SARS-CoV-2 Variants from Long-Term, Persistently Infected Immunocompromised Patients Have Altered Syncytia Formation, Temperature-Dependent Replication, and Serum Neutralizing Antibody Escape.

SARS-CoV-2 infection of immunocompromised individuals often leads to prolonged detection of viral RNA and infectious virus in nasal specimens, presumably due to the lack of induction of an appropriate adaptive immune response. Mutations identified in virus sequences obtained from persistently infected patients bear signatures of immune evasion and have some overlap with sequences present in variants of concern. We characterized virus isolates obtained greater than 100 days after the initial COVID-19 diagnosis from two COVID-19 patients undergoing immunosuppressive cancer therapy, wand compared them to an isolate from the start of the infection. Isolates from an individual who never mounted an antibody response specific to SARS-CoV-2 despite the administration of convalescent plasma showed slight reductions in plaque size and some showed temperature-dependent replication attenuation on human nasal epithelial cell culture compared to the virus that initiated infection. An isolate from another patient-who did mount a SARS-CoV-2 IgM response-showed temperature-dependent changes in plaque size as well as increased syncytia formation and escape from serum-neutralizing antibodies. Our results indicate that not all virus isolates from immunocompromised COVID-19 patients display clear signs of phenotypic change, but increased attention should be paid to monitoring virus evolution in this patient population.

Bystander monocytic cells drive infection-independent NLRP3 inflammasome response to SARS-CoV-2.

The pathogenesis of COVID-19 is associated with a hyperinflammatory immune response. Monocytes and macrophages play a central role in this hyperinflammatory response to SARS-CoV-2. NLRP3 inflammasome activation has been observed in monocytes of patients with COVID-19, but the mechanism and consequences of inflammasome activation require further investigation. In this study, we inoculated a macrophage-like THP-1 cell line, primary differentiated human nasal epithelial cell (hNEC) cultures, and primary monocytes with SARS-CoV-2. We found that the activation of the NLRP3 inflammasome in macrophages does not rely on viral replication, receptor-mediated entry, or actin-dependent entry. SARS-CoV-2 productively infected hNEC cultures without triggering the production of inflammasome cytokines IL-18 and IL-1ß. Importantly, these cytokines did not inhibit viral replication in hNEC cultures. SARS-CoV-2 inoculation of primary monocytes led to inflammasome activation and induced a macrophage phenotype in these cells. Monocytic cells from bronchoalveolar lavage (BAL) fluid, but not from peripheral blood, of patients with COVID-19, showed evidence of inflammasome activation, expressed the proinflammatory marker CD11b, and displayed oxidative burst. These findings highlight the central role of activated macrophages, as a result of direct viral sensing, in COVID-19 and support the inhibition of IL-1ß and IL-18 as potential therapeutic strategies to reduce immunopathology without increasing viral replication. IMPORTANCE: Inflammasome activation is associated with severe COVID-19. The impact of inflammasome activation on viral replication and mechanistic details of this activation are not clarified. This study advances our understanding of the role of inflammasome activation in macrophages by identifying TLR2, NLRP3, ASC, and caspase-1 as dependent factors in this activation. Further, it highlights that SARS-CoV-2 inflammasome activation is not a feature of nasal epithelial cells but rather activation of bystander macrophages in the airway. Finally, we demonstrate that two pro inflammatory cytokines produced by inflammasome activation, IL-18 and IL-1ß, do not restrict viral replication and are potential targets to ameliorate pathological inflammation in severe COVID-19.

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.

The harms of promoting the lab leak hypothesis for SARS-CoV-2 origins without evidence.

Science is humanity's best insurance against threats from nature, but it is a fragile enterprise that must be nourished and protected. The preponderance of scientific evidence indicates a natural origin for SARS-CoV-2. Yet, the theory that SARS-CoV-2 was engineered in and escaped from a lab dominates media attention, even in the absence of strong evidence. We discuss how the resulting anti-science movement puts the research community, scientific research, and pandemic preparedness at risk.

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.

Genomic evolution of influenza during the 2023-2024 season, the johns hopkins health system.

