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

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

Longitudinal analyses reveal immunological misfiring in severe COVID-19.

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

Phase 1 trial to model primary, secondary, and tertiary dengue using a monovalent vaccine.

BACKGROUND: The four co-circulating and immunologically interactive dengue virus serotypes (DENV1-4) pose a unique challenge to vaccine design because sub-protective immunity can increase the risk of severe dengue disease. Existing dengue vaccines have lower efficacy in DENV seronegative individuals but higher efficacy in DENV exposed individuals. There is an urgent need to identify immunological measures that are strongly associated with protection against viral replication and disease following sequential exposure to distinct serotypes. METHODS/DESIGN: This is a phase 1 trial wherein healthy adults with neutralizing antibodies to zero (seronegative), one non-DENV3 (heterotypic), or more than one (polytypic) DENV serotype will be vaccinated with the live attenuated DENV3 monovalent vaccine rDEN3Δ30/31-7164. We will examine how pre-vaccine host immunity influences the safety and immunogenicity of DENV3 vaccination in a non-endemic population. We hypothesize that the vaccine will be safe and well tolerated, and all groups will have a significant increase in the DENV1-4 neutralizing antibody geometric mean titer between days 0 and 28. Compared to the seronegative group, the polytypic group will have lower mean peak vaccine viremia, due to protection conferred by prior DENV exposure, while the heterotypic group will have higher mean peak viremia, due to mild enhancement. Secondary and exploratory endpoints include characterizing serological, innate, and adaptive cell responses; evaluating proviral or antiviral contributions of DENV-infected cells; and immunologically profiling the transcriptome, surface proteins, and B and T cell receptor sequences and affinities of single cells in both peripheral blood and draining lymph nodes sampled via serial image-guided fine needle aspiration. DISCUSSION: This trial will compare the immune responses after primary, secondary, and tertiary DENV exposure in naturally infected humans living in non-endemic areas. By evaluating dengue vaccines in a new population and modeling the induction of cross-serotypic immunity, this work may inform vaccine evaluation and broaden potential target populations. TRIAL REGISTRATION: NCT05691530 registered on January 20, 2023.

Acute encephalopathy with elevated CSF inflammatory markers as the initial presentation of COVID-19.

BACKGROUND: COVID-19 is caused by the severe acute respiratory syndrome virus SARS-CoV-2. It is widely recognized as a respiratory pathogen, but neurologic complications can be the presenting manifestation in a subset of infected patients. CASE PRESENTATION: We describe a 78-year old immunocompromised woman who presented with altered mental status after witnessed seizure-like activity at home. She was found to have SARS-CoV-2 infection and associated neuroinflammation. In this case, we undertake the first detailed analysis of cerebrospinal fluid (CSF) cytokines during COVID-19 infection and find a unique pattern of inflammation in CSF, but no evidence of viral neuroinvasion. CONCLUSION: Our findings suggest that neurologic symptoms such as encephalopathy and seizures may be the initial presentation of COVID-19. Central nervous system inflammation may associate with neurologic manifestations of disease.

Risk Factors for Disseminated Coccidioidomycosis, United States.

Of 150,000 new coccidioidomycosis infections that occur annually in the United States, ≈1% disseminate; one third of those cases are fatal. Immunocompromised hosts have higher rates of dissemination. We identified 8 patients with disseminated coccidioidomycosis who had defects in the interleukin-12/interferon-γ and STAT3 axes, indicating that these are critical host defense pathways.

Dengue virus IgG and serotype-specific neutralizing antibody titers measured with standard and mature viruses are associated with protection.

Recent work demonstrates the limitations of the standard dengue virus (DENV) neutralization assay to predict protection against dengue. We perform studies to compare how a commercial IgG ELISA, envelope domain III (EDIII) or non-structural protein 1 (NS1) binding antibodies, and titers from plaque reduction neutralization tests (PRNTs) using reference standard and clinical mature viruses are associated with dengue disease. Healthy children (n = 1,206) in Cebu, Philippines were followed for 5 years. High ELISA values (≥3) were associated with reduced dengue probability relative to naïve children (3% vs. 10%, p = 0.008), but antibody binding EDIII or NS1 from each serotype had no association. High standard and mature geometric mean PRNT titers were associated with reduced dengue disease overall (p < 0.01), and high DENV2 and DENV3 titers in both assays provided protection against the matched serotype (p < 0.02). However, while 52% of dengue cases had standard virus PRNT titers > 100, only 2% of cases had mature virus PRNT titers > 100 (p < 0.001), indicating a lower, more consistent threshold for protection. Each assay may be useful for different purposes as correlates of protection in population and vaccine trials.

Envelope-dimer epitope-like broadly protective antibodies against dengue in children following natural infection and vaccination.

