Induction of HIV Neutralizing Antibody Lineages in Mice with Diverse Precursor Repertoires.

The design of immunogens that elicit broadly reactive neutralizing antibodies (bnAbs) has been a major obstacle to HIV-1 vaccine development. One approach to assess potential immunogens is to use mice expressing precursors of human bnAbs as vaccination models. The bnAbs of the VRC01-class derive from the IGHV1-2 immunoglobulin heavy chain and neutralize a wide spectrum of HIV-1 strains via targeting the CD4 binding site of the envelope glycoprotein gp120. We now describe a mouse vaccination model that allows a germline human IGHV1-2(∗)02 segment to undergo normal V(D)J recombination and, thereby, leads to the generation of peripheral B cells that express a highly diverse repertoire of VRC01-related receptors. When sequentially immunized with modified gp120 glycoproteins designed to engage VRC01 germline and intermediate antibodies, IGHV1-2(∗)02-rearranging mice, which also express a VRC01-antibody precursor light chain, can support the affinity maturation of VRC01 precursor antibodies into HIV-neutralizing antibody lineages.

Glycan Masking Focuses Immune Responses to the HIV-1 CD4-Binding Site and Enhances Elicitation of VRC01-Class Precursor Antibodies.

An important class of HIV-1 broadly neutralizing antibodies, termed the VRC01 class, targets the conserved CD4-binding site (CD4bs) of the envelope glycoprotein (Env). An engineered Env outer domain (OD) eOD-GT8 60-mer nanoparticle has been developed as a priming immunogen for eliciting VRC01-class precursors and is planned for clinical trials. However, a substantial portion of eOD-GT8-elicited antibodies target non-CD4bs epitopes, potentially limiting its efficacy. We introduced N-linked glycans into non-CD4bs surfaces of eOD-GT8 to mask irrelevant epitopes and evaluated these mutants in a mouse model that expressed diverse immunoglobulin heavy chains containing human IGHV1-2∗02, the germline VRC01 VH segment. Compared to the parental eOD-GT8, a mutant with five added glycans stimulated significantly higher proportions of CD4bs-specific serum responses and CD4bs-specific immunoglobulin G+ B cells including VRC01-class precursors. These results demonstrate that glycan masking can limit elicitation of off-target antibodies and focus immune responses to the CD4bs, a major target of HIV-1 vaccine design.

COVID-19 tissue atlases reveal SARS-CoV-2 pathology and cellular targets.

COVID-19, which is caused by SARS-CoV-2, can result in acute respiratory distress syndrome and multiple organ failure1-4, but little is known about its pathophysiology. Here we generated single-cell atlases of 24 lung, 16 kidney, 16 liver and 19 heart autopsy tissue samples and spatial atlases of 14 lung samples from donors who died of COVID-19. Integrated computational analysis uncovered substantial remodelling in the lung epithelial, immune and stromal compartments, with evidence of multiple paths of failed tissue regeneration, including defective alveolar type 2 differentiation and expansion of fibroblasts and putative TP63+ intrapulmonary basal-like progenitor cells. Viral RNAs were enriched in mononuclear phagocytic and endothelial lung cells, which induced specific host programs. Spatial analysis in lung distinguished inflammatory host responses in lung regions with and without viral RNA. Analysis of the other tissue atlases showed transcriptional alterations in multiple cell types in heart tissue from donors with COVID-19, and mapped cell types and genes implicated with disease severity based on COVID-19 genome-wide association studies. Our foundational dataset elucidates the biological effect of severe SARS-CoV-2 infection across the body, a key step towards new treatments.

