Longitudinal analyses reveal immunological misfiring in severe COVID-19.

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

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

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

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

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

Claspin haploinsufficiency leads to defects in fertility, hyperplasia and an increased oncogenic potential.

Claspin is an adaptor protein required for ATR-dependent phosphorylation of CHK1 during S-phase following DNA replication stress. Claspin expression is highly variable in cancer, with low levels frequently correlating with poor patient survival. To learn more about the biological consequences of reduced Claspin expression and its effects on tumorigenesis, we investigated mice with a heterozygous knockout of the Clspn gene. Claspin haploinsufficiency resulted in reduced female fertility and a maternally inherited defect in oocyte meiosis I cell cycle progression. Furthermore, aged Clspn+/- mice developed spontaneous lymphoid hyperplasia and increased susceptibility to non-alcoholic fatty liver disease. Importantly, we demonstrate a tumour suppressor role for Claspin. Reduced Claspin levels result in increased liver damage and tumourigenesis in the DEN model of hepatocellular carcinoma. These data reveal that Clspn haploinsufficiency has widespread unanticipated biological effects and establishes the importance of Claspin as a regulatory node controlling tumorigenesis and multiple disease aetiologies.

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

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

Stability of SARS-CoV-2 RNA in Nonsupplemented Saliva.

The expense of saliva collection devices designed to stabilize severe acute respiratory syndrome coronavirus 2 RNA is prohibitive to mass testing. However, virus RNA in nonsupplemented saliva is stable for extended periods and at elevated temperatures. Simple plastic tubes for saliva collection will make large-scale testing and continued surveillance easier.

Herd Immunity Likely Protected the Men Who Have Sex With Men in the Recent Hepatitis A Outbreak in San Diego, California.

A high seroprevalence of hepatitis A virus (81%) among human immunodeficiency virus-negative high-risk men who have sex with men is likely why this community was largely spared from a recent hepatitis A virus outbreak in San Diego, California.

Structure-guided insights into potential function of novel genetic variants in the malaria vaccine candidate PfRh5.

The recent stall in the global reduction of malaria deaths has made the development of a highly effective vaccine essential. A major challenge to developing an efficacious vaccine is the extensive diversity of Plasmodium falciparum antigens. While genetic diversity plays a major role in immune evasion and is a barrier to the development of both natural and vaccine-induced protective immunity, it has been under-prioritized in the evaluation of malaria vaccine candidates. This study uses genomic approaches to evaluate genetic diversity in next generation malaria vaccine candidate PfRh5. We used targeted deep amplicon sequencing to identify non-synonymous Single Nucleotide Polymorphisms (SNPs) in PfRh5 (Reticulocyte-Binding Protein Homologue 5) in 189 P. falciparum positive samples from Southern Senegal and identified 74 novel SNPs. We evaluated the population prevalence of these SNPs as well as the frequency in individual samples and found that only a single SNP, C203Y, was present at every site. Many SNPs were unique to the individual sampled, with over 90% of SNPs being found in just one infected individual. In addition to population prevalence, we assessed individual level SNP frequencies which revealed that some SNPs were dominant (frequency of greater than 25% in a polygenomic sample) whereas most were rare, present at 2% or less of total reads mapped to the reference at the given position. Structural modeling uncovered 3 novel SNPs occurring under epitopes bound by inhibitory monoclonal antibodies, potentially impacting immune evasion, while other SNPs were predicted to impact PfRh5 structure or interactions with the receptor or binding partners. Our data demonstrate that PfRh5 exhibits greater genetic diversity than previously described, with the caveat that most of the uncovered SNPs are at a low overall frequency in the individual and prevalence in the population. The structural studies reveal that novel SNPs could have functional implications on PfRh5 receptor binding, complex formation, or immune evasion, supporting continued efforts to validate PfRh5 as an effective malaria vaccine target and development of a PfRh5 vaccine.

Plasmodium infection is associated with cross-reactive antibodies to carbohydrate epitopes on the SARS-CoV-2 Spike protein.

