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The emergence of SARS-CoV-2 variants with mutations in major neutralizing antibody-binding sites can affect humoral immunity induced by infection or vaccination(1–6). We analysed the development of anti-SARS-CoV-2 antibody and T cell responses in previously infected (recovered) or uninfected (naive) individuals that received mRNA vaccines to SARS-CoV-2. While previously infected individuals sustained higher antibody titers than uninfected individuals 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 (e.g., B.1.351 and P.1) had the greatest reduction, followed by lineages with L452R (e.g., B.1.617.2). While both groups retained neutralization capacity against all variants, plasma from previously infected vaccinated individuals displayed overall better neutralization capacity when compared to plasma from uninfected individuals that also received two vaccine doses, pointing to vaccine boosters as a relevant future strategy to alleviate the impact of emerging variants on antibody neutralizing activity.

SARS-CoV-2 infection can result in the development of a constellation of persistent sequelae following acute disease called post-acute sequelae of COVID-19 (PASC) or Long COVID1–3. Individuals diagnosed with Long COVID frequently report unremitting fatigue, post-exertional malaise, and a variety of cognitive and autonomic dysfunctions1–3; however, the basic biological mechanisms responsible for these debilitating symptoms are unclear. Here, 215 individuals were included in an exploratory, cross-sectional study to perform multi-dimensional immune phenotyping in conjunction with machine learning methods to identify key immunological features distinguishing Long COVID. Marked differences were noted in specific circulating myeloid and lymphocyte populations relative to matched control groups, as well as evidence of elevated humoral responses directed against SARS-CoV-2 among participants with Long COVID. Further, unexpected increases were observed in antibody responses directed against non-SARS-CoV-2 viral pathogens, particularly Epstein-Barr virus. Analysis of circulating immune mediators and various hormones also revealed pronounced differences, with levels of cortisol being uniformly lower among participants with Long COVID relative to matched control groups. Integration of immune phenotyping data into unbiased machine learning models identified significant distinguishing features critical in accurate classification of Long COVID, with decreased levels of cortisol being the most significant individual predictor. These findings will help guide additional studies into the pathobiology of Long COVID and may aid in the future development of objective biomarkers for Long COVID.

Cerebral small vessel disease (CSVD) is a common disease among elderly individuals and recognized as a major cause of vascular cognitive impairment. Recent studies demonstrated that CSVD is a disconnection syndrome. However, due to the covert neurological symptoms and subtle changes in clinical performance, the connection alterations during the stage of preclinical cognitive impairment (PCI) and mild cognitive impairment (MCI) are usually neglected and still largely unknown. Using diffusion tensor imaging (DTI), we investigated the early structural network changes in PCI and MCI patients. The PCI group demonstrated well preserved rich-club organization, less nodal strength loss, and disruption of connections centered in the feeder and local connections. Nevertheless, the MCI group manifested a disruption in the rich-club organization, a worse nodal strength loss especially in hub nodes, and an overall disturbance in rich-club, feeder and local connections. Moreover, in all CSVD patients, the strength of the rich-club, feeder and local connections was significantly correlated with multiple cognitive scores, especially in attention, executive, and memory domains; while in MCI patients, only the strength of the rich-club connections was significantly correlated with cognition. Furthermore, based on the network-based statistic analysis, we also identified distinct network component disruption pattern between the PCI group and the MCI group, validating the results described above. These results suggest a disruption pattern from peripheral to central connections with the change of cognitive status, shedding light on the early identification and the underlying disruption of CSVD.

Saliva Specimens to Detect SARS-CoV-2 Infection In this letter, the investigators report that saliva specimens and nasopharyngeal swab specimens had similar sensitivity in the detection of SARS-CoV...

The presence of tissue-resident memory T cells at barrier tissues is critical for long-lasting protective immune responses. Previous work has shown that tissue-resident memory T cells can be established by “pulling” virus-specific effector T cells from circulation to the genital mucosa via topical vaginal application of chemokines in mice. Once established, these cells protect hosts against genital herpes infection. We recently showed that vaginal application of aminoglycoside antibiotics induces robust activation of the IFN signaling pathway, including upregulation of chemokine expression within the tissue in mice. In this study, we show that a single topical application of neomycin, an inexpensive and vaginally nontoxic antibiotic, is sufficient to pull CD8 T cells to the vaginal mucosa and provide protection against genital herpes infection in mice.