Neuropathological features of SARS-CoV-2 delta and omicron variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is continually evolving resulting in variants with increased transmissibility, more severe disease, reduced effectiveness of treatments or vaccines, or diagnostic detection failure. The SARS-CoV-2 Delta variant (B.1.617.2 and AY lineages) was the dominant circulating strain in the United States from July to mid-December 2021, followed by the Omicron variant (B.1.1.529 and BA lineages). Coronavirus disease 2019 (COVID-19) has been associated with neurological sequelae including loss of taste/smell, headache, encephalopathy, and stroke, yet little is known about the impact of viral strain on neuropathogenesis. Detailed postmortem brain evaluations were performed for 22 patients from Massachusetts, including 12 who died following infection with Delta variant and 5 with Omicron variant, compared to 5 patients who died earlier in the pandemic. Diffuse hypoxic injury, occasional microinfarcts and hemorrhage, perivascular fibrinogen, and rare lymphocytes were observed across the 3 groups. SARS-CoV-2 protein and RNA were not detected in any brain samples by immunohistochemistry, in situ hybridization, or real-time quantitative PCR. These results, although preliminary, demonstrate that, among a subset of severely ill patients, similar neuropathological features are present in Delta, Omicron, and non-Delta/non-Omicron variant patients, suggesting that SARS-CoV-2 variants are likely to affect the brain by common neuropathogenic mechanisms.

High-depth sequencing characterization of viral dynamics across tissues in fatal COVID-19 reveals compartmentalized infection.

SARS-CoV-2 distribution and circulation dynamics are not well understood due to challenges in assessing genomic data from tissue samples. We develop experimental and computational workflows for high-depth viral sequencing and high-resolution genomic analyses from formalin-fixed, paraffin-embedded tissues and apply them to 120 specimens from six subjects with fatal COVID-19. To varying degrees, viral RNA is present in extrapulmonary tissues from all subjects. The majority of the 180 viral variants identified within subjects are unique to individual tissue samples. We find more high-frequency (>10%) minor variants in subjects with a longer disease course, with one subject harboring ten such variants, exclusively in extrapulmonary tissues. One tissue-specific high-frequency variant was a nonsynonymous mutation in the furin-cleavage site of the spike protein. Our findings suggest adaptation and/or compartmentalized infection, illuminating the basis of extrapulmonary COVID-19 symptoms and potential for viral reservoirs, and have broad utility for investigating human pathogens.

Phylodynamics of deer tick virus in North America.

The burden of ticks and the pathogens they carry is increasing worldwide. Powassan virus (POWV; Flaviviridae: Flavivirus), the only known North American tick-borne flavivirus, is of particular concern due to rising cases and the severe morbidity of POWV encephalitis. Here, we use a multifaceted approach to evaluate the emergence of the II POWV lineage, known as deer tick virus (DTV), in parts of North America where human cases occur. We detected DTV-positive ticks from eight of twenty locations in the Northeast USA with an average infection rate of 1.4 per cent. High-depth, whole-genome sequencing of eighty-four POWV and DTV samples allowed us to assess geographic and temporal phylodynamics. We observed both stable infection in the Northeast USA and patterns of geographic dispersal within and between regions. A Bayesian skyline analysis demonstrated DTV population expansion over the last 50 years. This is concordant with the documented expansion of Ixodes scapularis tick populations and suggests an increasing risk of human exposure as the vector spreads. Finally, we isolated sixteen novel viruses in cell culture and demonstrated limited genetic change after passage, a valuable resource for future studies investigating this emerging virus.

Natural history of Ebola virus disease in rhesus monkeys shows viral variant emergence dynamics and tissue-specific host responses.

Ebola virus (EBOV) causes Ebola virus disease (EVD), marked by severe hemorrhagic fever; however, the mechanisms underlying the disease remain unclear. To assess the molecular basis of EVD across time, we performed RNA sequencing on 17 tissues from a natural history study of 21 rhesus monkeys, developing new methods to characterize host-pathogen dynamics. We identified alterations in host gene expression with previously unknown tissue-specific changes, including downregulation of genes related to tissue connectivity. EBOV was widely disseminated throughout the body; using a new, broadly applicable deconvolution method, we found that viral load correlated with increased monocyte presence. Patterns of viral variation between tissues differentiated primary infections from compartmentalized infections, and several variants impacted viral fitness in a EBOV/Kikwit minigenome system, suggesting that functionally significant variants can emerge during early infection. This comprehensive portrait of host-pathogen dynamics in EVD illuminates new features of pathogenesis and establishes resources to study other emerging pathogens.

Synthetic DNA spike-ins (SDSIs) enable sample tracking and detection of inter-sample contamination in SARS-CoV-2 sequencing workflows.

