Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy.

The December 2019 outbreak of a novel respiratory virus, SARS-CoV-2, has become an ongoing global pandemic due in part to the challenge of identifying symptomatic, asymptomatic, and pre-symptomatic carriers of the virus. CRISPR diagnostics can augment gold-standard PCR-based testing if they can be made rapid, portable, and accurate. Here, we report the development of an amplification-free CRISPR-Cas13a assay for direct detection of SARS-CoV-2 from nasal swab RNA that can be read with a mobile phone microscope. The assay achieved ∼100 copies/µL sensitivity in under 30 min of measurement time and accurately detected pre-extracted RNA from a set of positive clinical samples in under 5 min. We combined crRNAs targeting SARS-CoV-2 RNA to improve sensitivity and specificity and directly quantified viral load using enzyme kinetics. Integrated with a reader device based on a mobile phone, this assay has the potential to enable rapid, low-cost, point-of-care screening for SARS-CoV-2.

Autoantibodies against type I IFNs in humans with alternative NF-κB pathway deficiency.

Patients with autoimmune polyendocrinopathy syndrome type 1 (APS-1) caused by autosomal recessive AIRE deficiency produce autoantibodies that neutralize type I interferons (IFNs)1,2, conferring a predisposition to life-threatening COVID-19 pneumonia3. Here we report that patients with autosomal recessive NIK or RELB deficiency, or a specific type of autosomal-dominant NF-κB2 deficiency, also have neutralizing autoantibodies against type I IFNs and are at higher risk of getting life-threatening COVID-19 pneumonia. In patients with autosomal-dominant NF-κB2 deficiency, these autoantibodies are found only in individuals who are heterozygous for variants associated with both transcription (p52 activity) loss of function (LOF) due to impaired p100 processing to generate p52, and regulatory (IκBδ activity) gain of function (GOF) due to the accumulation of unprocessed p100, therefore increasing the inhibitory activity of IκBδ (hereafter, p52LOF/IκBδGOF). By contrast, neutralizing autoantibodies against type I IFNs are not found in individuals who are heterozygous for NFKB2 variants causing haploinsufficiency of p100 and p52 (hereafter, p52LOF/IκBδLOF) or gain-of-function of p52 (hereafter, p52GOF/IκBδLOF). In contrast to patients with APS-1, patients with disorders of NIK, RELB or NF-κB2 have very few tissue-specific autoantibodies. However, their thymuses have an abnormal structure, with few AIRE-expressing medullary thymic epithelial cells. Human inborn errors of the alternative NF-κB pathway impair the development of AIRE-expressing medullary thymic epithelial cells, thereby underlying the production of autoantibodies against type I IFNs and predisposition to viral diseases.

Clonally expanded B cells in multiple sclerosis bind EBV EBNA1 and GlialCAM.

Multiple sclerosis (MS) is a heterogenous autoimmune disease in which autoreactive lymphocytes attack the myelin sheath of the central nervous system. B lymphocytes in the cerebrospinal fluid (CSF) of patients with MS contribute to inflammation and secrete oligoclonal immunoglobulins1,2. Epstein-Barr virus (EBV) infection has been epidemiologically linked to MS, but its pathological role remains unclear3. Here we demonstrate high-affinity molecular mimicry between the EBV transcription factor EBV nuclear antigen 1 (EBNA1) and the central nervous system protein glial cell adhesion molecule (GlialCAM) and provide structural and in vivo functional evidence for its relevance. A cross-reactive CSF-derived antibody was initially identified by single-cell sequencing of the paired-chain B cell repertoire of MS blood and CSF, followed by protein microarray-based testing of recombinantly expressed CSF-derived antibodies against MS-associated viruses. Sequence analysis, affinity measurements and the crystal structure of the EBNA1-peptide epitope in complex with the autoreactive Fab fragment enabled tracking of the development of the naive EBNA1-restricted antibody to a mature EBNA1-GlialCAM cross-reactive antibody. Molecular mimicry is facilitated by a post-translational modification of GlialCAM. EBNA1 immunization exacerbates disease in a mouse model of MS, and anti-EBNA1 and anti-GlialCAM antibodies are prevalent in patients with MS. Our results provide a mechanistic link for the association between MS and EBV and could guide the development of new MS therapies.