Survivors of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection frequently experience lingering neurological symptoms, including impairment in attention, concentration, speed of information processing and memory. This long-COVID cognitive syndrome shares many features with the syndrome of cancer therapy-related cognitive impairment (CRCI). Neuroinflammation, particularly microglial reactivity and consequent dysregulation of hippocampal neurogenesis and oligodendrocyte lineage cells, is central to CRCI. We hypothesized that similar cellular mechanisms may contribute to the persistent neurological symptoms associated with even mild SARS-CoV-2 respiratory infection. Here, we explored neuroinflammation caused by mild respiratory SARS-CoV-2 infection – without neuroinvasion - and effects on hippocampal neurogenesis and the oligodendroglial lineage. Using a mouse model of mild respiratory SARS-CoV-2 infection induced by intranasal SARS-CoV-2 delivery, we found white matter-selective microglial reactivity, a pattern observed in CRCI. Human brain tissue from 9 individuals with COVID-19 or SARS-CoV-2 infection exhibits the same pattern of prominent white matter-selective microglial reactivity. In mice, pro-inflammatory CSF cytokines/chemokines were elevated for at least 7-weeks post-infection; among the chemokines demonstrating persistent elevation is CCL11, which is associated with impairments in neurogenesis and cognitive function. Humans experiencing long-COVID with cognitive symptoms (48 subjects) similarly demonstrate elevated CCL11 levels compared to those with long-COVID who lack cognitive symptoms (15 subjects). Impaired hippocampal neurogenesis, decreased oligodendrocytes and myelin loss in subcortical white matter were evident at 1 week, and persisted until at least 7 weeks, following mild respiratory SARS-CoV-2 infection in mice. Taken together, the findings presented here illustrate striking similarities between neuropathophysiology after cancer therapy and after SARS-CoV-2 infection, and elucidate cellular deficits that may contribute to lasting neurological symptoms following even mild 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.

SARS-CoV-2 remdesivir resistance mutations have been generated in vitro but have not been reported in patients receiving treatment with the antiviral agent. We present a case of an immunocompromised patient with acquired B-cell deficiency who developed an indolent, protracted course of SARS-CoV-2 infection. Remdesivir therapy alleviated symptoms and produced a transient virologic response, but her course was complicated by recrudescence of high-grade viral shedding. Whole genome sequencing identified a mutation, E802D, in the nsp12 RNA-dependent RNA polymerase, which was not present in pre-treatment specimens. In vitro experiments demonstrated that the mutation conferred a ~6-fold increase in remdesivir IC50 but resulted in a fitness cost in the absence of remdesivir. Sustained clinical and virologic response was achieved after treatment with casirivimab-imdevimab. Although the fitness cost observed in vitro may limit the risk posed by E802D, this case illustrates the importance of monitoring for remdesivir resistance and the potential benefit of combinatorial therapies in immunocompromised patients with SARS-CoV-2 infection.

SARS-CoV-2 remdesivir resistance mutations have been generated in vitro but have not been reported in patients receiving treatment with the antiviral agent. We present a case of an immunocompromised patient with acquired B-cell deficiency who developed an indolent, protracted course of SARS-CoV-2 infection. Remdesivir therapy alleviated symptoms and produced a transient virologic response, but her course was complicated by recrudescence of high-grade viral shedding. Whole genome sequencing identified a mutation, E802D, in the nsp12 RNA-dependent RNA polymerase, which was not present in pre-treatment specimens. In vitro experiments demonstrated that the mutation conferred a ∼6-fold increase in remdesivir IC50 but resulted in a fitness cost in the absence of remdesivir. Sustained clinical and virologic response was achieved after treatment with casirivimab-imdevimab. Although the fitness cost observed in vitro may limit the risk posed by E802D, this case illustrates the importance of monitoring for remdesivir resistance and the potential benefit of combinatorial therapies in immunocompromised patients with SARS-CoV-2 infection.

Widespread impairments in white matter and cerebrovascular integrity have been consistently implicated in the pathophysiology of patients with small vessel disease (SVD). However, the neural circuit mechanisms that underlie the developing progress of clinical cognitive symptoms remain largely elusive. Here, we conducted cross-modal MRI scanning including diffusion tensor imaging and arterial spin labeling in a cohort of 113 patients with SVD, which included 74 patients with vascular mild cognitive impairment (vMCI) and 39 patients without vMCI symptoms, and hence developed multimode imaging-based machine learning models to identify markers that discriminated SVD subtypes. Diffusion and perfusion features, respectively, extracted from individual white matter and gray matter regions were used to train three sets of classifiers in a nested 10-fold fashion: diffusion-based, perfusion-based, and combined diffusion-perfusion-based classifiers. We found that the diffusion-perfusion combined classifier achieved the highest accuracy of 72.57% with leave-one-out cross-validation, with the diffusion features largely spanning the capsular lateral pathway of the cholinergic tracts, and the perfusion features mainly distributed in the frontal-subcortical-limbic areas. Furthermore, diffusion-based features within vMCI group were associated with performance on executive function tests. We demonstrated the superior accuracy of using diffusion-perfusion combined multimode imaging features for classifying vMCI subtype out of a cohort of patients with SVD. Disruption of white matter integrity might play a critical role in the progression of cognitive impairment in patients with SVD, while malregulation of coritcal perfusion needs further study.

