Coast-to-Coast Spread of SARS-CoV-2 during the Early Epidemic in the United States.

The novel coronavirus SARS-CoV-2 was first detected in the Pacific Northwest region of the United States in January 2020, with subsequent COVID-19 outbreaks detected in all 50 states by early March. To uncover the sources of SARS-CoV-2 introductions and patterns of spread within the United States, we sequenced nine viral genomes from early reported COVID-19 patients in Connecticut. Our phylogenetic analysis places the majority of these genomes with viruses sequenced from Washington state. By coupling our genomic data with domestic and international travel patterns, we show that early SARS-CoV-2 transmission in Connecticut was likely driven by domestic introductions. Moreover, the risk of domestic importation to Connecticut exceeded that of international importation by mid-March regardless of our estimated effects of federal travel restrictions. This study provides evidence of widespread sustained transmission of SARS-CoV-2 within the United States and highlights the critical need for local surveillance.

Recurrent Potent Human Neutralizing Antibodies to Zika Virus in Brazil and Mexico.

Antibodies to Zika virus (ZIKV) can be protective. To examine the antibody response in individuals who develop high titers of anti-ZIKV antibodies, we screened cohorts in Brazil and Mexico for ZIKV envelope domain III (ZEDIII) binding and neutralization. We find that serologic reactivity to dengue 1 virus (DENV1) EDIII before ZIKV exposure is associated with increased ZIKV neutralizing titers after exposure. Antibody cloning shows that donors with high ZIKV neutralizing antibody titers have expanded clones of memory B cells that express the same immunoglobulin VH3-23/VK1-5 genes. These recurring antibodies cross-react with DENV1, but not other flaviviruses, neutralize both DENV1 and ZIKV, and protect mice against ZIKV challenge. Structural analyses reveal the mechanism of recognition of the ZEDIII lateral ridge by VH3-23/VK1-5 antibodies. Serologic testing shows that antibodies to this region correlate with serum neutralizing activity to ZIKV. Thus, high neutralizing responses to ZIKV are associated with pre-existing reactivity to DENV1 in humans.

Impact of circulating SARS-CoV-2 variants on mRNA vaccine-induced immunity.

The emergence of SARS-CoV-2 variants with mutations in major neutralizing antibody-binding sites can affect humoral immunity induced by infection or vaccination1-6. Here we analysed the development of anti-SARS-CoV-2 antibody and T cell responses in individuals who were previously infected (recovered) or uninfected (naive) and received mRNA vaccines to SARS-CoV-2. While individuals who were previously infected sustained higher antibody titres than individuals who were uninfected post-vaccination, the latter reached comparable levels of neutralization responses to the ancestral strain after the second vaccine dose. T cell activation markers measured upon spike or nucleocapsid peptide in vitro stimulation showed a progressive increase after vaccination. Comprehensive analysis of plasma neutralization using 16 authentic isolates of distinct locally circulating SARS-CoV-2 variants revealed a range of reduction in the neutralization capacity associated with specific mutations in the spike gene: lineages with E484K and N501Y/T (for example, B.1.351 and P.1) had the greatest reduction, followed by lineages with L452R (for example, B.1.617.2). While both groups retained neutralization capacity against all variants, plasma from individuals who were previously infected and vaccinated displayed overall better neutralization capacity than plasma from individuals who were uninfected and also received two vaccine doses, pointing to vaccine boosters as a relevant future strategy to alleviate the effect of emerging variants on antibody neutralizing activity.

Diverse functional autoantibodies in patients with COVID-19.