Discovering disease-causing pathogens in resource-scarce Southeast Asia using a global metagenomic pathogen monitoring system.

SignificanceMetagenomic pathogen sequencing offers an unbiased approach to characterizing febrile illness. In resource-scarce settings with high biodiversity, it is critical to identify disease-causing pathogens in order to understand burden and to prioritize efforts for control. Here, metagenomic next-generation sequencing (mNGS) characterization of the pathogen landscape in Cambodia revealed diverse vector-borne and zoonotic pathogens irrespective of age and gender as risk factors. Identification of key pathogens led to changes in national program surveillance. This study is a "real world" example of the use of mNGS surveillance of febrile individuals, executed in-country, to identify outbreaks of vector-borne, zoonotic, and other emerging pathogens in a resource-scarce setting.

Detection of High-Risk Paraneoplastic Antibodies against TRIM9 and TRIM67 Proteins.

OBJECTIVE: Co-occurring anti-tripartite motif-containing protein 9 and 67 autoantibodies (TRIM9/67-IgG) have been reported in only a very few cases of paraneoplastic cerebellar syndrome. The value of these biomarkers and the most sensitive methods of TRIM9/67-IgG detection are not known. METHODS: We performed a retrospective, multicenter study to evaluate the cerebrospinal fluid and serum of candidate TRIM9/67-IgG cases by tissue-based immunofluorescence, peptide phage display immunoprecipitation sequencing, overexpression cell-based assay (CBA), and immunoblot. Cases in which TRIM9/67-IgG was detected by at least 2 assays were considered TRIM9/67-IgG positive. RESULTS: Among these cases (n = 13), CBA was the most sensitive (100%) and revealed that all cases had TRIM9 and TRIM67 autoantibodies. Of TRIM9/67-IgG cases with available clinical history, a subacute cerebellar syndrome was the most common presentation (n = 7/10), followed by encephalitis (n = 3/10). Of these 10 patients, 70% had comorbid cancer (7/10), 85% of whom (n = 6/7) had confirmed metastatic disease. All evaluable cancer biopsies expressed TRIM9 protein (n = 5/5), whose expression was elevated in the cancerous regions of the tissue in 4 of 5 cases. INTERPRETATION: TRIM9/67-IgG is a rare but likely high-risk paraneoplastic biomarker for which CBA appears to be the most sensitive diagnostic assay. ANN NEUROL 2023;94:1086-1101.

Zika virus alters centrosome organization to suppress the innate immune response.

Zika virus (ZIKV) is a flavivirus transmitted via mosquitoes and sex to cause congenital neurodevelopmental defects, including microcephaly. Inherited forms of microcephaly (MCPH) are associated with disrupted centrosome organization. Similarly, we found that ZIKV infection disrupted centrosome organization. ZIKV infection disrupted the organization of centrosomal proteins including CEP63, a MCPH-associated protein. The ZIKV nonstructural protein NS3 bound CEP63, and expression of NS3 was sufficient to alter centrosome architecture and CEP63 localization. Loss of CEP63 suppressed ZIKV-induced centrosome disorganization, indicating that ZIKV requires CEP63 to disrupt centrosome organization. ZIKV infection or CEP63 loss decreased the centrosomal localization and stability of TANK-binding kinase 1 (TBK1), a regulator of the innate immune response. ZIKV infection also increased the centrosomal accumulation of the CEP63 interactor DTX4, a ubiquitin ligase that degrades TBK1. Therefore, we propose that ZIKV disrupts CEP63 function to increase centrosomal DTX4 localization and destabilization of TBK1, thereby tempering the innate immune response.

Genomic analysis, immunomodulation and deep phenotyping of patients with nodding syndrome.