The global spread and continued evolution of SARS-CoV-2 has driven an unprecedented surge in viral genomic surveillance. Amplicon-based sequencing methods provide a sensitive, low-cost and rapid approach but suffer a high potential for contamination, which can undermine laboratory processes and results. This challenge will increase with the expanding global production of sequences across a variety of laboratories for epidemiological and clinical interpretation, as well as for genomic surveillance of emerging diseases in future outbreaks. We present SDSI + AmpSeq, an approach that uses 96 synthetic DNA spike-ins (SDSIs) to track samples and detect inter-sample contamination throughout the sequencing workflow. We apply SDSIs to the ARTIC Consortium's amplicon design, demonstrate their utility and efficiency in a real-time investigation of a suspected hospital cluster of SARS-CoV-2 cases and validate them across 6,676 diagnostic samples at multiple laboratories. We establish that SDSI + AmpSeq provides increased confidence in genomic data by detecting and correcting for relatively common, yet previously unobserved modes of error, including spillover and sample swaps, without impacting genome recovery.

Susceptibility-weighted imaging reveals cerebral microvascular injury in severe COVID-19.

We evaluated the incidence, distribution, and histopathologic correlates of microvascular brain lesions in patients with severe COVID-19. Sixteen consecutive patients admitted to the intensive care unit with severe COVID-19 undergoing brain MRI for evaluation of coma or neurologic deficits were retrospectively identified. Eleven patients had punctate susceptibility-weighted imaging (SWI) lesions in the subcortical and deep white matter, eight patients had >10 SWI lesions, and four patients had lesions involving the corpus callosum. The distribution of SWI lesions was similar to that seen in patients with hypoxic respiratory failure, sepsis, and disseminated intravascular coagulation. Brain autopsy in one patient revealed that SWI lesions corresponded to widespread microvascular injury, characterized by perivascular and parenchymal petechial hemorrhages and microscopic ischemic lesions. Collectively, these radiologic and histopathologic findings add to growing evidence that patients with severe COVID-19 are at risk for multifocal microvascular hemorrhagic and ischemic lesions in the subcortical and deep white matter.

Intrathecal inflammatory responses in the absence of SARS-CoV-2 nucleic acid in the CSF of COVID-19 hospitalized patients.

OBJECTIVE: Little is known about CSF profiles in patients with acute COVID-19 infection and neurological symptoms. Here, CSF was tested for SARS-CoV-2 RNA and inflammatory cytokines and chemokines and compared to controls and patients with known neurotropic pathogens. METHODS: CSF from twenty-seven consecutive patients with COVID-19 and neurological symptoms was assayed for SARS-CoV-2 RNA using quantitative reverse transcription PCR (RT-qPCR) and unbiased metagenomic sequencing. Assays for blood brain barrier (BBB) breakdown (CSF:serum albumin ratio (Q-Alb)), and proinflammatory cytokines and chemokines (IL-6, IL-8, IL-15, IL-16, monocyte chemoattractant protein -1 (MCP-1) and monocyte inhibitory protein - 1ß (MIP-1ß)) were performed in 23 patients and compared to CSF from patients with HIV-1 (16 virally suppressed, 5 unsuppressed), West Nile virus (WNV) (n = 4) and 16 healthy controls (HC). RESULTS: Median CSF cell count for COVID-19 patients was 1 white blood cell/µL; two patients were infected with a second pathogen (Neisseria, Cryptococcus neoformans). No CSF samples had detectable SARS-CoV-2 RNA by either detection method. In patients with COVID-19 only, CSF IL-6, IL-8, IL-15, and MIP-1ß levels were higher than HC and suppressed HIV (corrected-p < 0.05). MCP-1 and MIP-1ß levels were higher, while IL-6, IL-8, IL-15 were similar in COVID-19 compared to WNV patients. Q-Alb correlated with all proinflammatory markers, with IL-6, IL-8, and MIP-1ß (r ≥ 0.6, p < 0.01) demonstrating the strongest associations. CONCLUSIONS: Lack of SARS-CoV-2 RNA in CSF is consistent with pre-existing literature. Evidence of intrathecal proinflammatory markers in a subset of COVID-19 patients with BBB breakdown despite minimal CSF pleocytosis is atypical for neurotropic pathogens.