COVID-19 manifests with a wide spectrum of clinical phenotypes that are characterized by exaggerated and misdirected host immune responses1-6. Although pathological innate immune activation is well-documented in severe disease1, the effect of autoantibodies on disease progression is less well-defined. Here we use a high-throughput autoantibody discovery technique known as rapid extracellular antigen profiling7 to screen a cohort of 194 individuals infected with SARS-CoV-2, comprising 172 patients with COVID-19 and 22 healthcare workers with mild disease or asymptomatic infection, for autoantibodies against 2,770 extracellular and secreted proteins (members of the exoproteome). We found that patients with COVID-19 exhibit marked increases in autoantibody reactivities as compared to uninfected individuals, and show a high prevalence of autoantibodies against immunomodulatory proteins (including cytokines, chemokines, complement components and cell-surface proteins). We established that these autoantibodies perturb immune function and impair virological control by inhibiting immunoreceptor signalling and by altering peripheral immune cell composition, and found that mouse surrogates of these autoantibodies increase disease severity in a mouse model of SARS-CoV-2 infection. Our analysis of autoantibodies against tissue-associated antigens revealed associations with specific clinical characteristics. Our findings suggest a pathological role for exoproteome-directed autoantibodies in COVID-19, with diverse effects on immune functionality and associations with clinical outcomes.

Sex differences in immune responses that underlie COVID-19 disease outcomes.

There is increasing evidence that coronavirus disease 2019 (COVID-19) produces more severe symptoms and higher mortality among men than among women1-5. However, whether immune responses against severe acute respiratory syndrome coronavirus (SARS-CoV-2) differ between sexes, and whether such differences correlate with the sex difference in the disease course of COVID-19, is currently unknown. Here we examined sex differences in viral loads, SARS-CoV-2-specific antibody titres, plasma cytokines and blood-cell phenotyping in patients with moderate COVID-19 who had not received immunomodulatory medications. Male patients had higher plasma levels of innate immune cytokines such as IL-8 and IL-18 along with more robust induction of non-classical monocytes. By contrast, female patients had more robust T cell activation than male patients during SARS-CoV-2 infection. Notably, we found that a poor T cell response negatively correlated with patients' age and was associated with worse disease outcome in male patients, but not in female patients. By contrast, higher levels of innate immune cytokines were associated with worse disease progression in female patients, but not in male patients. These findings provide a possible explanation for the observed sex biases in COVID-19, and provide an important basis for the development of a sex-based approach to the treatment and care of male and female patients with COVID-19.

Longitudinal analyses reveal immunological misfiring in severe COVID-19.

Recent studies have provided insights into the pathogenesis of coronavirus disease 2019 (COVID-19)1-4. However, the longitudinal immunological correlates of disease outcome remain unclear. Here we serially analysed immune responses in 113 patients with moderate or severe COVID-19. Immune profiling revealed an overall increase in innate cell lineages, with a concomitant reduction in T cell number. An early elevation in cytokine levels was associated with worse disease outcomes. Following an early increase in cytokines, patients with moderate COVID-19 displayed a progressive reduction in type 1 (antiviral) and type 3 (antifungal) responses. By contrast, patients with severe COVID-19 maintained these elevated responses throughout the course of the disease. Moreover, severe COVID-19 was accompanied by an increase in multiple type 2 (anti-helminths) effectors, including interleukin-5 (IL-5), IL-13, immunoglobulin E and eosinophils. Unsupervised clustering analysis identified four immune signatures, representing growth factors (A), type-2/3 cytokines (B), mixed type-1/2/3 cytokines (C), and chemokines (D) that correlated with three distinct disease trajectories. The immune profiles of patients who recovered from moderate COVID-19 were enriched in tissue reparative growth factor signature A, whereas the profiles of those with who developed severe disease had elevated levels of all four signatures. Thus, we have identified a maladapted immune response profile associated with severe COVID-19 and poor clinical outcome, as well as early immune signatures that correlate with divergent disease trajectories.

MPL36, a major plasminogen (PLG) receptor in pathogenic Leptospira, has an essential role during infection.