The aetiology of nodding syndrome remains unclear, and comprehensive genotyping and phenotyping data from patients remain sparse. Our objectives were to characterize the phenotype of patients with nodding syndrome, investigate potential contributors to disease aetiology, and evaluate response to immunotherapy. This cohort study investigated members of a single-family unit from Lamwo District, Uganda. The participants for this study were selected by the Ugandan Ministry of Health as representative for nodding syndrome and with a conducive family structure for genomic analyses. Of the eight family members who participated in the study at the National Institutes of Health (NIH) Clinical Center, three had nodding syndrome. The three affected patients were extensively evaluated with metagenomic sequencing for infectious pathogens, exome sequencing, spinal fluid immune analyses, neurometabolic and toxicology testing, continuous electroencephalography and neuroimaging. Five unaffected family members underwent a subset of testing for comparison. A distinctive interictal pattern of sleep-activated bursts of generalized and multifocal epileptiform discharges and slowing was observed in two patients. Brain imaging showed two patients had mild generalized cerebral atrophy, and both patients and unaffected family members had excessive metal deposition in the basal ganglia. Trace metal biochemical evaluation was normal. CSF was non-inflammatory and one patient had CSF-restricted oligoclonal bands. Onchocerca volvulus-specific antibodies were present in all patients and skin snips were negative for active onchocerciasis. Metagenomic sequencing of serum and CSF revealed hepatitis B virus in the serum of one patient. Vitamin B6 metabolites were borderline low in all family members and CSF pyridoxine metabolites were normal. Mitochondrial DNA testing was normal. Exome sequencing did not identify potentially causal candidate gene variants. Nodding syndrome is characterized by a distinctive pattern of sleep-activated epileptiform activity. The associated growth stunting may be due to hypothalamic dysfunction. Extensive testing years after disease onset did not clarify a causal aetiology. A trial of immunomodulation (plasmapheresis in two patients and intravenous immunoglobulin in one patient) was given without short-term effect, but longer-term follow-up was not possible to fully assess any benefit of this intervention.

Viral Detection by Reverse Transcriptase Polymerase Chain Reaction in Upper Respiratory Tract and Metagenomic RNA Sequencing in Lower Respiratory Tract in Critically Ill Children With Suspected Lower Respiratory Tract Infection.

OBJECTIVES: Viral lower respiratory tract infection (vLRTI) contributes to substantial morbidity and mortality in children. Diagnosis is typically confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) of nasopharyngeal specimens in hospitalized patients; however, it is unknown whether nasopharyngeal detection accurately reflects presence of virus in the lower respiratory tract (LRT). This study evaluates agreement between viral detection from nasopharyngeal specimens by RT-PCR compared with metagenomic next-generation RNA sequencing (RNA-Seq) from tracheal aspirates (TAs). DESIGN: This is an analysis of of a seven-center prospective cohort study. SETTING: Seven PICUs within academic children's hospitals in the United States. PATIENTS: Critically ill children (from 1 mo to 18 yr) who required mechanical ventilation via endotracheal tube for greater than or equal to 72 hours. INTERVENTIONS: We evaluated agreement in viral detection between paired upper and LRT samples. Results of clinical nasopharyngeal RT-PCR were compared with TA RNA-Seq. Positive and negative predictive agreement and Cohen's Kappa were used to assess agreement. MEASUREMENTS AND MAIN RESULTS: Of 295 subjects with paired testing available, 200 (68%) and 210 (71%) had positive viral testing by RT-PCR from nasopharyngeal and RNA-Seq from TA samples, respectively; 184 (62%) were positive by both nasopharyngeal RT-PCR and TA RNA-Seq for a virus, and 69 (23%) were negative by both methods. Nasopharyngeal RT-PCR detected the most abundant virus identified by RNA-Seq in 92.4% of subjects. Among the most frequent viruses detected, respiratory syncytial virus demonstrated the highest degree of concordance (κ = 0.89; 95% CI, 0.83-0.94), whereas rhinovirus/enterovirus demonstrated lower concordance (κ = 0.55; 95% CI, 0.44-0.66). Nasopharyngeal PCR was more likely to detect multiple viruses than TA RNA-Seq (54 [18.3%] vs 24 [8.1%], p ≤ 0.001). CONCLUSIONS: Viral nucleic acid detection in the upper versus LRT reveals good overall agreement, but concordance depends on the virus. Further studies are indicated to determine the utility of LRT sampling or the use of RNA-Seq to determine LRTI etiology.

NFKB2 haploinsufficiency identified via screening for IFN-α2 autoantibodies in children and adolescents hospitalized with SARS-CoV-2-related complications.