Cross-reactive antibodies (Abs) to epitopes that span envelope proteins on the virion surface are hypothesized to protect against dengue. Here, we measured Abs targeting the quaternary envelope dimer epitope (EDE) as well as neutralizing and binding Abs and evaluate their association with dengue virus (DENV) infection, vaccine response, and disease outcome in dengue vaccinated and unvaccinated children (n=252) within a longitudinal cohort in Cebu, Philippines (n=2,996). Abs targeting EDE were prevalent and strongly associated with broad neutralization of DENV1-4 in those with baseline multitypic immunity. Subsequent natural infection and vaccination boosted EDE-like, neutralizing, and binding Abs. EDE-like Abs were associated with reduced dengue risk and mediated the protective effect of binding and neutralizing Abs on symptomatic and severe dengue. Thus, Abs targeting quaternary epitopes help explain broad cross protection in those with multiple prior DENV exposures, making them useful for evaluation and development of future vaccines and therapeutics.

Specific ligand binding domain residues confer low dioxin responsiveness to AHR1ß of Xenopus laevis.

The aryl hydrocarbon receptor (AHR) is a Per-ARNT-Sim (PAS) family protein that mediates the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in vertebrates. Frogs are remarkably insensitive to TCDD, and AHRs from Xenopus laevis bind TCDD with low affinity. We sought to identify structural features of X. laevis AHR1ß associated with low TCDD sensitivity. Substitution of the entire ligand binding domain (LBD) with the corresponding sequence from mouse AHR(b-1) dramatically increased TCDD responsiveness in transactivation assays. To identify the amino acid residues responsible, we constructed a comparative model of the AHR1ß LBD using homologous domains of PAS proteins HIF2α and ARNT. The model revealed an internal cavity with dimensions similar to those of the putative binding cavity of mouse AHR(b-1), suggesting the importance of side chain interactions over cavity size. Of residues with side chains clearly pointing into the cavity, only two differed from the mouse sequence. When A354, located within a conserved ß-strand, was changed to serine, the corresponding mouse residue, the EC50 for TCDD decreased more than 15-fold. When N325 was changed to serine, the EC50 decreased 3-fold. When the mutations were combined, the EC50 decreased from 18.6 to 0.8 nM, the value nearly matching the TCDD sensitivity of mouse AHR. Velocity sedimentation analysis confirmed that mutant frog AHRs exhibited correspondingly increased levels of TCDD binding. We also assayed mutant AHRs for responsiveness to a candidate endogenous ligand, 6-formylindolo[3,2-b]carbazole (FICZ). Mutations that increased sensitivity to TCDD also increased sensitivity to FICZ. This comparative study represents a novel approach to discerning fundamental information about the structure of AHR and its interactions with biologically important agonists.

Immunity to non-dengue flaviviruses impacts dengue virus IgG ELISA specificity in Cambodia.

Seroprevalence studies are the gold standard for disease surveillance, and serology was used to determine eligibility for the first licensed dengue vaccine. However, expanding flavivirus endemicity, co-circulation, and vaccination complicate serology results. Among 713 healthy Cambodian children, a commonly used indirect dengue virus IgG ELISA (PanBio) had a lower specificity than previously reported (94% vs. 100%). Of those with false positive PanBio results, 46% had detectable neutralizing antibodies against other flaviviruses, with the highest frequency against West Nile virus (WNV). Immunity to non-dengue flaviviruses can impact dengue surveillance and potentially pre-vaccine screening efforts.

The Effect of Age on Dengue Presentation and the Diagnostic Accuracy of the 2015 Pan American Health Organization Case Criteria in a Puerto Rican Cohort.

Background: We evaluated dengue presentation by age, the performance of the 2015 Pan American Health Organization (PAHO) case criteria in identifying dengue cases, and variables to improve specificity. Methods: Patients with fever ≤7 days (N = 10 408) were recruited from 2 emergency departments from May 2012 through December 2015. Serum samples were tested for dengue, chikungunya, and nasopharyngeal swabs for respiratory viruses. Smoothing splines assessed differences in the frequencies of signs/symptoms by age. Least absolute shrinkage and selection operator regressions identified the variables that best predicted dengue. Results: Among 985 dengue cases, children aged <5 years were least likely to have leukopenia, but most likely to have rash and petechiae. Adults had the highest odds of aches/pains and headaches/retro-orbital pain. The 2015 PAHO criteria had sensitivity of 93% and specificity of 25%. Specificity could be improved by requiring at least 2 of the following criteria: vomiting/nausea, petechiae, rash, or leukopenia (specificity 68%, sensitivity 71%) or by using 2015 PAHO criteria plus either (1) aspartate aminotransferase >50 IU/L or platelet count <100 000 platelets/µL (specificity 81%, sensitivity 56%) or (2) itchy skin or absence of rhinorrhea or cough (specificity 51%, sensitivity 82%). Conclusions: The 2015 PAHO dengue case criteria had excellent sensitivity but poor specificity. This can be improved by adding signs/symptoms associated with dengue diagnosis.

'Mix and Match' vaccination: Is dengue next?