Sero-surveillance can monitor and project disease burden and risk. However, SARS-CoV-2 antibody test results can produce false positive results, limiting their efficacy as a sero-surveillance tool. False positive SARS-CoV-2 antibody results are associated with malaria exposure, and understanding this association is essential to interpret sero-surveillance results from malaria-endemic countries. Here, pre-pandemic samples from eight malaria endemic and non-endemic countries and four continents were tested by ELISA to measure SARS-CoV-2 Spike S1 subunit reactivity. Individuals with acute malaria infection generated substantial SARS-CoV-2 reactivity. Cross-reactivity was not associated with reactivity to other human coronaviruses or other SARS-CoV-2 proteins, as measured by peptide and protein arrays. ELISAs with deglycosylated and desialated Spike S1 subunits revealed that cross-reactive antibodies target sialic acid on N-linked glycans of the Spike protein. The functional activity of cross-reactive antibodies measured by neutralization assays showed that cross-reactive antibodies did not neutralize SARS-CoV-2 in vitro. Since routine use of glycosylated or sialated assays could result in false positive SARS-CoV-2 antibody results in malaria endemic regions, which could overestimate exposure and population-level immunity, we explored methods to increase specificity by reducing cross-reactivity. Overestimating population-level exposure to SARS-CoV-2 could lead to underestimates of risk of continued COVID-19 transmission in sub-Saharan Africa.

Assessing the functional impact of PfRh5 genetic diversity on ex vivo erythrocyte invasion inhibition.

The PfRh5-Basigin ligand-receptor interaction is an essential step in the merozoite invasion process and represents an attractive vaccine target. To reveal genotype-phenotype associations between naturally occurring allelic variants of PfRh5 and invasion inhibition, we performed ex vivo invasion inhibition assays with monoclonal antibodies targeting basigin coupled with PfRh5 next-generation amplicon sequencing. We found dose-dependent inhibition of invasion across all isolates tested, and no statistically significant difference in invasion inhibition for any single nucleotide polymorphisms. This study demonstrates that PfRh5 remains highly conserved and functionally essential, even in a highly endemic setting, supporting continued development as a strain-transcendent malaria vaccine target.

Kynurenic acid may underlie sex-specific immune responses to COVID-19.

Coronavirus disease 2019 (COVID-19) has poorer clinical outcomes in males than in females, and immune responses underlie these sex-related differences. Because immune responses are, in part, regulated by metabolites, we examined the serum metabolomes of COVID-19 patients. In male patients, kynurenic acid (KA) and a high KA-to-kynurenine (K) ratio (KA:K) positively correlated with age and with inflammatory cytokines and chemokines and negatively correlated with T cell responses. Males that clinically deteriorated had a higher KA:K than those that stabilized. KA inhibits glutamate release, and glutamate abundance was lower in patients that clinically deteriorated and correlated with immune responses. Analysis of data from the Genotype-Tissue Expression (GTEx) project revealed that the expression of the gene encoding the enzyme that produces KA, kynurenine aminotransferase, correlated with cytokine abundance and activation of immune responses in older males. This study reveals that KA has a sex-specific link to immune responses and clinical outcomes in COVID-19, suggesting a positive feedback between metabolites and immune responses in males.

Tracking smell loss to identify healthcare workers with SARS-CoV-2 infection.

INTRODUCTION: Healthcare workers (HCW) treating COVID-19 patients are at high risk for infection and may also spread infection through their contact with vulnerable patients. Smell loss has been associated with SARS-CoV-2 infection, but it is unknown whether monitoring for smell loss can be used to identify asymptomatic infection among high risk individuals. In this study we sought to determine if tracking smell sensitivity and loss using an at-home assessment could identify SARS-CoV-2 infection in HCW. METHODS AND FINDINGS: We performed a prospective cohort study tracking 473 HCW across three months to determine if smell loss could predict SARS-CoV-2 infection in this high-risk group. HCW subjects completed a longitudinal, behavioral at-home assessment of olfaction with household items, as well as detailed symptom surveys that included a parosmia screening questionnaire, and real-time quantitative polymerase chain reaction testing to identify SARS-CoV-2 infection. Our main measures were the prevalence of smell loss in SARS-CoV-2-positive HCW versus SARS-CoV-2-negative HCW, and timing of smell loss relative to SARS-CoV-2 test positivity. SARS-CoV-2 was identified in 17 (3.6%) of 473 HCW. HCW with SARS-CoV-2 infection were more likely to report smell loss than SARS-CoV-2-negative HCW on both the at-home assessment and the screening questionnaire (9/17, 53% vs 105/456, 23%, P < .01). 6/9 (67%) of SARS-CoV-2-positive HCW reporting smell loss reported smell loss prior to having a positive SARS-CoV-2 test, and smell loss was reported a median of two days before testing positive. Neurological symptoms were reported more frequently among SARS-CoV-2-positive HCW who reported smell loss compared to those without smell loss (9/9, 100% vs 3/8, 38%, P < .01). CONCLUSIONS: In this prospective study of HCW, self-reported changes in smell using two different measures were predictive of SARS-CoV-2 infection. Smell loss frequently preceded a positive test and was associated with neurological symptoms.