Functional Profiling of Antibody Immune Repertoires in Convalescent Zika Virus Disease Patients.

The re-emergence of Zika virus (ZIKV) caused widespread infections that were linked to Guillain-Barré syndrome in adults and congenital malformation in fetuses, and epidemiological data suggest that ZIKV infection can induce protective antibody responses. A more detailed understanding of anti-ZIKV antibody responses may lead to enhanced antibody discovery and improved vaccine designs against ZIKV and related flaviviruses. Here, we applied recently-invented library-scale antibody screening technologies to determine comprehensive functional molecular and genetic profiles of naturally elicited human anti-ZIKV antibodies in three convalescent individuals. We leveraged natively paired antibody yeast display and NGS to predict antibody cross-reactivities and coarse-grain antibody affinities, to perform in-depth immune profiling of IgM, IgG, and IgA antibody repertoires in peripheral blood, and to reveal virus maturation state-dependent antibody interactions. Repertoire-scale comparison of ZIKV VLP-specific and non-specific antibodies in the same individuals also showed that mean antibody somatic hypermutation levels were substantially influenced by donor-intrinsic characteristics. These data provide insights into antiviral antibody responses to ZIKV disease and outline systems-level strategies to track human antibody immune responses to emergent viral infections.

DNA spike-ins enable confident interpretation of SARS-CoV-2 genomic data from amplicon-based sequencing.

The rapid global spread and continued evolution of SARS-CoV-2 has highlighted an unprecedented need for viral genomic surveillance and clinical viral sequencing. Amplicon-based sequencing methods provide a sensitive, low-cost and rapid approach but suffer a high potential for contamination, which can undermine lab processes and results. This challenge will only increase with expanding global production of sequences by diverse research groups for epidemiological and clinical interpretation. We present an approach which uses synthetic DNA spike-ins (SDSIs) to track samples and detect inter-sample contamination through a sequencing workflow. Applying this approach to the ARTIC Consortium's amplicon design, we define a series of best practices for Illumina-based sequencing and provide a detailed characterization of approaches to increase sensitivity for low-viral load samples incorporating the SDSIs. We demonstrate the utility and efficiency of the SDSI method amidst a real-time investigation of a suspected hospital cluster of SARS-CoV-2 cases.

A single-cell and spatial atlas of autopsy tissues reveals pathology and cellular targets of SARS-CoV-2.

The SARS-CoV-2 pandemic has caused over 1 million deaths globally, mostly due to acute lung injury and acute respiratory distress syndrome, or direct complications resulting in multiple-organ failures. Little is known about the host tissue immune and cellular responses associated with COVID-19 infection, symptoms, and lethality. To address this, we collected tissues from 11 organs during the clinical autopsy of 17 individuals who succumbed to COVID-19, resulting in a tissue bank of approximately 420 specimens. We generated comprehensive cellular maps capturing COVID-19 biology related to patients' demise through single-cell and single-nucleus RNA-Seq of lung, kidney, liver and heart tissues, and further contextualized our findings through spatial RNA profiling of distinct lung regions. We developed a computational framework that incorporates removal of ambient RNA and automated cell type annotation to facilitate comparison with other healthy and diseased tissue atlases. In the lung, we uncovered significantly altered transcriptional programs within the epithelial, immune, and stromal compartments and cell intrinsic changes in multiple cell types relative to lung tissue from healthy controls. We observed evidence of: alveolar type 2 (AT2) differentiation replacing depleted alveolar type 1 (AT1) lung epithelial cells, as previously seen in fibrosis; a concomitant increase in myofibroblasts reflective of defective tissue repair; and, putative TP63+ intrapulmonary basal-like progenitor (IPBLP) cells, similar to cells identified in H1N1 influenza, that may serve as an emergency cellular reserve for severely damaged alveoli. Together, these findings suggest the activation and failure of multiple avenues for regeneration of the epithelium in these terminal lungs. SARS-CoV-2 RNA reads were enriched in lung mononuclear phagocytic cells and endothelial cells, and these cells expressed distinct host response transcriptional programs. We corroborated the compositional and transcriptional changes in lung tissue through spatial analysis of RNA profiles in situ and distinguished unique tissue host responses between regions with and without viral RNA, and in COVID-19 donor tissues relative to healthy lung. Finally, we analyzed genetic regions implicated in COVID-19 GWAS with transcriptomic data to implicate specific cell types and genes associated with disease severity. Overall, our COVID-19 cell atlas is a foundational dataset to better understand the biological impact of SARS-CoV-2 infection across the human body and empowers the identification of new therapeutic interventions and prevention strategies.