Antibodies secreted into mucosal barriers serve to protect the host from a variety of pathogens, and are the basis for successful vaccines1. In type I mucosa (such as the intestinal tract), dimeric IgA secreted by local plasma cells is transported through polymeric immunoglobulin receptors2 and mediates robust protection against viruses3,4. However, owing to the paucity of polymeric immunoglobulin receptors and plasma cells, how and whether antibodies are delivered to the type II mucosa represented by the lumen of the lower female reproductive tract remains unclear. Here, using genital herpes infection in mice, we show that primary infection does not establish plasma cells in the lamina propria of the female reproductive tract. Instead, upon secondary challenge with herpes simplex virus 2, circulating memory B cells that enter the female reproductive tract serve as the source of rapid and robust antibody secretion into the lumen of this tract. CD4 tissue-resident memory T cells secrete interferon-γ, which induces expression of chemokines, including CXCL9 and CXCL10. Circulating memory B cells are recruited to the vaginal mucosa in a CXCR3-dependent manner, and secrete virus-specific IgG2b, IgG2c and IgA into the lumen. These results reveal that circulating memory B cells act as a rapidly inducible source of mucosal antibodies in the female reproductive tract.

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. We analysed the development of anti-SARS-CoV-2 antibody and T cell responses in previously infected (recovered) or uninfected (naive) individuals that received mRNA vaccines to SARS-CoV-2. While previously infected individuals sustained higher antibody titers than uninfected individuals post-vaccination, the latter reached comparable levels of neutralization responses to the ancestral strain than previously infected individuals 7 days after the second vaccine dose. T cell activation markers measured upon spike or nucleocapsid peptide in vitro stimulation showed a progressive increase after vaccination in the time-points analysed. 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 (e.g., B.1.351 and P.1) had the greatest reduction, followed by lineages with L452R (e.g., B.1.617.2) or with E484K (without N501Y/T). While both groups retained neutralization capacity against all variants, plasma from previously infected vaccinated individuals displayed overall better neutralization capacity when compared to plasma from uninfected individuals that also received two vaccine doses, pointing to vaccine boosters as a relevant future strategy to alleviate the impact of emerging variants on antibody neutralizing activity.

BackgroundThe 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).MethodsA 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.ResultsWhole 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.ConclusionsThe 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.

The underlying immunologic deficiencies enabling SARS-CoV-2 reinfections are currently unknown. Here we describe a renal-transplant recipient who developed recurrent, symptomatic SARS-CoV-2 infection 7 months after primary infection. To elucidate the immunological mechanisms responsible for reinfection, we performed longitudinal profiling of cellular and humoral responses during both primary and recurrent SARS-CoV-2 infection. We found that the patient responded to the primary infection with transient, poor-quality adaptive immune responses that was further compromised by intervening treatment for acute rejection of the renal allograft prior to reinfection. Importantly, we identified the development of neutralizing antibodies and humoral memory responses prior to SARS-CoV-2 reinfection. However, these neutralizing antibodies failed to confer protection against reinfection, suggesting that additional factors are required for efficient prevention of SARS-CoV-2 reinfection. Further, we found no evidence supporting viral evasion of primary adaptive immune responses, suggesting that susceptibility to reinfection may be determined by host factors rather than pathogen adaptation.

Recent studies have provided insights into innate and adaptive immune dynamics in coronavirus disease 2019 (COVID-19). However, the exact features of antibody responses that govern COVID-19 disease outcomes remain unclear. In this study, we analyzed humoral immune responses in 229 patients with asymptomatic, mild, moderate and severe COVID-19 over time to probe the nature of antibody responses in disease severity and mortality. We observed a correlation between anti-spike (S) immunoglobulin G (IgG) levels, length of hospitalization and clinical parameters associated with worse clinical progression. Although high anti-S IgG levels correlated with worse disease severity, such correlation was time dependent. Deceased patients did not have higher overall humoral response than discharged patients. However, they mounted a robust, yet delayed, response, measured by anti-S, anti-receptor-binding domain IgG and neutralizing antibody (NAb) levels compared to survivors. Delayed seroconversion kinetics correlated with impaired viral control in deceased patients. Finally, although sera from 85% of patients displayed some neutralization capacity during their disease course, NAb generation before 14 d of disease onset emerged as a key factor for recovery. These data indicate that COVID-19 mortality does not correlate with the cross-sectional antiviral antibody levels per se but, rather, with the delayed kinetics of NAb production.

SummaryPrior to the emergence of antigenically distinct SARS-CoV-2 variants, reinfections were reported infrequently - presumably due to the generation of durable and protective immune responses. However, case reports also suggested that rare, repeated infections may occur as soon as 48 days following initial disease onset. The underlying immunologic deficiencies enabling SARS-CoV-2 reinfections are currently unknown. Here we describe a renal transplant recipient who developed recurrent, symptomatic SARS-CoV-2 infection - confirmed by whole virus genome sequencing - 7 months after primary infection. To elucidate the immunological mechanisms responsible for SARS-CoV-2 reinfection, we performed longitudinal profiling of cellular and humoral responses during both primary and recurrent SARS-CoV-2 infection. We found that the patient responded to the primary infection with transient, poor-quality adaptive immune responses. The patient’s immune system was further compromised by intervening treatment for acute rejection of the renal allograft prior to reinfection. Importantly, we also identified the development of neutralizing antibodies and the formation of humoral memory responses prior to SARS-CoV-2 reinfection. However, these neutralizing antibodies failed to confer protection against reinfection, suggesting that additional factors are required for efficient prevention of SARS-CoV-2 reinfection. Further, we found no evidence supporting viral evasion of primary adaptive immune responses, suggesting that susceptibility to reinfection may be determined by host factors rather than pathogen adaptation in this patient. In summary, our study suggests that a low neutralizing antibody presence alone is not sufficient to confer resistance against reinfection. Thus, patients with solid organ transplantation, or patients who are otherwise immunosuppressed, who recover from infection with SARS-CoV-2 may not develop sufficient protective immunity and are at risk of reinfection.