Case Study: Longitudinal immune profiling of a SARS-CoV-2 reinfection in a solid organ transplant recipient.

Prior 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.

SalivaDirect: A simplified and flexible platform to enhance SARS-CoV-2 testing capacity.

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 (<0.05%) results. CONCLUSIONS: We demonstrate that saliva is a valid alternative to swabs for SARS-CoV-2 screening and that SalivaDirect can make large-scale testing more accessible and affordable. Uniquely, we can designate other laboratories to use our sensitive, flexible, and simplified platform under our EUA (https://publichealth.yale.edu/salivadirect/). FUNDING: This study was funded by the NBA and NBPA (N.D.G.), the Huffman Family Donor Advised Fund (N.D.G.), a Fast Grant from Emergent Ventures at the Mercatus Center at George Mason University (N.D.G.), the Yale Institute for Global Health (N.D.G.), and the Beatrice Kleinberg Neuwirth Fund (A.I.K.). C.B.F.V. is supported by NWO Rubicon 019.181EN.004.

Maternal respiratory SARS-CoV-2 infection in pregnancy is associated with a robust inflammatory response at the maternal-fetal interface.

BACKGROUND: Pregnant women are at increased risk for severe outcomes from coronavirus disease 2019 (COVID-19), but the pathophysiology underlying this increased morbidity and its potential effect on the developing fetus is not well understood. METHODS: We assessed placental histology, ACE2 expression, and viral and immune dynamics at the term placenta in pregnant women with and without respiratory severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. FINDINGS: The majority (13 of 15) of placentas analyzed had no detectable viral RNA. ACE2 was detected by immunohistochemistry in syncytiotrophoblast cells of the normal placenta during early pregnancy but was rarely seen in healthy placentas at full term, suggesting that low ACE2 expression may protect the term placenta from viral infection. Using immortalized cell lines and primary isolated placental cells, we found that cytotrophoblasts, the trophoblast stem cells and precursors to syncytiotrophoblasts, rather than syncytiotrophoblasts or Hofbauer cells, are most vulnerable to SARS-CoV-2 infection in vitro. To better understand potential immune mechanisms shielding placental cells from infection in vivo, we performed bulk and single-cell transcriptomics analyses and found that the maternal-fetal interface of SARS-CoV-2-infected women exhibited robust immune responses, including increased activation of natural killer (NK) and T cells, increased expression of interferon-related genes, as well as markers associated with pregnancy complications such as preeclampsia. CONCLUSIONS: SARS-CoV-2 infection in late pregnancy is associated with immune activation at the maternal-fetal interface even in the absence of detectable local viral invasion. FUNDING: NIH (T32GM007205, F30HD093350, K23MH118999, R01AI157488, U01DA040588) and Fast Grant funding support from Emergent Ventures at the Mercatus Center.

Saliva viral load is a dynamic unifying correlate of COVID-19 severity and mortality.

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.

SARS-CoV-2 infection in pregnancy is associated with robust inflammatory response at the maternal-fetal interface.

Pregnant women appear to be at increased risk for severe outcomes associated with COVID-19, but the pathophysiology underlying this increased morbidity and its potential impact on the developing fetus is not well understood. In this study of pregnant women with and without COVID-19, we assessed viral and immune dynamics at the placenta during maternal SARS-CoV-2 infection. Amongst uninfected women, ACE2 was detected by immunohistochemistry in syncytiotrophoblast cells of the normal placenta during early pregnancy but was rarely seen in healthy placentas at full term. Term placentas from women infected with SARS-CoV-2, however, displayed a significant increase in ACE2 levels. Using immortalized cell lines and primary isolated placental cells, we determined the vulnerability of various placental cell types to direct infection by SARS-CoV-2 in vitro. Yet, despite the susceptibility of placental cells to SARS-CoV-2 infection, viral RNA was detected in the placentas of only a subset (~13%) of women in this cohort. Through single cell transcriptomic analyses, we found that the maternal-fetal interface of SARS-CoV-2-infected women exhibited markers associated with pregnancy complications, such as preeclampsia, and robust immune responses, including increased activation of placental NK and T cells and increased expression of interferon-related genes. Overall, this study suggests that SARS-CoV-2 is associated with immune activation at the maternal-fetal interface even in the absence of detectable local viral invasion. While this likely represents a protective mechanism shielding the placenta from infection, inflammatory changes in the placenta may also contribute to poor pregnancy outcomes and thus warrant further investigation.