Phylogenetic analysis of SARS-CoV-2 in Boston highlights the impact of superspreading events.

Analysis of 772 complete severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes from early in the Boston-area epidemic revealed numerous introductions of the virus, a small number of which led to most cases. The data revealed two superspreading events. One, in a skilled nursing facility, led to rapid transmission and significant mortality in this vulnerable population but little broader spread, whereas other introductions into the facility had little effect. The second, at an international business conference, produced sustained community transmission and was exported, resulting in extensive regional, national, and international spread. The two events also differed substantially in the genetic variation they generated, suggesting varying transmission dynamics in superspreading events. Our results show how genomic epidemiology can help to understand the link between individual clusters and wider community spread.

Powassan Virus Neuropathology and Genomic Diversity in Patients With Fatal Encephalitis.

BACKGROUND: Powassan virus (POWV) is an emerging cause of severe encephalitis; very little is known about human pathogenicity due to challenges in diagnosis and viral RNA recovery. We present 3 patients with fatal encephalitis due to POWV lineage II (deer tick virus). METHODS: We obtained 27 unique samples, including from brain biopsy and autopsy, and used metagenomic sequencing, quantitative reverse transcriptase polymerase chain reaction, and a newly developed CRISPR-based diagnostic assay to perform the first detailed characterization of POWV compartmentalization and genomics between and within human subjects. RESULTS: In all 3 patients, imaging and histopathology findings were notable for profound cerebellar involvement. All patients were initially diagnosed with POWV by metagenomic sequencing, and 2 of the 3 had negative clinical testing by serology. We detected POWV RNA in 13 clinical samples; levels were highest in the cerebellum, and there was very little involvement of peripheral tissue. We assembled complete POWV genomes from 8 samples, providing unique information about the strains of POWV lineage II (deer tick virus) that infect humans. CONCLUSIONS: We demonstrate the utility of molecular assays for detecting POWV infection, including in seronegative patients, and nominate viral genomic features that may relate to human infection and neuropathogenicity. The cerebellum was identified as a key target POWV in fatal infection, by radiological and histopathological findings as well as molecular testing.

Cerebral Microvascular Injury in Severe COVID-19.

IMPORTANCE: Microvascular lesions are common in patients with severe COVID-19. Radiologic-pathologic correlation in one case suggests a combination of microvascular hemorrhagic and ischemic lesions that may reflect an underlying hypoxic mechanism of injury, which requires validation in larger studies. OBJECTIVE: To determine the incidence, distribution, and clinical and histopathologic correlates of microvascular lesions in patients with severe COVID-19. DESIGN: Observational, retrospective cohort study: March to May 2020. SETTING: Single academic medical center. PARTICIPANTS: Consecutive patients (16) admitted to the intensive care unit with severe COVID-19, undergoing brain MRI for evaluation of coma or focal neurologic deficits. EXPOSURES: Not applicable. MAIN OUTCOME AND MEASURES: Hypointense microvascular lesions identified by a prototype ultrafast high-resolution susceptibility-weighted imaging (SWI) MRI sequence, counted by two neuroradiologists and categorized by neuroanatomic location. Clinical and laboratory data (most recent measurements before brain MRI). Brain autopsy and cerebrospinal fluid PCR for SARS-CoV 2 in one patient who died from severe COVID-19. RESULTS: Eleven of 16 patients (69%) had punctate and linear SWI lesions in the subcortical and deep white matter, and eight patients (50%) had >10 SWI lesions. In 4/16 patients (25%), lesions involved the corpus callosum. Brain autopsy in one patient revealed that SWI lesions corresponded to widespread microvascular injury, characterized by perivascular and parenchymal petechial hemorrhages and microscopic ischemic lesions. CONCLUSIONS AND RELEVANCE: SWI lesions are common in patients with neurological manifestations of severe COVID-19 (coma and focal neurologic deficits). The distribution of lesions is similar to that seen in patients with hypoxic respiratory failure, sepsis, and disseminated intravascular coagulation. Collectively, these radiologic and histopathologic findings suggest that patients with severe COVID-19 are at risk for multifocal microvascular hemorrhagic and ischemic lesions in the subcortical and deep white matter.