Influenza, a human disease caused by viruses in the Orthomyxoviridae family, is estimated to infect 5% -10 % of adults and 20% -30 % of children annually. Influenza A (IAV) and Influenza B (IBV) viruses accumulate amino acid substitutions (AAS) in the hemagglutinin (HA) and neuraminidase (NA) proteins seasonally. These changes, as well as the dominating viral subtypes, vary depending on geographical location, which may impact disease prevalence and the severity of the season. Genomic surveillance is crucial for capturing circulation patterns and characterizing AAS that may affect disease outcomes, vaccine efficacy, or antiviral drug activities. In this study, whole-genome sequencing of IAV and IBV was attempted on positive remnant clinical samples (587) collected from 580 patients between June 2023 and February 2024 in the Johns Hopkins Health System (JHHS). Full-length HA segments were obtained from 424 (72.2 %) samples. H1N1pdm09 (71.7 %) was the predominant IAV subtype, followed by H3N2 (16.7 %) and IBV-Victoria clade V1A.3a.2 (11.6 %). Within H1N1pdm09 HA sequences, the 6B1A.5a.2a.1 (60.5 %) clade was the most represented. Full-length NA segments were obtained from 421 (71.7 %) samples. Within H1N1pdm09 and IBV, AAS previously proposed to change susceptibility to NA inhibitors were infrequently detected. Phylogeny of HA and NA demonstrated heterogeneous HA and NA H1N1pdm09 and IBV subclades. No significant differences were observed in admission rates or use of supplemental oxygen between different subtypes or clades. Influenza virus genomic surveillance is essential for understanding the seasonal evolution of influenza viruses and their association with disease prevalence and outcomes.

A scoping review of global COVID-19 vaccine hesitancy among pregnant persons.

Uptake of the COVID-19 vaccine among pregnant persons is lower than the general population. This scoping review explored pregnant people's attitudes towards the COVID-19 vaccine, reasons for vaccine hesitancy, and whether attitudes about COVID-19 vaccines differ by country of origin. A scoping review was conducted across PubMed, Embase, CINHAL, and Scopus. Inclusion criteria were articles published in English from 2019-2022 focused on attitudes towards COVID-19 vaccination among pregnant persons. Data analysis was done via the 5Cs framework for vaccine hesitancy: Constraints, Complacency, Calculation, Confidence, and Collective Responsibility. 44 articles were extracted. A lack of confidence in vaccine safety was the most prevalent theme of hesitancy among pregnant persons. This was largely driven by a lack of access to information about the vaccine as well as mistrust of the vaccine and medical professionals. Meanwhile, vaccine acceptance was mostly driven by a desire to protect themselves and their loved ones. Overall, COVID-19 vaccine hesitancy among pregnant persons continues to be high. Vaccine hesitancy is primarily driven by fear of the unknown side effects of the vaccine on pregnant persons and their fetuses along with a lack of information and medical mistrust. Some differences can be seen between high income and low- and middle-income countries regarding vaccine hesitancy, showing that a single solution cannot be applied to all who are vaccine hesitant. General strategies, however, can be utilized to reduce vaccine hesitancy, including advocating for inclusion of pregnant persons in clinical trials and incorporating consistent COVID-19 vaccine counseling during prenatal appointments.

Vaccinating the Frontlines: A Qualitative Exploration of Hospital Healthcare Worker Perspectives on Influenza and COVID-19 Immunization.