The severity of the COVID-19 pandemic and the development of multiple SARS-CoV-2 vaccines expedited vaccine 'mix and match' trials in humans and demonstrated the benefits of mixing vaccines that vary in formulation, strength, and immunogenicity. Heterologous sequential vaccination may be an effective approach for protecting against dengue, as this strategy would mimic the natural route to broad dengue protection and may overcome the imbalances in efficacy of the individual leading live attenuated dengue vaccines. Here we review 'mix and match' vaccination trials against SARS-CoV-2, HIV, and dengue virus and discuss the possible advantages and concerns of future heterologous immunization with the leading dengue vaccines. COVID-19 trials suggest that priming with a vaccine that induces strong cellular responses, such as an adenoviral vectored product, followed by heterologous boost may optimize T cell immunity. Moreover, heterologous vaccination may induce superior humoral immunity compared to homologous vaccination when the priming vaccine induces a narrower response than the boost. The HIV trials reported that heterologous vaccination was associated with broadened antigen responses and that the sequence of the vaccines significantly impacts the regimen's immunogenicity and efficacy. In heterologous dengue immunization trials, where at least one dose was with a live attenuated vaccine, all reported equivalent or increased immunogenicity compared to homologous boost, although one study reported increased reactogenicity. The three leading dengue vaccines have been evaluated for safety and efficacy in thousands of study participants but not in combination in heterologous dengue vaccine trials. Various heterologous regimens including different combinations and sequences should be trialed to optimize cellular and humoral immunity and the breadth of the response while limiting reactogenicity. A blossoming field dedicated to more accurate correlates of protection and enhancement will help confirm the safety and efficacy of these strategies.

Multiscale PHATE identifies multimodal signatures of COVID-19.

As the biomedical community produces datasets that are increasingly complex and high dimensional, there is a need for more sophisticated computational tools to extract biological insights. We present Multiscale PHATE, a method that sweeps through all levels of data granularity to learn abstracted biological features directly predictive of disease outcome. Built on a coarse-graining process called diffusion condensation, Multiscale PHATE learns a data topology that can be analyzed at coarse resolutions for high-level summarizations of data and at fine resolutions for detailed representations of subsets. We apply Multiscale PHATE to a coronavirus disease 2019 (COVID-19) dataset with 54 million cells from 168 hospitalized patients and find that patients who die show CD16hiCD66blo neutrophil and IFN-γ+ granzyme B+ Th17 cell responses. We also show that population groupings from Multiscale PHATE directly fed into a classifier predict disease outcome more accurately than naive featurizations of the data. Multiscale PHATE is broadly generalizable to different data types, including flow cytometry, single-cell RNA sequencing (scRNA-seq), single-cell sequencing assay for transposase-accessible chromatin (scATAC-seq), and clinical variables.

Macrophage Migration Inhibitory Factor is not Associated with Sarcoidosis Susceptibility or Severity in Whites or Blacks.

BACKGROUND: Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine, and increased MIF expression has been associated with the development and severity of multiple granulomatous, autoimmune diseases. However, MIF association studies have been discordant in sarcoidosis. OBJECTIVE: To evaluate associations between macrophage migration inhibitory factor (MIF) promoter polymorphisms and sarcoidosis susceptibility and severity. METHODS: Three hundred and fifty one patients with sarcoidosis were recruited through the Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis (GRADS) study. Genomic DNA was isolated from serum, and the MIF -173G/C SNP [rs755622] and MIF -794 CATT5-8 microsatellite repeat [rs5844572] were genotyped. Allelic frequencies were compared between cases and healthy controls and associations between MIF alleles and sarcoidosis severity were assessed. RESULTS: The frequencies of the high expression -173C SNP and the low expression -794 CATT5 containing genotypes in white and black sarcoidosis patients were the same as those of healthy controls. High expression MIF alleles were not associated with sarcoidosis severity. Associations between MIF alleles and extrapulmonary sarcoidosis phenotypes were limited by small sample sizes. CONCLUSIONS: High expression MIF genotypes were not associated with the susceptibility to or severity of pulmonary sarcoidosis in a large North American cohort. (Sarcoidosis Vasc Diffuse Lung Dis 2020; 37 (3): e2020004).

Acute encephalopathy with elevated CSF inflammatory markers as the initial presentation of COVID-19.

BACKGROUND: COVID-19 is caused by the severe acute respiratory syndrome virus SARS-CoV-2. It is widely recognized as a respiratory pathogen, but neurologic complications can be the presenting manifestation in a subset of infected patients. CASE PRESENTATION: We describe a 78-year old immunocompromised woman who presented with altered mental status after witnessed seizure-like activity at home. She was found to have SARS-CoV-2 infection and associated neuroinflammation. In this case, we undertake the first detailed analysis of cerebrospinal fluid (CSF) cytokines during COVID-19 infection and find a unique pattern of inflammation in CSF, but no evidence of viral neuroinvasion. CONCLUSION: Our findings suggest that neurologic symptoms such as encephalopathy and seizures may be the initial presentation of COVID-19. Central nervous system inflammation may associate with neurologic manifestations of disease.

Analytical sensitivity and efficiency comparisons of SARS-CoV-2 RT-qPCR primer-probe sets.

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 (Charité) 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.