Leptospirosis, a zoonosis with worldwide distribution, is caused by pathogenic spirochetes belonging to the genus Leptospira. Bacterial outer membrane proteins (OMPs), particularly those with surface-exposed regions, play crucial roles in pathogen dissemination and virulence mechanisms. Here we characterized the leptospiral Membrane Protein L36 (MPL36), a rare lipoprotein A (RlpA) homolog with a C-terminal Sporulation related (SPOR) domain, as an important virulence factor in pathogenic Leptospira. Our results confirmed that MPL36 is surface exposed and expressed during infection. Using recombinant MPL36 (rMPL36) we also confirmed previous findings of its high plasminogen (PLG)-binding ability determined by lysine residues of the C-terminal region of the protein, with ability to convert bound-PLG to active plasmin. Using Koch's molecular postulates, we determined that a mutant of mpl36 has a reduced PLG-binding ability, leading to a decreased capacity to adhere and translocate MDCK cell monolayers. Using recombinant protein and mutant strains, we determined that the MPL36-bound plasmin (PLA) can degrade fibrinogen. Finally, our mpl36 mutant had a significant attenuated phenotype in the hamster model for acute leptospirosis. Our data indicates that MPL36 is the major PLG binding protein in pathogenic Leptospira, and crucial to the pathogen's ability to attach and interact with host tissues during infection. The MPL36 characterization contributes to the expanding field of bacterial pathogens that explore PLG for their virulence, advancing the goal to close the knowledge gap regarding leptospiral pathogenesis while offering a novel potential candidate to improve diagnostic and prevention of this important zoonotic neglected disease.

No evidence of fetal defects or anti-syncytin-1 antibody induction following COVID-19 mRNA vaccination.

The impact of Coronavirus Disease 2019 (COVID-19) mRNA vaccination on pregnancy and fertility has become a major topic of public interest. We investigated 2 of the most widely propagated claims to determine (1) whether COVID-19 mRNA vaccination of mice during early pregnancy is associated with an increased incidence of birth defects or growth abnormalities; and (2) whether COVID-19 mRNA-vaccinated human volunteers exhibit elevated levels of antibodies to the human placental protein syncytin-1. Using a mouse model, we found that intramuscular COVID-19 mRNA vaccination during early pregnancy at gestational age E7.5 did not lead to differences in fetal size by crown-rump length or weight at term, nor did we observe any gross birth defects. In contrast, injection of the TLR3 agonist and double-stranded RNA mimic polyinosinic-polycytidylic acid, or poly(I:C), impacted growth in utero leading to reduced fetal size. No overt maternal illness following either vaccination or poly(I:C) exposure was observed. We also found that term fetuses from these murine pregnancies vaccinated prior to the formation of the definitive placenta exhibit high circulating levels of anti-spike and anti-receptor-binding domain (anti-RBD) antibodies to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) consistent with maternal antibody status, indicating transplacental transfer in the later stages of pregnancy after early immunization. Finally, we did not detect increased levels of circulating anti-syncytin-1 antibodies in a cohort of COVID-19 vaccinated adults compared to unvaccinated adults by ELISA. Our findings contradict popular claims associating COVID-19 mRNA vaccination with infertility and adverse neonatal outcomes.

Lack of association between pandemic chilblains and SARS-CoV-2 infection.

An increased incidence of chilblains has been observed during the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic and attributed to viral infection. Direct evidence of this relationship has been limited, however, as most cases do not have molecular evidence of prior SARS-CoV-2 infection with PCR or antibodies. We enrolled a cohort of 23 patients who were diagnosed and managed as having SARS-CoV-2-associated skin eruptions (including 21 pandemic chilblains [PC]) during the first wave of the pandemic in Connecticut. Antibody responses were determined through endpoint titration enzyme-linked immunosorbent assay and serum epitope repertoire analysis. T cell responses to SARS-CoV-2 were assessed by T cell receptor sequencing and in vitro SARS-CoV-2 antigen-specific peptide stimulation assays. Immunohistochemical and PCR studies of PC biopsies and tissue microarrays for evidence of SARS-CoV-2 were performed. Among patients diagnosed and managed as "covid toes" during the pandemic, we find a percentage of prior SARS-CoV-2 infection (9.5%) that approximates background seroprevalence (8.5%) at the time. Immunohistochemistry studies suggest that SARS-CoV-2 staining in PC biopsies may not be from SARS-CoV-2. Our results do not support SARS-CoV-2 as the causative agent of pandemic chilblains; however, our study does not exclude the possibility of SARS-CoV-2 seronegative abortive infections.