COVID survivors frequently experience lingering neurological symptoms that resemble cancer-therapy-related cognitive impairment, a syndrome for which white matter microglial reactivity and consequent neural dysregulation is central. Here, we explored the neurobiological effects of respiratory SARS-CoV-2 infection and found white-matter-selective microglial reactivity in mice and humans. Following mild respiratory COVID in mice, persistently impaired hippocampal neurogenesis, decreased oligodendrocytes, and myelin loss were evident together with elevated CSF cytokines/chemokines including CCL11. Systemic CCL11 administration specifically caused hippocampal microglial reactivity and impaired neurogenesis. Concordantly, humans with lasting cognitive symptoms post-COVID exhibit elevated CCL11 levels. Compared with SARS-CoV-2, mild respiratory influenza in mice caused similar patterns of white-matter-selective microglial reactivity, oligodendrocyte loss, impaired neurogenesis, and elevated CCL11 at early time points, but after influenza, only elevated CCL11 and hippocampal pathology persisted. These findings illustrate similar neuropathophysiology after cancer therapy and respiratory SARS-CoV-2 infection which may contribute to cognitive impairment following even mild COVID.

SARS-CoV-2 infection has so far affected over 42 million people worldwide, causing over 1.1 million deaths. With the large majority of SARS-CoV-2 infected individuals being asymptomatic, major concerns have been raised about possible long-term consequences of the infection. We developed an antigen capture assay to detect SARS-CoV-2 spike protein in urine samples from COVID-19 patients whose diagnosis was confirmed by PCR from nasopharyngeal swabs (NP-PCR+). The study used a collection of 233 urine samples from 132 participants from Yale New Haven Hospital and the Children’s Hospital of Philadelphia obtained during the pandemic (106 NP-PCR+ and 26 NP-PCR-) as well as a collection of 20 urine samples from 20 individuals collected before the pandemic. Our analysis identified 23 out of 91 (25%) NP-PCR+ adult participants with SARS-CoV-2 spike S1 protein in urine (Ur-S+). Interestingly, although all NP-PCR+ children were Ur-S-, 1 NP-PCR-child was found to be positive for spike protein in urine. Of the 23 Ur-S+ adults, only 1 individual showed detectable viral RNA in urine. Our analysis further showed that 24% and 21% of NP-PCR+ adults have high levels of albumin and cystatin C in urine, respectively. Among individuals with albuminuria (>0.3 mg/mg of creatinine) statistical correlation could be found between albumin and spike protein in urine. Together, our data showe that 1 of 4 of SARS-CoV-2 infected individuals develop renal abnormalities such as albuminuria. Awareness about the long-term impact of these findings is warranted.

While several clinical and immunological parameters correlate with disease severity and mortality in SARS-CoV-2 infection, work remains in identifying unifying correlates of coronavirus disease 2019 (COVID-19) that can be used to guide clinical practice. Here, we examine saliva and nasopharyngeal (NP) viral load over time and correlate them with patient demographics, and cellular and immune profiling. We found that saliva viral load was significantly higher in those with COVID-19 risk factors; that it correlated with increasing levels of disease severity and showed a superior ability over nasopharyngeal viral load as a predictor of mortality over time (AUC=0.90). A comprehensive analysis of immune factors and cell subsets revealed strong predictors of high and low saliva viral load, which were associated with increased disease severity or better overall outcomes, respectively. Saliva viral load was positively associated with many known COVID-19 inflammatory markers such as IL-6, IL-18, IL-10, and CXCL10, as well as type 1 immune response cytokines. Higher saliva viral loads strongly correlated with the progressive depletion of platelets, lymphocytes, and effector T cell subsets including circulating follicular CD4 T cells (cTfh). Anti-spike (S) and anti-receptor binding domain (RBD) IgG levels were negatively correlated with saliva viral load showing a strong temporal association that could help distinguish severity and mortality in COVID-19. Finally, patients with fatal COVID-19 exhibited higher viral loads, which correlated with the depletion of cTfh cells, and lower production of anti-RBD and anti-S IgG levels. Together these results demonstrated that viral load – as measured by saliva but not nasopharyngeal — is a dynamic unifying correlate of disease presentation, severity, and mortality over time.