Introduction: Although they face higher occupational risk of contracting viral respiratory infections, hospital healthcare worker vaccine hesitancy persists. While most studies have used survey methods to quantify the prevalence of and reasons for healthcare worker vaccine hesitancy, this study employs a qualitative approach to understand their attitudes and beliefs associated with influenza and COVID-19 vaccination. Methods: To understand frontline healthcare worker experiences and perspectives on influenza and COVID-19 vaccination, 30 semi-structured interviews were conducted in summer/fall 2022 with staff recruited from two Johns Hopkins hospitals in Maryland. An in-depth, key informant interview was conducted with an expert in public health audience engagement. Interviews were audio recorded and transcribed for thematic and Framework analysis using NVivo software (QSR International, Melbourne, Australia). Results: Healthcare workers engaged in little influenza vaccine information seeking due to their familiarity with the disease and low perceived disease severity. Approximately half (n=16) of healthcare workers reported no vaccine hesitancy towards influenza or COVID-19 vaccines. No physicians or physician assistants expressed any vaccine hesitancy, while most nurses expressed some (n=10). More than half of the women (n=14) expressed COVID-19 vaccine hesitancy compared to none of the men. Structural factors including hospital tier, unit assignment, and professional role influenced perceived risk of disease exposure and subsequent healthcare worker vaccination decisions. Institutional policies, including mandates and a pro-vaccine environment encouraged vaccination uptake. Healthcare workers reported being more receptive to vaccine messaging that focused on protection from disease, scientific and public health data and their heightened occupational exposure to pathogens. Conclusions: Despite their medical knowledge, healthcare workers are susceptible to vaccine hesitancy. Strategies to address specific concerns are needed and can be informed by our findings. A flexible and multi-pronged approach that considers individual anxieties, workplace structures, and the need for open communication with tailored messaging is necessary to promote vaccine acceptance in healthcare settings. KEY MESSAGES: What is already known on this topic: Healthcare worker vaccine hesitancy has been associated with many factors including race, gender, age and concerns about vaccine safety.What this study adds: Much of the research on healthcare worker vaccine hesitancy has used surveys and questionnaires giving a broad description of the prevalence and patterns of vaccine hesitancy in the healthcare workforce. This qualitative study examines vaccine behavior (rather than merely intent) through a cross comparison of healthcare workers' experiences and attitudes towards influenza and COVID-19 vaccination.How this study might affect research, practice or policy: Study findings can be used to help tailor vaccine messaging to hospital healthcare workers which could offset concerns regarding vaccine efficacy and risk, to promote vaccine uptake.

Genomic Evolution and Surveillance of Respiratory Syncytial Virus during the 2023-2024 Season.

Respiratory syncytial virus (RSV) is a significant cause of morbidity, particularly in infants. This study describes RSV genomic diversity and disease outcomes during the 2023-2024 season in the Johns Hopkins Hospital System (JHHS). Between August and December 2023, 406 patient samples were sequenced, showing that RSV-B GB5.0.5a was the dominant genotype detected. RSV-A genotype GA2.3.5 was detected less frequently. Metadata analysis of patient data revealed that, although RSV-B was more commonly detected, patients with RSV-A infections were more frequently hospitalized. Analysis of both the G- and F-genes revealed multiple amino acid substitutions in both RSV-A and RSV-B, with some positions within the F-protein that could be associated with evasion of antibody responses. Phylogenetic analysis revealed the genetic diversity of circulating GB5.0.5a and GA2.3.5 genotypes. This study serves as an important baseline for genomic surveillance of RSV within the JHHS and will assist in characterizing the impact of the newly approved RSV vaccines on RSV genomic evolution and the emergence of escape mutations.

Longer interval between maternal RSV vaccination and birth increases placental transfer efficiency.

Background: Respiratory Syncytial Virus (RSV) is associated with significant neonatal and infant morbidity and mortality. Maternal bivalent RSVpreF RSV vaccination to protect neonates and infants was approved in September 2023 for administration between 32+0 and 36+6 weeks to protect neonates and infants. This approved timeframe is narrower than the 24-36 week window evaluated in the clinical trial, due to the possible association between preterm birth and vaccine administration. Currently, data are lacking on how maternal vaccine timing within the approved window affects the transfer of antibodies from mother to fetus, critical information that could influence clinical practice. Objectives: We sought to examine how gestational age at vaccination and time elapsed from maternal RSV vaccination to delivery impacted transfer of maternal antibodies measured in the umbilical cord at delivery and in peripheral blood of 2-month infants. We also examined differences in maternal and cord RSV antibody levels achieved by vaccination versus natural RSV infection. Study Design: A prospective cohort study was conducted at two academic medical centers between September 20, 2023 and March 21, 2024, enrolling 124 individuals who received the RSV vaccine during pregnancy. Infant capillary blood was collected at 2 months of age from 29 of the infants. Maternal and cord IgG levels achieved by RSV vaccination were compared to those associated with maternal natural RSV infection, using banked blood from 20 maternal:cord dyads collected prior to the availability of the maternal RSV vaccine. Levels of IgG against RSV strain A2 and B fusion (F) and attachment (G) proteins and against pertussis toxin (as a comparator antigen from a vaccine routinely administered earlier in pregnancy) were measured using a Binding Antibody Multiplex Assay. Differences in titers between vaccination and natural infection were examined using Wilcoxon rank sum test. Differences in cord:maternal transfer ratios and 2-month infant antibody levels by timing of maternal vaccination were evaluated by Kruskal-Wallis testing. Results: Maternal RSV vaccination resulted in significantly higher maternal and cord anti-F RSV antibody levels than natural infection (5.72 vs 4.82 log 10 MFI, p < 0.0001 maternal; 5.81 vs 5.03 log 10 MFI, p < 0.0001 cord). Maternal vaccination 2-3 weeks and 3-4 weeks prior to delivery was associated with significantly lower cord:maternal transfer ratios than were observed when vaccination occurred > 5 weeks prior to delivery (p = 0.03 for 2-3 weeks, p = 0.007 for 3-4 weeks), and significantly lower transfer ratios than observed for pertussis vaccination administered prior to 30 weeks' gestation (p = 0.008 for 2-3 weeks, p = 0.03 for 3-4 weeks, similar at > 4 weeks). Conclusions: Vaccine administration earlier in the approved 32-36 week window (at least 5 weeks prior to delivery) results in the highest transplacental transfer of maternal antibodies to the neonate. These results should inform the counseling of pregnant individuals on optimal vaccination timing.