COVID-19 annual update: a narrative review.

Three and a half years after the pandemic outbreak, now that WHO has formally declared that the emergency is over, COVID-19 is still a significant global issue. Here, we focus on recent developments in genetic and genomic research on COVID-19, and we give an outlook on state-of-the-art therapeutical approaches, as the pandemic is gradually transitioning to an endemic situation. The sequencing and characterization of rare alleles in different populations has made it possible to identify numerous genes that affect either susceptibility to COVID-19 or the severity of the disease. These findings provide a beginning to new avenues and pan-ethnic therapeutic approaches, as well as to potential genetic screening protocols. The causative virus, SARS-CoV-2, is still in the spotlight, but novel threatening virus could appear anywhere at any time. Therefore, continued vigilance and further research is warranted. We also note emphatically that to prevent future pandemics and other world-wide health crises, it is imperative to capitalize on what we have learnt from COVID-19: specifically, regarding its origins, the world's response, and insufficient preparedness. This requires unprecedented international collaboration and timely data sharing for the coordination of effective response and the rapid implementation of containment measures.

An AI-powered patient triage platform for future viral outbreaks using COVID-19 as a disease model.

Over the last century, outbreaks and pandemics have occurred with disturbing regularity, necessitating advance preparation and large-scale, coordinated response. Here, we developed a machine learning predictive model of disease severity and length of hospitalization for COVID-19, which can be utilized as a platform for future unknown viral outbreaks. We combined untargeted metabolomics on plasma data obtained from COVID-19 patients (n = 111) during hospitalization and healthy controls (n = 342), clinical and comorbidity data (n = 508) to build this patient triage platform, which consists of three parts: (i) the clinical decision tree, which amongst other biomarkers showed that patients with increased eosinophils have worse disease prognosis and can serve as a new potential biomarker with high accuracy (AUC = 0.974), (ii) the estimation of patient hospitalization length with ± 5 days error (R2 = 0.9765) and (iii) the prediction of the disease severity and the need of patient transfer to the intensive care unit. We report a significant decrease in serotonin levels in patients who needed positive airway pressure oxygen and/or were intubated. Furthermore, 5-hydroxy tryptophan, allantoin, and glucuronic acid metabolites were increased in COVID-19 patients and collectively they can serve as biomarkers to predict disease progression. The ability to quickly identify which patients will develop life-threatening illness would allow the efficient allocation of medical resources and implementation of the most effective medical interventions. We would advocate that the same approach could be utilized in future viral outbreaks to help hospitals triage patients more effectively and improve patient outcomes while optimizing healthcare resources.

Use of Whole-Genome Sequencing to Estimate the Contribution of Immune Evasion and Waning Immunity on Decreasing COVID-19 Vaccine Effectiveness.

BACKGROUND: The impact variant-specific immune evasion and waning protection have on declining coronavirus disease 2019 (COVID-19) vaccine effectiveness (VE) remains unclear. Using whole-genome sequencing (WGS), we examined the contribution these factors had on the decline that followed the introduction of the Delta variant. Furthermore, we evaluated calendar-period-based classification as a WGS alternative. METHODS: We conducted a test-negative case-control study among people tested for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) between 1 April and 24 August 2021. Variants were classified using WGS and calendar period. RESULTS: We included 2029 cases (positive, sequenced samples) and 343 727 controls (negative tests). VE 14-89 days after second dose was significantly higher against Alpha (84.4%; 95% confidence interval [CI], 75.6%-90.0%) than Delta infection (68.9%; 95% CI, 58.0%-77.1%). The odds of Delta infection were significantly higher 90-149 than 14-89 days after second dose (P value = .003). Calendar-period-classified VE estimates approximated WGS-classified estimates; however, calendar-period-based classification was subject to misclassification (35% Alpha, 4% Delta). CONCLUSIONS: Both waning protection and variant-specific immune evasion contributed to the lower effectiveness. While calendar-period-classified VE estimates mirrored WGS-classified estimates, our analysis highlights the need for WGS when variants are cocirculating and misclassification is likely.