Neurological symptoms are frequently observed in COVID-19. Here, we examine the neuroinvasive potential of SARS-CoV-2 and demonstrate infection of neurons in three separate approaches: mouse model, human brain organoid, and autopsy of COVID-19 patients., Although COVID-19 is considered to be primarily a respiratory disease, SARS-CoV-2 affects multiple organ systems including the central nervous system (CNS). Yet, there is no consensus on the consequences of CNS infections. Here, we used three independent approaches to probe the capacity of SARS-CoV-2 to infect the brain. First, using human brain organoids, we observed clear evidence of infection with accompanying metabolic changes in infected and neighboring neurons. However, no evidence for type I interferon responses was detected. We demonstrate that neuronal infection can be prevented by blocking ACE2 with antibodies or by administering cerebrospinal fluid from a COVID-19 patient. Second, using mice overexpressing human ACE2, we demonstrate SARS-CoV-2 neuroinvasion in vivo. Finally, in autopsies from patients who died of COVID-19, we detect SARS-CoV-2 in cortical neurons and note pathological features associated with infection with minimal immune cell infiltrates. These results provide evidence for the neuroinvasive capacity of SARS-CoV-2 and an unexpected consequence of direct infection of neurons by SARS-CoV-2., Graphical Abstract

SummaryRecent studies have provided insights into innate and adaptive immune dynamics in coronavirus disease 2019 (COVID-19). Yet, the exact feature of antibody responses that governs COVID-19 disease outcomes remain unclear. Here, we analysed humoral immune responses in 209 asymptomatic, mild, moderate and severe COVID-19 patients over time to probe the nature of antibody responses in disease severity and mortality. We observed a correlation between anti-Spike (S) IgG levels, length of hospitalization and clinical parameters associated with worse clinical progression. While high anti-S IgG levels correlated with worse disease severity, such correlation was time-dependent. Deceased patients did not have higher overall humoral response than live discharged patients. However, they mounted a robust, yet delayed response, measured by anti-S, anti-RBD IgG, and neutralizing antibody (NAb) levels, compared to survivors. Delayed seroconversion kinetics correlated with impaired viral control in deceased patients. Finally, while sera from 89% of patients displayed some neutralization capacity during their disease course, NAb generation prior to 14 days of disease onset emerged as a key factor for recovery. These data indicate that COVID-19 mortality does not correlate with the cross-sectional antiviral antibody levelsper se, but rather with the delayed kinetics of NAb production.

The recent spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exemplifies the critical need for accurate and rapid diagnostic assays to prompt clinical and public health interventions. Currently, several quantitative reverse transcription–PCR (RT–qPCR) assays are being used by clinical, research and public health laboratories. However, it is currently unclear whether results from different tests are comparable. Our goal was to make independent evaluations of primer–probe sets used in four common SARS-CoV-2 diagnostic assays. From our comparisons of RT–qPCR analytical efficiency and sensitivity, we show that all primer–probe sets can be used to detect SARS-CoV-2 at 500 viral RNA copies per reaction. The exception for this is the RdRp-SARSr (Charite) confirmatory primer–probe set which has low sensitivity, probably due to a mismatch to circulating SARS-CoV-2 in the reverse primer. We did not find evidence for background amplification with pre-COVID-19 samples or recent SARS-CoV-2 evolution decreasing sensitivity. Our recommendation for SARS-CoV-2 diagnostic testing is to select an assay with high sensitivity and that is regionally used, to ease comparability between outcomes. This is a comparative analysis of the performance of the primer–probe sets from four open-source molecular diagnostic assays for SARS-CoV-2 recommended by the World Health Organization.

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., The current RT-qPCR assay recommended for SARS-CoV-2 testing in the United States requires analysis of three genomic targets per sample: two viral and one host. This study describes a multiplex PCR that saves both reagents and labor while accurately detecting SARS-CoV-2 RNA.

Israelow et al. show that AAV-mediated expression of human ACE2 allows for SARS-CoV-2 infection and disease investigation in mice. This pathology is in part driven by type I interferon signaling, which recruits inflammatory immune cells without aborting viral replication., Severe acute respiratory syndrome–coronavirus 2 (SARS-Cov-2) has caused over 13,000,000 cases of coronavirus disease (COVID-19) with a significant fatality rate. Laboratory mice have been the stalwart of therapeutic and vaccine development; however, they do not support infection by SARS-CoV-2 due to the virus’s inability to use the mouse orthologue of its human entry receptor angiotensin-converting enzyme 2 (hACE2). While hACE2 transgenic mice support infection and pathogenesis, these mice are currently limited in availability and are restricted to a single genetic background. Here we report the development of a mouse model of SARS-CoV-2 based on adeno-associated virus (AAV)–mediated expression of hACE2. These mice support viral replication and exhibit pathological findings found in COVID-19 patients. Moreover, we show that type I interferons do not control SARS-CoV-2 replication in vivo but are significant drivers of pathological responses. Thus, the AAV-hACE2 mouse model enables rapid deployment for in-depth analysis following robust SARS-CoV-2 infection with authentic patient-derived virus in mice of diverse genetic backgrounds., Graphical Abstract