BACKGROUND: Autoantibodies against type I IFNs occur in approximately 10% of adults with life-threatening coronavirus disease 2019 (COVID-19). The frequency of anti-IFN autoantibodies in children with severe sequelae of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is unknown. OBJECTIVE: We quantified anti-type I IFN autoantibodies in a multicenter cohort of children with severe COVID-19, multisystem inflammatory syndrome in children (MIS-C), and mild SARS-CoV-2 infections. METHODS: Circulating anti-IFN-α2 antibodies were measured by a radioligand binding assay. Whole-exome sequencing, RNA sequencing, and functional studies of peripheral blood mononuclear cells were used to study any patients with levels of anti-IFN-α2 autoantibodies exceeding the assay's positive control. RESULTS: Among 168 patients with severe COVID-19, 199 with MIS-C, and 45 with mild SARS-CoV-2 infections, only 1 had high levels of anti-IFN-α2 antibodies. Anti-IFN-α2 autoantibodies were not detected in patients treated with intravenous immunoglobulin before sample collection. Whole-exome sequencing identified a missense variant in the ankyrin domain of NFKB2, encoding the p100 subunit of nuclear factor kappa-light-chain enhancer of activated B cells, aka NF-κB, essential for noncanonical NF-κB signaling. The patient's peripheral blood mononuclear cells exhibited impaired cleavage of p100 characteristic of NFKB2 haploinsufficiency, an inborn error of immunity with a high prevalence of autoimmunity. CONCLUSIONS: High levels of anti-IFN-α2 autoantibodies in children and adolescents with MIS-C, severe COVID-19, and mild SARS-CoV-2 infections are rare but can occur in patients with inborn errors of immunity.

Low Prevalence of Interferon α Autoantibodies in People Experiencing Symptoms of Post-Coronavirus Disease 2019 (COVID-19) Conditions, or Long COVID.

Interferon (IFN)-specific autoantibodies have been implicated in severe coronavirus disease 2019 (COVID-19) and have been proposed as a potential driver of the persistent symptoms characterizing "long COVID," a type of postacute sequelae of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We report that only 2 of 215 participants with convalescent SARS-CoV-2 infection tested over 394 time points, including 121 people experiencing long COVID symptoms, had detectable IFN-α2 antibodies. Both had been hospitalized during the acute phase of the infection. These data suggest that persistent anti-IFN antibodies, although a potential driver of severe COVID-19, are unlikely to contribute to long COVID symptoms in the postacute phase of the infection.

Successful Treatment of Balamuthia mandrillaris Granulomatous Amebic Encephalitis with Nitroxoline.

A patient in California, USA, with rare and usually fatal Balamuthia mandrillaris granulomatous amebic encephalitis survived after receiving treatment with a regimen that included the repurposed drug nitroxoline. Nitroxoline, which is a quinolone typically used to treat urinary tract infections, was identified in a screen for drugs with amebicidal activity against Balamuthia.

Discovery and Genomic Characterization of a Novel Henipavirus, Angavokely Virus, from Fruit Bats in Madagascar.

The genus Henipavirus (family Paramyxoviridae) currently comprises seven viruses, four of which have demonstrated prior evidence of zoonotic capacity. These include the biosafety level 4 agents Hendra (HeV) and Nipah (NiV) viruses, which circulate naturally in pteropodid fruit bats. Here, we describe and characterize Angavokely virus (AngV), a divergent henipavirus identified in urine samples from wild, Madagascar fruit bats. We report the nearly complete 16,740-nucleotide genome of AngV, which encodes the six major henipavirus structural proteins (nucleocapsid, phosphoprotein, matrix, fusion, glycoprotein, and L polymerase). Within the phosphoprotein (P) gene, we identify an alternative start codon encoding the AngV C protein and a putative mRNA editing site where the insertion of one or two guanine residues encodes, respectively, additional V and W proteins. In other paramyxovirus systems, C, V, and W are accessory proteins involved in antagonism of host immune responses during infection. Phylogenetic analysis suggests that AngV is ancestral to all four previously described bat henipaviruses-HeV, NiV, Cedar virus (CedV), and Ghanaian bat virus (GhV)-but evolved more recently than rodent- and shrew-derived henipaviruses, Mojiang (MojV), Gamak (GAKV), and Daeryong (DARV) viruses. Predictive structure-based alignments suggest that AngV is unlikely to bind ephrin receptors, which mediate cell entry for all other known bat henipaviruses. Identification of the AngV receptor is needed to clarify the virus's potential host range. The presence of V and W proteins in the AngV genome suggest that the virus could be pathogenic following zoonotic spillover. IMPORTANCE Henipaviruses include highly pathogenic emerging zoonotic viruses, derived from bat, rodent, and shrew reservoirs. Bat-borne Hendra (HeV) and Nipah (NiV) are the most well-known henipaviruses, for which no effective antivirals or vaccines for humans have been described. Here, we report the discovery and characterization of a novel henipavirus, Angavokely virus (AngV), isolated from wild fruit bats in Madagascar. Genomic characterization of AngV reveals all major features associated with pathogenicity in other henipaviruses, suggesting that AngV could be pathogenic following spillover to human hosts. Our work suggests that AngV is an ancestral bat henipavirus that likely uses viral entry pathways distinct from those previously described for HeV and NiV. In Madagascar, bats are consumed as a source of human food, presenting opportunities for cross-species transmission. Characterization of novel henipaviruses and documentation of their pathogenic and zoonotic potential are essential to predicting and preventing the emergence of future zoonoses that cause pandemics.