Phylogenetic analysis of SARS-CoV-2 in the Boston area highlights the role of recurrent importation and superspreading events.

SARS-CoV-2 has caused a severe, ongoing outbreak of COVID-19 in Massachusetts with 111,070 confirmed cases and 8,433 deaths as of August 1, 2020. To investigate the introduction, spread, and epidemiology of COVID-19 in the Boston area, we sequenced and analyzed 772 complete SARS-CoV-2 genomes from the region, including nearly all confirmed cases within the first week of the epidemic and hundreds of cases from major outbreaks at a conference, a nursing facility, and among homeless shelter guests and staff. The data reveal over 80 introductions into the Boston area, predominantly from elsewhere in the United States and Europe. We studied two superspreading events covered by the data, events that led to very different outcomes because of the timing and populations involved. One produced rapid spread in a vulnerable population but little onward transmission, while the other was a major contributor to sustained community transmission, including outbreaks in homeless populations, and was exported to several other domestic and international sites. The same two events differed significantly in the number of new mutations seen, raising the possibility that SARS-CoV-2 superspreading might encompass disparate transmission dynamics. Our results highlight the failure of measures to prevent importation into MA early in the outbreak, underscore the role of superspreading in amplifying an outbreak in a major urban area, and lay a foundation for contact tracing informed by genetic data.

VRC34-Antibody Lineage Development Reveals How a Required Rare Mutation Shapes the Maturation of a Broad HIV-Neutralizing Lineage.

Rare mutations have been proposed to restrict the development of broadly neutralizing antibodies against HIV-1, but this has not been explicitly demonstrated. We hypothesized that such rare mutations might be identified by comparing broadly neutralizing and non-broadly neutralizing branches of an antibody-developmental tree. Because sequences of antibodies isolated from the fusion peptide (FP)-targeting VRC34-antibody lineage suggested it might be suitable for such rare mutation analysis, we carried out next-generation sequencing (NGS) on B cell transcripts from donor N123, the source of the VRC34 lineage, and functionally and structurally characterized inferred intermediates along broadly neutralizing and poorly neutralizing developmental branches. The broadly neutralizing VRC34.01 branch required the rare heavy-chain mutation Y33P to bind FP, whereas the early bifurcated VRC34.05 branch did not require this rare mutation and evolved less breadth. Our results demonstrate how a required rare mutation can restrict development and shape the maturation of a broad HIV-1-neutralizing antibody lineage.

Computational Optics Enables Breast Cancer Profiling in Point-of-Care Settings.

The global burden of cancer, severe diagnostic bottlenecks in underserved regions, and underfunded health care systems are fueling the need for inexpensive, rapid, and treatment-informative diagnostics. On the basis of advances in computational optics and deep learning, we have developed a low-cost digital system, termed AIDA (artificial intelligence diffraction analysis), for breast cancer diagnosis of fine needle aspirates. Here, we show high accuracy (>90%) in (i) recognizing cells directly from diffraction patterns and (ii) classifying breast cancer types using deep-learning-based analysis of sample aspirates. The image algorithm is fast, enabling cellular analyses at high throughput (∼3 s per 1000 cells), and the unsupervised processing allows use by lower skill health care workers. AIDA can perform quantitative molecular profiling on individual cells, revealing intratumor molecular heterogeneity, and has the potential to improve cancer diagnosis and treatment. The system could be further developed for other cancers and thus find widespread use in global health.