A growing body of evidence indicates sex differences in the clinical outcomes of coronavirus disease 2019 (COVID-19)1-4. However, whether immune responses against SARS-CoV-2 differ between sexes, and whether such differences explain male susceptibility to COVID-19, is currently unknown. In this study, we examined sex differences in viral loads, SARS-CoV-2-specific antibody titers, plasma cytokines, as well as blood cell phenotyping in COVID-19 patients. By focusing our analysis on patients with mild to moderate disease who had not received immunomodulatory medications, our results revealed that male patients had higher plasma levels of innate immune cytokines and chemokines including IL-8, IL-18, and CCL5, along with more robust induction of non-classical monocytes. In contrast, female patients mounted significantly more robust T cell activation than male patients during SARS-CoV-2 infection, which was sustained in old age. Importantly, we found that a poor T cell response negatively correlated with patients’ age and was predictive of worse disease outcome in male patients, but not in female patients. Conversely, higher innate immune cytokines in female patients associated with worse disease progression, but not in male patients. These findings reveal a possible explanation underlying observed sex biases in COVID-19, and provide important basis for the development of sex-based approach to the treatment and care of men and women with COVID-19.

The recent spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exemplifies the critical need for accurate and rapid diagnostic assays to prompt clinical and public health interventions. Currently, several quantitative reverse-transcription polymerase chain reaction (qRT-PCR) assays are being used by clinical, research, and public health laboratories. However, it is currently unclear if results from different tests are comparable. Our goal was to evaluate the primer-probe sets used in four common diagnostic assays available on the World Health Organization (WHO) website. To facilitate this effort, we generated RNA transcripts to be used as assay standards and distributed them to other laboratories for internal validation. We then used these (1) RNA transcript standards, (2) full-length SARS-CoV-2 RNA, and (3) pre-COVID-19 nasopharyngeal swabs, and (4) clinical samples from COVID-19 patients to determine analytical efficiency and sensitivity of the qRT-PCR primer-probe sets. We show that all primer-probe sets can be used to detect SARS-CoV-2, but there are clear differences in the ability to differentiate between true negatives and positives with low amounts of virus. We found that several primer-probe sets cross-react with SARS-CoV-2-negative nasopharyngeal swabs. However, background cross-reactivity by the 2019-nCoV_N2 set issued by the US Centers for Disease Control and Prevention did not interfere with outcomes of the combined 9N19 and 9N29 assay when testing COVID-19 clinical samples. Our findings characterize the limitations of currently used primer-probe sets and can assist other laboratories in selecting appropriate assays for the detection of SARS-CoV-2.

One promising approach for a herpes simplex virus vaccine uses a vaccine to prime and a chemoattractant to pull immune cells into the genital tract. We evaluated subunit vaccines (prime) and imiquimod (pull) in the guinea pig (gp) model of recurrent Herpes simplex virus type-2 (HSV-2). Following vaginal HSV-2 infection, gps were vaccinated with various combination of glycoproteins and adjuvant with or without subcutaneous or local applications of imiquimod after infection. Animals were examined daily for recurrent lesions and vaginal swabs collected for recurrent shedding. Although both the vaccines alone and imiquimod alone reduced recurrent HSV disease, the combination of local imiquimod and vaccine (Prime and Pull) was the most effective. In the first study, immunization with the trivalent vaccine alone or imiquimod alone decreased recurrent disease. However, the largest decrease was with the combination of vaccine and local imiquimod (P, Herpes simplex virus: prime and pull vaccination “Prime and pull” vaccination strategies involve an initial vaccination followed by the local application of a stimulant such as chemokines to recruit immune cells to infection target areas such as the mucosa. David Bernstein and colleagues investigate the efficacy of a prime and pull approach in a guinea pig model of recurrent genital Herpes simplex virus 2 (HSV-2) infection. Animals were vaccinated with HSV-2 glycoproteins in adjuvant with or without subcutaneous or topical (vaginal) exposure to the immune stimulant imiquimod to act as the “pull”. Animals with topical imiquimod show superior control of HSV-2 infection and improved recruitment of CD8+ T cells to the vaginal epithelium. Topical application of imiquimod demonstrates better control than subcutaneous imiquimod suggesting that “pulling” might be a useful approach in HSV vaccination.

Recent studies have provided insights into innate and adaptive immune dynamics in coronavirus disease 2019 (COVID-19). However, the exact features of antibody responses that govern COVID-19 disease outcomes remain unclear. In this study, we analyzed humoral immune responses in 229 patients with asymptomatic, mild, moderate and severe COVID-19 over time to probe the nature of antibody responses in disease severity and mortality. We observed a correlation between anti-spike (S) immunoglobulin G (IgG) levels, length of hospitalization and clinical parameters associated with worse clinical progression. Although high anti-S IgG levels correlated with worse disease severity, such correlation was time dependent. Deceased patients did not have higher overall humoral response than discharged patients. However, they mounted a robust, yet delayed, response, measured by anti-S, anti-receptor-binding domain IgG and neutralizing antibody (NAb) levels compared to survivors. Delayed seroconversion kinetics correlated with impaired viral control in deceased patients. Finally, although sera from 85% of patients displayed some neutralization capacity during their disease course, NAb generation before 14 d of disease onset emerged as a key factor for recovery. These data indicate that COVID-19 mortality does not correlate with the cross-sectional antiviral antibody levels per se but, rather, with the delayed kinetics of NAb production.