ZSCAN1 Autoantibodies Are Associated with Pediatric Paraneoplastic ROHHAD.

OBJECTIVE: Rapid-onset Obesity with Hypothalamic Dysfunction, Hypoventilation and Autonomic Dysregulation (ROHHAD), is a severe pediatric disorder of uncertain etiology resulting in hypothalamic dysfunction and frequent sudden death. Frequent co-occurrence of neuroblastic tumors have fueled suspicion of an autoimmune paraneoplastic neurological syndrome (PNS); however, specific anti-neural autoantibodies, a hallmark of PNS, have not been identified. Our objective is to determine if an autoimmune paraneoplastic etiology underlies ROHHAD. METHODS: Immunoglobulin G (IgG) from pediatric ROHHAD patients (n = 9), non-inflammatory individuals (n = 100) and relevant pediatric controls (n = 25) was screened using a programmable phage display of the human peptidome (PhIP-Seq). Putative ROHHAD-specific autoantibodies were orthogonally validated using radioactive ligand binding and cell-based assays. Expression of autoantibody targets in ROHHAD tumor and healthy brain tissue was assessed with immunohistochemistry and mass spectrometry, respectively. RESULTS: Autoantibodies to ZSCAN1 were detected in ROHHAD patients by PhIP-Seq and orthogonally validated in 7/9 ROHHAD patients and 0/125 controls using radioactive ligand binding and cell-based assays. Expression of ZSCAN1 in ROHHAD tumor and healthy human brain tissue was confirmed. INTERPRETATION: Our results support the notion that tumor-associated ROHHAD syndrome is a pediatric PNS, potentially initiated by an immune response to peripheral neuroblastic tumor. ZSCAN1 autoantibodies may aid in earlier, accurate diagnosis of ROHHAD syndrome, thus providing a means toward early detection and treatment. This work warrants follow-up studies to test sensitivity and specificity of a novel diagnostic test. Last, given the absence of the ZSCAN1 gene in rodents, our study highlights the value of human-based approaches for detecting novel PNS subtypes. ANN NEUROL 2022;92:279-291.

The pulmonary metatranscriptome prior to pediatric HCT identifies post-HCT lung injury.

Lung injury after pediatric allogeneic hematopoietic cell transplantation (HCT) is a common and disastrous complication that threatens long-term survival. To develop strategies to prevent lung injury, novel tools are needed to comprehensively assess lung health in HCT candidates. Therefore, this study analyzed biospecimens from 181 pediatric HCT candidates who underwent routine pre-HCT bronchoalveolar lavage (BAL) at the University Medical Center Utrecht between 2005 and 2016. BAL fluid underwent metatranscriptomic sequencing of microbial and human RNA, and unsupervised clustering and generalized linear models were used to associate microbiome gene expression data with the development of post-HCT lung injury. Microbe-gene correlations were validated using a geographically distinct cohort of 18 pediatric HCT candidates. The cumulative incidence of post-HCT lung injury varied significantly according to 4 pre-HCT pulmonary metatranscriptome clusters, with the highest incidence observed in children with pre-HCT viral enrichment and innate immune activation, as well as in children with profound microbial depletion and concomitant natural killer/T-cell activation (P < .001). In contrast, children with pre-HCT pulmonary metatranscriptomes containing diverse oropharyngeal taxa and lacking inflammation rarely developed post-HCT lung injury. In addition, activation of epithelial-epidermal differentiation, mucus production, and cellular adhesion were associated with fatal post-HCT lung injury. In a separate validation cohort, associations among pulmonary respiratory viral load, oropharyngeal taxa, and pulmonary gene expression were recapitulated; the association with post-HCT lung injury needs to be validated in an independent cohort. This analysis suggests that assessment of the pre-HCT BAL fluid may identify high-risk pediatric HCT candidates who may benefit from pathobiology-targeted interventions.