Sex and gender differences in adverse events following influenza and COVID-19 vaccination.

INTRODUCTION: Active 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. In a predominately young adult female population of healthcare workers, we sought to determine the intersection of biological sex and sociocultural gender differences in prospective active reporting of vaccine outcomes, which remains poorly characterized. METHODS: This cohort study enrolled Johns Hopkins Health System healthcare workers (HCWs) who were recruited from the mandatory 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 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. RESULTS: Females were more likely to report local AEs after either 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 either influenza (OR = 1.18, p = 0.552) or COVID-19 (OR = 0.96, p = 0.907) vaccination. Hormonal birth control use did not impact the rates of reported AEs following influenza 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. CONCLUSIONS: Our findings highlight the need for sex- and gender-inclusive policies to inform more effective mandatory occupational health vaccination strategies. Further research is needed to evaluate the potential disruption of AEs on occupational responsibilities following mandated vaccination for healthcare workers, a predominately female population, and to more fully characterize the post-vaccination behavioral differences between men and women.

Research that addresses both the sex and gender differences of vaccine outcomes and behaviors is lacking. In this survey study of healthcare workers, comprised of mostly reproductive-aged females/women, we investigated biological sex (male/female) and gender (man/woman) differences in vaccine adverse events and outcomes following either influenza or bivalent COVID-19 vaccination.Regardless of age or race, females were more likely to report local (at injection site), but not systemic (whole body), adverse events than males, consistent across influenza and bivalent COVID-19 vaccine cohorts. Sex hormones are hypothesized to play a role in the differences in immune response following vaccination between males and females. We investigated if hormonal birth control use among females may be associated with differences in vaccine adverse events among the influenza vaccine cohort. However, there was no difference in the likelihood of reporting adverse events between birth control users and non-users. Based on open-ended responses to survey questions, women were found to report more interruptions to their daily routine than men following COVID-19 vaccination. Women were also more likely to seek out self-treatment with over-the-counter medication and intentionally schedule their vaccination around days off in anticipation of adverse events.With nearly 80% of healthcare jobs held by women, even higher for direct patient care positions like nursing, females/women may be disproportionately impacted by mandated annual vaccinations. Vaccinations are necessary for the prevention of disease transmission; however, our findings highlight a need for more equitable occupational vaccine strategies that consider both sex and gender differences.

Effect of human H3N2 influenza virus reassortment on influenza incidence and severity during the 2017-18 influenza season in the USA: a retrospective observational genomic analysis.