The role of type I interferon (IFN-I) in SARS-CoV-2 infection has remained controversial. Elevated IFN-I levels are associated with increased COVID-19 morbidity and mortality; however, impaired IFN-I signaling, either genetic or via autoantibodies, is also associated with worse outcomes. While these studies have focused on the role of IFN-I in COVID-19 pathogenesis, its role in modulating adaptive immunity to SARS-CoV-2 remains unknown. Here we investigate the role of IFN-I in shaping the humoral immune response to SARS-CoV-2 infection in both COVID-19 patients and mouse models of SARS-CoV-2 infection. We find that patients with COVID-19 who have higher levels of plasma IFN-I develop lower anti-spike (S1) and anti-receptor binding domain (RBD) IgG levels. Similarly, we find that mice lacking the interferon-α/β receptor (IFNAR) or treated with IFNAR blocking antibodies develop higher levels of anti-SARS-CoV-2 S1 and RBD IgG, as well as higher levels of anti-S1 IgA, IgG1, and IgG2. Furthermore, we find that IFNAR deficiency promotes the development of follicular helper T cells, germinal center B cells, and antibody-secreting cells in the draining lymph node. Accordingly, patients with higher serum IFN-I levels have reduced circulating follicular helper T cells. Despite higher levels of anti-SARS-CoV-2 IgG, sera from IFN-I sufficient or deficient mice harbored comparable neutralizing activities, suggesting reduced antibody potency (IC50/IgG) in IFNAR-deficient mice. Similarly, patients with higher serum IFN-I levels produced higher-potency anti-SARS-CoV-2 antibodies. These findings highlight a beneficial role for IFN-I in modulating cellular responses critical for promoting a potent antibody response to SARS-CoV-2.

Background Scaling SARS-CoV-2 testing to meet demands of safe reopenings continues to be plagued by assay costs and supply chain shortages. In response, we developed SalivaDirect, which received Emergency Use Authorization (EUA) from the U.S. Food and Drug Administration (FDA). Methods We simplified our saliva-based diagnostic test by (1) not requiring collection tubes with preservatives, (2) replacing nucleic acid extraction with a simple enzymatic and heating step, and (3) testing specimens with a dualplex qRT-PCR assay. Moreover, we validated SalivaDirect with reagents and instruments from multiple vendors to minimize supply chain issues. Findings From our hospital cohort, we show a high positive agreement (94%) between saliva tested with SalivaDirect and nasopharyngeal swabs tested with a commercial qRT-PCR kit. In partnership with the National Basketball Association (NBA) and National Basketball Players Association (NBPA), we tested 3,779 saliva specimens from healthy individuals and detected low rates of invalid (0.3%) and false-positive (, Context and Significance Frequent testing is critical to limit SARS-CoV-2 transmission. In response to this need, we developed SalivaDirect, a sensitive, simplified, and flexible testing framework, which received Emergency Use Authorization (EUA) from the U.S. Food and Drug Administration (FDA). We tested saliva collected from a hospital cohort and showed a high positive agreement (94%) as compared to paired nasopharyngeal swabs tested with a commercial diagnostic kit. Then, we partnered with the National Basketball Association (NBA) to test a large cohort of mostly healthy individuals, and we detected low rates of invalid (0.3%) and false-positive (0.03%–0.05%) results. Our study shows that SalivaDirect can help to increase testing capacity by providing access to an affordable framework that is less prone to supply chain shortages., Graphical Abstract, Highlights SalivaDirect is a simplified saliva-based test for detection of SARS-CoV-2 The testing framework is flexible to minimize the risk of supply chain issues SalivaDirect is sensitive, with low rates of invalid and false-positive results Laboratories can be designated to use SalivaDirect to increase testing capacity, SalivaDirect is a sensitive saliva-based COVID-19 diagnostic test, which received Emergency Use Authorization from the U.S. FDA. With the ability to designate other laboratories, the flexible and simplified framework helps to increase the capacity of existing laboratory infrastructure for SARS-CoV-2 testing.