Elevated N-Linked Glycosylation of IgG V Regions in Myasthenia Gravis Disease Subtypes.

Elevated N-linked glycosylation of IgG V regions (IgG-VN-Glyc) is an emerging molecular phenotype associated with autoimmune disorders. To test the broader specificity of elevated IgG-VN-Glyc, we studied patients with distinct subtypes of myasthenia gravis (MG), a B cell-mediated autoimmune disease. Our experimental design focused on examining the B cell repertoire and total IgG. It specifically included adaptive immune receptor repertoire sequencing to quantify and characterize N-linked glycosylation sites in the circulating BCR repertoire, proteomics to examine glycosylation patterns of the total circulating IgG, and an exploration of human-derived recombinant autoantibodies, which were studied with mass spectrometry and Ag binding assays to respectively confirm occupation of glycosylation sites and determine whether they alter binding. We found that the frequency of IgG-VN-Glyc motifs was increased in the total BCR repertoire of patients with MG when compared with healthy donors. The elevated frequency was attributed to both biased V gene segment usage and somatic hypermutation. IgG-VN-Glyc could be observed in the total circulating IgG in a subset of patients with MG. Autoantigen binding, by four patient-derived MG autoantigen-specific mAbs with experimentally confirmed presence of IgG-VN-Glyc, was not altered by the glycosylation. Our findings extend prior work on patterns of Ig V region N-linked glycosylation in autoimmunity to MG subtypes.

Deep profiling of protease substrate specificity enabled by dual random and scanned human proteome substrate phage libraries.

Proteolysis is a major posttranslational regulator of biology inside and outside of cells. Broad identification of optimal cleavage sites and natural substrates of proteases is critical for drug discovery and to understand protease biology. Here, we present a method that employs two genetically encoded substrate phage display libraries coupled with next generation sequencing (SPD-NGS) that allows up to 10,000-fold deeper sequence coverage of the typical six- to eight-residue protease cleavage sites compared to state-of-the-art synthetic peptide libraries or proteomics. We applied SPD-NGS to two classes of proteases, the intracellular caspases, and the ectodomains of the sheddases, ADAMs 10 and 17. The first library (Lib 10AA) allowed us to identify 104 to 105 unique cleavage sites over a 1,000-fold dynamic range of NGS counts and produced consensus and optimal cleavage motifs based position-specific scoring matrices. A second SPD-NGS library (Lib hP), which displayed virtually the entire human proteome tiled in contiguous 49 amino acid sequences with 25 amino acid overlaps, enabled us to identify candidate human proteome sequences. We identified up to 104 natural linear cut sites, depending on the protease, and captured most of the examples previously identified by proteomics and predicted 10- to 100-fold more. Structural bioinformatics was used to facilitate the identification of candidate natural protein substrates. SPD-NGS is rapid, reproducible, simple to perform and analyze, inexpensive, and renewable, with unprecedented depth of coverage for substrate sequences, and is an important tool for protease biologists interested in protease specificity for specific assays and inhibitors and to facilitate identification of natural protein substrates.

A pathogenic and clonally expanded B cell transcriptome in active multiple sclerosis.