BACKGROUND: During the 2017-18 influenza season in the USA, there was a high incidence of influenza illness and mortality. However, no apparent antigenic change was identified in the dominant H3N2 viruses, and the severity of the season could not be solely attributed to a vaccine mismatch. We aimed to investigate whether the altered virus properties resulting from gene reassortment were underlying causes of the increased case number and disease severity associated with the 2017-18 influenza season. METHODS: Samples included were collected from patients with influenza who were prospectively recruited during the 2016-17 and 2017-18 influenza seasons at the Johns Hopkins Hospital Emergency Departments in Baltimore, MD, USA, as well as from archived samples from Johns Hopkins Health System sites. Among 647 recruited patients with influenza A virus infection, 411 patients with whole-genome sequences were available in the Johns Hopkins Center of Excellence for Influenza Research and Surveillance network during the 2016-17 and 2017-18 seasons. Phylogenetic trees were constructed based on viral whole-genome sequences. Representative viral isolates of the two seasons were characterised in immortalised cell lines and human nasal epithelial cell cultures, and patients' demographic data and clinical outcomes were analysed. FINDINGS: Unique H3N2 reassortment events were observed, resulting in two predominant strains in the 2017-18 season: HA clade 3C.2a2 and clade 3C.3a, which had novel gene segment constellations containing gene segments from HA clade 3C.2a1 viruses. The reassortant re3C.2a2 viruses replicated with faster kinetics and to a higher peak titre compared with the parental 3C.2a2 and 3C.2a1 viruses (48 h vs 72 h). Furthermore, patients infected with reassortant 3C.2a2 viruses had higher Influenza Severity Scores than patients infected with the parental 3C.2a2 viruses (median 3·00 [IQR 1·00-4·00] vs 1·50 [1·00-2·00]; p=0·018). INTERPRETATION: Our findings suggest that the increased severity of the 2017-18 influenza season was due in part to two intrasubtypes, cocirculating H3N2 reassortant viruses with fitness advantages over the parental viruses. This information could help inform future vaccine development and public health policies. FUNDING: The Center of Excellence for Influenza Research and Response in the US, National Science and Technology Council, and Chang Gung Memorial Hospital in Taiwan.

Clade-defining mutations in human H1N1 hemagglutinin protein from 2021-2023 have opposing effects on in vitro fitness and antigenic drift.

Seasonal influenza viruses frequently acquire mutations that have the potential to alter both virus replication and antigenic profile. Recent seasonal H1N1 viruses have acquired mutations to their hemagglutinin (HA) protein receptor binding site (RBS) and antigenic sites, and have branched into the clades 5a.2a and 5a.2a.1. Both clades demonstrated improved in vitro fitness compared with the parental 5a.2 clade as measured through plaque formation, infectious virus production in human nasal epithelial cells, and receptor binding diversity. Both clades also showed reduced neutralization by serum from healthcare workers vaccinated in the 2022-23 Northern Hemisphere influenza season compared to the vaccine strain. To investigate the phenotypic impact of individual clade-defining mutations, recombinant viruses containing single HA mutations were generated on a 5a.2 genetic background. The 5a.2a mutation Q189E improved plaque formation and virus replication, but was more efficiently neutralized by serum from individuals vaccinated in 2022-23. In contrast, the 5a.2a mutation E224A and both 5a.2a.1 mutations P137S and K142R impaired aspects of in vitro fitness but contributed significantly to antigenic drift. Surprisingly, the E224A mutation and not Q189E caused broader receptor binding diversity seen in clinical isolates of 5a.2a and 5a.2a.1, suggesting that receptor binding diversity alone may not be responsible for the phenotypic effects of the Q189E mutation. These data document an evolutionary trade-off between mutations that improve viral fitness and those that allow for the evasion of existing host immunity.

Multi-center evaluation of the Research Use Only NeuMoDx monkeypox virus (MPXV) fully automated real-time PCR assay.

The mpox outbreak, caused by monkeypox virus (MPXV), accelerated the development of molecular diagnostics. In this study, we detail the evaluation of the Research Use Only (RUO) NeuMoDx MPXV assay by multiple European and US sites. The assay was designed and developed by Qiagen for the NeuMoDx Molecular Systems. Primers and probes were tested for specificity and inclusivity in silico. The analytical sensitivity of the assay was determined by testing dilutions of synthetic and genomic MPXV DNA. A total of 296 clinical samples were tested by three sites; the Johns Hopkins University (US), UZ Gent (Belgium, Europe), and Hospital Universitario San Cecilio (Spain, Europe). The analytical sensitivity of the assay was 50 copies/mL for both clades I and II. The assay showed 100% in silico identity for 80 clade I and 99.98% in silico identity for 5,162 clade II genomes. Clade II primers and probes showed 100% in silico specificity; however, identity of at least one of the two sets of clade I primers and probes with variola, cowpox, camelpox, and vaccinia viruses was noticed. The clinical validation showed sensitivity of 99.21% [95% confidence interval (CI): 95.66-99.98%] and specificity of 96.64% (95% CI: 91.62-99.08%) for lesion swab samples. The NeuMoDx MPXV Test shows acceptable analytical and clinical performance. The assay improves the laboratory's workflow as it consolidates nucleic acid extraction, PCR, data analysis, and interpretation and can be interfaced. The Test Strip can differentiate clades I and II, which has important laboratory safety implications. IMPORTANCE: In this manuscript, we provide detailed in silico analysis and clinical evaluation of the assay using a large cohort of clinical samples across three academic centers in Europe and the United States. Because the assay differentiates MPXV clades I and II, this manuscript is timely due to the current need to rule out the regulated clade I by diagnostic clinical laboratories. In December 2023, and due to first report of cases of sexually transmitted clade I infections in the Democratic Republic of the Congo, when generic assays that do not differentiate the clades are used, samples are considered regulated. The assay meets the need of full automation and has a marked positive impact on the laboratory workflow.

