatives (specificity [Sp]=99.1%; 95% CI=97.3%, 100.0%) at 8-million beads coupling scale and 80/81 positives (Se=98.8%; 95% CI=93.3%, 100.0%] and 127/127 negatives (Sp=100%; 95% CI=97.1%, 100.0%) at 20-million beads coupling scale. Salivary SARS-CoV-2 IgG crossed the MIA cutoff of 0.1 BAU/mL on average 9 days post-COVID-19 symptom onset and peaked around day 30. Among n=30 matched saliva and plasma samples, salivary SARS-CoV-2 MIA IgG levels correlated with corresponding-antigen plasma ELISA IgG (N: {rho}=0.67, RBD: {rho}=0.76, S: {rho} =0.82; all p<0.0001). Correlations of plasma SARS-CoV-2 nAb assay area under the curve (AUC) with salivary MIA IgG (N: {rho}=0.68, RBD: {rho}=0.78, S: {rho} =0.79; all p<0.0001) and with plasma ELISA IgG (N: {rho}=0.76, RBD: {rho}=0.79, S: {rho} =0.76; p<0.0001) were similar. Conclusions. A salivary SARS-CoV-2 IgG MIA produced consistently high Se (>98.8%) and Sp (>99.1%) across two bead coupling scales and correlations with nAb responses that were similar to blood-based SARS-CoV-2 IgG ELISA data. This non-invasive salivary SARS-CoV-2 IgG MIA could increase engagement of vulnerable populations and improve broad understanding of humoral immunity (kinetics and gaps) within the evolving context of booster vaccination, viral variants and waning immunity.

Omicron Subvariants: Clinical, Laboratory, and Cell Culture Characterization (preprint)

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.

Characterization of SARS-CoV-2 Omicron BA.4 and BA.5 clinical isolates (preprint)

The BA.2 sublineage of the SARS-CoV-2 Omicron variant has become dominant in most countries around the world; however, the prevalence of BA.4 and BA.5 is increasing rapidly in several regions. BA.2 is less pathogenic in animal models than previously circulating variants of concern (VOC). Compared with BA.2, however, BA.4 and BA.5 possess additional substitutions in the spike protein, which play a key role in viral infectivity, raising concerns that the infectivity and pathogenicity of BA.4 and BA.5 are higher than those of BA.2. Here, we evaluated the replicative ability and pathogenicity of authentic BA.4 and BA.5 isolates in wild-type Syrian hamsters and human ACE2 (hACE2) transgenic hamsters. In contrast to recent data with a recombinant chimeric virus possessing the spike protein of BA.4/BA.5 in the background of a BA.2 strain, we observed no obvious differences among BA.2, BA.4, and BA.5 isolates in growth ability or pathogenicity in hamsters, and less pathogenicity compared to a previously circulating Delta (B.1.617.2 lineage) isolate. In addition, in vivo competition experiments revealed that BA.5 outcompeted BA.2 in hamsters, whereas BA.4 and BA.2 exhibited similar fitness. These findings suggest that BA.4 and BA.5 have similar pathogenicity to BA.2 in rodents and that BA.5 possesses viral fitness superior to that of BA.2. Our study highlights the importance of using authentic isolates when evaluating virological features.