Central nervous system B cells have several potential roles in multiple sclerosis (MS): secretors of proinflammatory cytokines and chemokines, presenters of autoantigens to T cells, producers of pathogenic antibodies, and reservoirs for viruses that trigger demyelination. To interrogate these roles, single-cell RNA sequencing (scRNA-Seq) was performed on paired cerebrospinal fluid (CSF) and blood from subjects with relapsing-remitting MS (RRMS; n = 12), other neurologic diseases (ONDs; n = 1), and healthy controls (HCs; n = 3). Single-cell immunoglobulin sequencing (scIg-Seq) was performed on a subset of these subjects and additional RRMS (n = 4), clinically isolated syndrome (n = 2), and OND (n = 2) subjects. Further, paired CSF and blood B cell subsets (RRMS; n = 7) were isolated using fluorescence activated cell sorting for bulk RNA sequencing (RNA-Seq). Independent analyses across technologies demonstrated that nuclear factor kappa B (NF-κB) and cholesterol biosynthesis pathways were activated, and specific cytokine and chemokine receptors were up-regulated in CSF memory B cells. Further, SMAD/TGF-ß1 signaling was down-regulated in CSF plasmablasts/plasma cells. Clonally expanded, somatically hypermutated IgM+ and IgG1+ CSF B cells were associated with inflammation, blood-brain barrier breakdown, and intrathecal Ig synthesis. While we identified memory B cells and plasmablast/plasma cells with highly similar Ig heavy-chain sequences across MS subjects, similarities were also identified with ONDs and HCs. No viral transcripts, including from Epstein-Barr virus, were detected. Our findings support the hypothesis that in MS, CSF B cells are driven to an inflammatory and clonally expanded memory and plasmablast/plasma cell phenotype.

SARS-CoV-2 Variant Exposures Elicit Antibody Responses With Differential Cross-Neutralization of Established and Emerging Strains Including Delta and Omicron.

The wide spectrum of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with phenotypes impacting transmission and antibody sensitivity necessitates investigation of immune responses to different spike protein versions. Here, we compare neutralization of variants of concern, including B.1.617.2 (delta) and B.1.1.529 (omicron), in sera from individuals exposed to variant infection, vaccination, or both. We demonstrate that neutralizing antibody responses are strongest against variants sharing certain spike mutations with the immunizing exposure, and exposure to multiple spike variants increases breadth of variant cross-neutralization. These findings contribute to understanding relationships between exposures and antibody responses and may inform booster vaccination strategies.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Transmission Dynamics and Immune Responses in a Household of Vaccinated Persons.

While SARS-CoV-2 vaccines prevent severe disease effectively, postvaccination "breakthrough" COVID-19 infections and transmission among vaccinated individuals remain ongoing concerns. We present an in-depth characterization of transmission and immunity among vaccinated individuals in a household, revealing complex dynamics and unappreciated comorbidities, including autoimmunity to type 1 interferon in the presumptive index case.

Estimation of Secondary Household Attack Rates for Emergent Spike L452R Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Variants Detected by Genomic Surveillance at a Community-Based Testing Site in San Francisco.

BACKGROUND: Sequencing of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral genome from patient samples is an important epidemiological tool for monitoring and responding to the pandemic, including the emergence of new mutations in specific communities. METHODS: SARS-CoV-2 genomic sequences were generated from positive samples collected, along with epidemiological metadata, at a walk-up, rapid testing site in the Mission District of San Francisco, California during 22 November to 1 December, 2020, and 10-29 January 2021. Secondary household attack rates and mean sample viral load were estimated and compared across observed variants. RESULTS: A total of 12 124 tests were performed yielding 1099 positives. From these, 928 high-quality genomes were generated. Certain viral lineages bearing spike mutations, defined in part by L452R, S13I, and W152C, comprised 54.4% of the total sequences from January, compared to 15.7% in November. Household contacts exposed to the "California" or "West Coast" variants (B.1.427 and B.1.429) were at higher risk of infection compared to household contacts exposed to lineages lacking these variants (0.36 vs 0.29, risk ratio [RR] = 1.28; 95% confidence interval [CI]: 1.00-1.64). The reproductive number was estimated to be modestly higher than other lineages spreading in California during the second half of 2020. Viral loads were similar among persons infected with West Coast versus non-West Coast strains, as was the proportion of individuals with symptoms (60.9% vs 64.3%). CONCLUSIONS: The increase in prevalence, relative household attack rates, and reproductive number are consistent with a modest transmissibility increase of the West Coast variants. Summary: We observed a growing prevalence and modestly elevated attack rate for "West Coast" severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants in a community testing setting in San Francisco during January 2021, suggesting its modestly higher transmissibility.