Passive immunotherapies for the next influenza pandemic.

Influenzavirus is among the most relevant candidates for a next pandemic. We review here the phylogeny of former influenza pandemics, and discuss candidate lineages. After briefly reviewing the other existing antiviral options, we discuss in detail the evidences supporting the efficacy of passive immunotherapies against influenzavirus, with a focus on convalescent plasma.

Variant mutation in SARS-CoV-2 nucleocapsid enhances viral infection via altered genomic encapsidation.

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.

Evolution of nasal and olfactory infection characteristics of SARS-CoV-2 variants.

SARS-CoV-2 infection of the upper airway and the subsequent immune response are early, critical factors in COVID-19 pathogenesis. By studying infection of human biopsies in vitro and in a hamster model in vivo, we demonstrated a transition in nasal tropism from olfactory to respiratory epithelium as the virus evolved. Analyzing each variant revealed that SARS-CoV-2 WA1 or Delta infect a proportion of olfactory neurons in addition to the primary target sustentacular cells. The Delta variant possessed broader cellular invasion capacity into the submucosa, while Omicron displayed enhanced nasal respiratory infection and longer retention in the sinonasal epithelium. The olfactory neuronal infection by WA1 and the subsequent olfactory bulb transport via axon were more pronounced in younger hosts. In addition, the observed viral clearance delay and phagocytic dysfunction in aged olfactory mucosa were accompanied by a decline of phagocytosis-related genes. Further, robust basal stem cell activation contributed to neuroepithelial regeneration and restored ACE2 expression postinfection. Together, our study characterized the nasal tropism of SARS-CoV-2 strains, immune clearance, and regeneration after infection. The shifting characteristics of viral infection at the airway portal provide insight into the variability of COVID-19 clinical features, particularly long COVID, and may suggest differing strategies for early local intervention.

Multisite development and validation of machine learning models to predict severe outcomes and guide decision-making for emergency department patients with influenza.

Objective: Millions of Americans are infected by influenza annually. A minority seek care in the emergency department (ED) and, of those, only a limited number experience severe disease or death. ED clinicians must distinguish those at risk for deterioration from those who can be safely discharged. Methods: We developed random forest machine learning (ML) models to estimate needs for critical care within 24 h and inpatient care within 72 h in ED patients with influenza. Predictor data were limited to those recorded prior to ED disposition decision: demographics, ED complaint, medical problems, vital signs, supplemental oxygen use, and laboratory results. Our study population was comprised of adults diagnosed with influenza at one of five EDs in our university health system between January 1, 2017 and May 18, 2022; visits were divided into two cohorts to facilitate model development and validation. Prediction performance was assessed by the area under the receiver operating characteristic curve (AUC) and the Brier score. Results: Among 8032 patients with laboratory-confirmed influenza, incidence of critical care needs was 6.3% and incidence of inpatient care needs was 19.6%. The most common reasons for ED visit were symptoms of respiratory tract infection, fever, and shortness of breath. Model AUCs were 0.89 (95% CI 0.86-0.93) for prediction of critical care and 0.90 (95% CI 0.88-0.93) for inpatient care needs; Brier scores were 0.026 and 0.042, respectively. Importantpredictors included shortness of breath, increasing respiratory rate, and a high number of comorbid diseases. Conclusions: ML methods can be used to accurately predict clinical deterioration in ED patients with influenza and have potential to support ED disposition decision-making.

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.

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.

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 of the RNA BNT162b2 vaccine encoding the Wuhan-like spike (WT) and were boosted with a fourth dose of the WT vaccine or the bivalent WT and BA.4/5 spike (WT + BA.4/5). While neutralizing antibody to wild-type virus was comparable in both vaccine groups, BNT162b2 (WT + BA.4/BA.5) 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.