INTRODUCTION Development of long-term antibody-mediated immunity relies on the germinal center (GC) reaction, in which CD4 + T cells help B cells to proliferate and differentiate into long-lived antibody-producing plasma cells or memory B cells. The GC reaction is orchestrated in a specialized microenvironment and critically depends on guided migratory dynamics of T and B cells. Follicular T helper (T FH ) cells, a subset of effector CD4 + helper T cells specialized in supporting the GC reaction, deliver contact-dependent help signals, such as the CD40 ligand, and cytokines, such as interleukin-21 (IL-21), to promote GC B cell proliferation and differentiation. However, exaggerated T FH accumulation and function can cause abnormal GC responses and potentiate autoantibody-mediated inflammatory diseases or even lymphomas. Understanding of how T FH cells are recruited, retained, and functionally regulated in the GC microenvironment is needed for mechanistic insights into long-lived antibody-mediated immunity. Such understanding will inform better design of antibody-based vaccines for microbial infections and therapies for autoimmune diseases. RATIONALE Local mechanisms that regulate T FH cell dynamics and helper functions in GCs are not well defined. Multiple soluble guidance cues sensed by heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors promote T FH confinement toward the center of the B cell follicle of the secondary lymphoid organ, where the GC is typically formed. Contact-dependent guidance mechanisms are also thought to help regulate recruitment and retention of T FH cells in GCs. To search for such mechanisms, we examined the erythropoietin-producing hepatocellular (EPH) receptors and EPH-interacting protein (ephrin, EFN) family members. These molecules are known to guide cell migration during tissue morphogenesis in a contact-dependent manner. RESULTS We found that ephrin-B1 (EFNB1), a class B ephrin, was highly expressed by GC B cells and demarcated the GC tissue domain boundary. Although EFNB1 was not required for initial GC formation or development of T FH cells, its ablation from B cells led to exaggerated local accumulation of T FH cells inside the GC. Dynamic imaging by intravital microscopy revealed that EFNB1 repulsively inhibited GC recruitment and retention of T FH cells, likely as a result of EFNB1-mediated suppression of antigen-specific GC B cell–T FH adhesion. This contact-dependent repulsion required forward signaling through T FH -expressed EPHB6 but not EPHB4 receptor. Unexpectedly, despite the exaggerated GC T FH accumulation, the magnitude of GC responses was largely unchanged, whereas development of plasma cells and affinities of antigen-specific antibodies were actually reduced. By contrast, in a GC mixed with EFNB1-sufficient and -deficient B cells, the latter cells had a competitive advantage in contributing to the plasma cell compartment. These apparently contradicting phenotypes revealed that, by EPHB4-dependent forward signaling, EFNB1 also promotes GC T FH production of IL-21, cytokine that is needed to drive plasma cell formation. Consequently, at the tissue level, EFNB1-deficient GCs lack sufficient IL-21 to support normal plasma cell formation and affinity maturation; in a competitive GC reaction where the same cohort of T FH cells were shared by EFNB1-deficient and -sufficient GC B cells, the role of EFNB1 in suppressing antigen-specific interactions between individual T FH and GC B cells dominates, affording the EFNB1-deficient GC B cells with more contact-dependent help and a competitive advantage. CONCLUSION By revealing a dual role for the GC-expressed EFNB1 molecule, our results uncover a regulatory system that controls GC T FH dynamics and function in the local tissue microenvironment. EFNB1 suppresses T-B adhesion and repulsively inhibits dynamic T FH recruitment and retention in the GC; yet, it also positively promotes IL-21 production by T FH cells as they dwell in the GC at any moment. This counterintuitive combination of negative regulation of residence but positive promotion of effector functions controlled by the same EFNB1 molecule likely helps to ensure that the potentially dangerous GC reaction is productive yet self-limiting. That helper T cell cytokine production is critically modulated in a tissue microenvironment–specific manner by dedicated receptor-ligand systems, the EFNB1-EPHB4 pair in the case of GCs, suggests strategies and targets of manipulation for the development of improved vaccines or therapies.

Germinal centers (GCs) support high-affinity, long-lived humoral immunity. How memory B cells develop in GCs is not clear. Through the use of a cell-cycle-reporting system, we identified GC-derived memory precursor cells (GC-MP cells) that had quit cycling and reached G0 phase while in the GC, exhibited memory-associated phenotypes with signs of affinity maturation and localized toward the GC border. After being transferred into adoptive hosts, GC-MP cells reconstituted a secondary response like genuine memory B cells. GC-MP cells expressed the interleukin 9 (IL-9) receptor and responded to IL-9. Acute treatment with IL-9 or antibody to IL-9 accelerated or retarded the positioning of GC-MP cells toward the GC edge and exit from the GC, and enhanced or inhibited the development of memory B cells, which required B cell-intrinsic responsiveness to IL-9. Follicular helper T cells (TFH cells) produced IL-9, and deletion of IL-9 from T cells or, more specifically, from GC TFH cells led to impaired memory formation of B cells. Therefore, the GC development of memory B cells is promoted by TFH cell-derived IL-9.

Affinity-matured, isotype-switched antibodies afford humoral protection against microbial infections. Cells capable of producing such antibodies are derived from the germinal center (GC) formed during a T-dependent B-cell response. Follicular T-helper (TFH) cells are a recently defined subset of CD4 T cells that are specialized in promoting the B-cell response and GC reaction. These cells exhibit a CXCR5(+)ICOS(hi)PD-1(hi) surface phenotype, express a high level of transcriptional repressor Bcl-6 and possess a unique ability to reside in the GC. Insights into how TFH cells develop and function promise to refine our strategies toward more effective antibody-based vaccines and therapies for humoral autoimmunity. In this review, we summarize the current understanding of how TFH-associated molecules regulate dynamic localization and B-cell-interacting properties of these cells, as both aspects are at the core of being TFH cells.