Testing Frequency Matters: An Evaluation of the Diagnostic Performance of a Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Rapid Antigen Test in US Correctional Facilities.

BACKGROUND: The Centers for Disease Control and Prevention recommends serial rapid antigen assay collection within congregate facilities. Although modeling and observational studies from communities and long-term care facilities have shown serial collection provides adequate sensitivity and specificity, the accuracy within correctional facilities remains unknown. METHODS: Using Connecticut Department of Correction data from 21 November 2020 to 15 June 2021, we estimated the accuracy of a rapid assay, BinaxNOW (Abbott), under 3 collection strategies: single test collection and serial collection of 2 and 3 tests separated by 1-4 days. The sensitivity and specificity of the first (including single), second, and third serially collected BinaxNOW tests were estimated relative to RT-PCRs collected ≤1 day of the BinaxNOW test. The accuracy metrics of the testing strategies were then estimated as the sum (sensitivity) and product (specificity) of tests in each strategy. RESULTS: Of the 13 112 residents who contributed ≥1 BinaxNOW test during the study period, 3825 contributed ≥1 RT-PCR paired BinaxNOW test. In relation to RT-PCR, the 3-rapid-antigen-test strategy had a sensitivity of 95.9% (95% CI: 93.6-97.5%) and specificity of 98.3% (95% CI: 96.7-99.1%). The sensitivities of the 2- and 1-rapid-antigen-test strategies were 88.8% and 66.8%, and the specificities were 98.5% and 99.4%, respectively. The sensitivity was higher among symptomatic residents and when RT-PCRs were collected before BinaxNOW tests. CONCLUSIONS: We found serial antigen test collection resulted in high diagnostic accuracy. These findings support serial collection for outbreak investigation, screening, and when rapid detection is required (such as intakes or transfers).

Rapid, reliable, and reproducible cell fusion assay to quantify SARS-Cov-2 spike interaction with hACE2.

COVID-19 is a global crisis of unimagined dimensions. Currently, Remedesivir is only fully licensed FDA therapeutic. A major target of the vaccine effort is the SARS-CoV-2 spike-hACE2 interaction, and assessment of efficacy relies on time consuming neutralization assay. Here, we developed a cell fusion assay based upon spike-hACE2 interaction. The system was tested by transient co-transfection of 293T cells, which demonstrated good correlation with standard spike pseudotyping for inhibition by sera and biologics. Then established stable cell lines were very well behaved and gave even better correlation with pseudotyping results, after a short, overnight co-incubation. Results with the stable cell fusion assay also correlated well with those of a live virus assay. In summary we have established a rapid, reliable, and reproducible cell fusion assay that will serve to complement the other neutralization assays currently in use, is easy to implement in most laboratories, and may serve as the basis for high throughput screens to identify inhibitors of SARS-CoV-2 virus-cell binding and entry.

Detection of SARS-CoV-2 RNA by multiplex RT-qPCR.

The current quantitative reverse transcription PCR (RT-qPCR) assay recommended for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing in the United States requires analysis of 3 genomic targets per sample: 2 viral and 1 host. To simplify testing and reduce the volume of required reagents, we devised a multiplex RT-qPCR assay to detect SARS-CoV-2 in a single reaction. We used existing N1, N2, and RP primer and probe sets by the Centers for Disease Control and Prevention, but substituted fluorophores to allow multiplexing of the assay. The cycle threshold (Ct) values of our multiplex RT-qPCR were comparable to those obtained by the single assay adapted for research purposes. Low copy numbers (≥500 copies/reaction) of SARS-CoV-2 RNA were consistently detected by the multiplex RT-qPCR. Our novel multiplex RT-qPCR improves upon current single diagnostics by saving reagents, costs, time, and labor.

Cutting Edge: Severe SARS-CoV-2 Infection in Humans Is Defined by a Shift in the Serum Lipidome, Resulting in Dysregulation of Eicosanoid Immune Mediators.

The COVID-19 pandemic has affected more than 20 million people worldwide, with mortality exceeding 800,000 patients. Risk factors associated with severe disease and mortality include advanced age, hypertension, diabetes, and obesity. Each of these risk factors pathologically disrupts the lipidome, including immunomodulatory eicosanoid and docosanoid lipid mediators (LMs). We hypothesized that dysregulation of LMs may be a defining feature of the severity of COVID-19. By examining LMs and polyunsaturated fatty acid precursor lipids in serum from hospitalized COVID-19 patients, we demonstrate that moderate and severe disease are separated by specific differences in abundance of immune-regulatory and proinflammatory LMs. This difference in LM balance corresponded with decreased LM products of ALOX12 and COX2 and an increase LMs products of ALOX5 and cytochrome p450. Given the important immune-regulatory role of LMs, these data provide mechanistic insight into an immuno-lipidomic imbalance in severe COVID-19.

Bartonella in Norway rats (Rattus norvegicus) from the urban slum environment in Brazil.

Bartonella are rodent-borne bacteria that cause varied human etiologies. Studies on synanthropic rodents are rare, causing gaps in epidemiological knowledge. We tested bloodclot samples from 79 rats from an urban slum in Salvador, Brazil through PCR targeting gltA gene. Nine samples (11.4%) were positive: six had 100% identity with Bartonella sp. isolate JF429580 and 99.5% with B. queenslandensis strain AUST/NH8; three were 100% identical to isolate JF429532 and 99.7% to B. tribocorum. This is the second report on urban rat Bartonella indicating bacterial circulation at detectable rates. Its presence in rats from vulnerable human settlements demands public health attention.

Genetic Evidence for a Potential Environmental Pathway to Spillover Infection of Rat-Borne Leptospirosis.

In this study, we genotyped samples from environmental reservoirs (surface water and soil), colonized rat specimens, and cases of human severe leptospirosis from an endemic urban slum in Brazil, to determine the molecular epidemiology of pathogenic Leptospira and identify pathways of leptospirosis infection. We identified a well-established population of Leptospira interrogans serovar Copenhageni common to human leptospirosis cases, and animal and environmental reservoirs. This finding provides genetic evidence for a potential environmental spillover pathway for rat-borne leptospirosis through the environment in this urban community and highlights the importance of environmental and social interventions to reduce spillover infections.

Longitudinal Immune Profiling of a Severe Acute Respiratory Syndrome Coronavirus 2 Reinfection in a Solid Organ Transplant Recipient.

BACKGROUND: The underlying immunologic deficiencies enabling severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reinfection are currently unknown. We describe deep longitudinal immune profiling of a transplant recipient hospitalized twice for coronavirus disease 2019 (COVID-19). METHODS: A 66-year-old male renal transplant recipient was hospitalized with COVID-19 March 2020 then readmitted to the hospital with COVID-19 233 days after initial diagnosis. Virologic and immunologic investigations were performed on samples from the primary and secondary infections. RESULTS: Whole viral genome sequencing and phylogenetic analysis revealed that viruses causing both infections were caused by distinct genetic lineages without evidence of immune escape mutations. Longitudinal comparison of cellular and humoral responses during primary SARS-CoV-2 infection revealed that this patient responded to the primary infection with low neutralization titer anti-SARS-CoV-2 antibodies that were likely present at the time of reinfection. CONCLUSIONS: The development of neutralizing antibodies and humoral memory responses in this patient failed to confer protection against reinfection, suggesting that they were below a neutralizing titer threshold or that additional factors may be required for efficient prevention of SARS-CoV-2 reinfection. Development of poorly neutralizing antibodies may have been due to profound and relatively specific reduction in naive CD4 T-cell pools. Seropositivity alone may not be a perfect correlate of protection in immunocompromised patients.

Rickettsial Infections Causing Acute Febrile Illness in Urban Slums, Brazil.

We conducted enhanced acute febrile illness surveillance in an urban slum community in Salvador, Brazil. We found that rickettsial infection accounted for 3.5% of urgent care visits for acute fever. Our results suggest that rickettsiae might be an underrecognized, treatable cause of acute febrile illness in impoverished urban populations in Brazil.