Deciphering bat influenza H18N11 infection dynamics in male Jamaican fruit bats on a single-cell level.

Jamaican fruit bats (Artibeus jamaicensis) naturally harbor a wide range of viruses of human relevance. These infections are typically mild in bats, suggesting unique features of their immune system. To better understand the immune response to viral infections in bats, we infected male Jamaican fruit bats with the bat-derived influenza A virus (IAV) H18N11. Using comparative single-cell RNA sequencing, we generated single-cell atlases of the Jamaican fruit bat intestine and mesentery. Gene expression profiling showed that H18N11 infection resulted in a moderate induction of interferon-stimulated genes and transcriptional activation of immune cells. H18N11 infection was predominant in various leukocytes, including macrophages, B cells, and NK/T cells. Confirming these findings, human leukocytes, particularly macrophages, were also susceptible to H18N11, highlighting the zoonotic potential of this bat-derived IAV. Our study provides insight into a natural virus-host relationship and thus serves as a fundamental resource for future in-depth characterization of bat immunology.

A Modified BPaL Regimen for Tuberculosis Treatment replaces Linezolid with Inhaled Spectinamides.

The Nix-TB clinical trial evaluated a new 6-month regimen containing three-oral-drugs; bedaquiline (B), pretomanid (Pa) and linezolid (L) (BPaL regimen) for treatment of tuberculosis (TB). This regimen achieved remarkable results as almost 90% of the multidrug resistant (MDR) or extensively drug resistant (XDR) TB participants were cured but many patients also developed severe adverse effects (AEs). The AEs were associated with the long-term administration of the protein synthesis inhibitor linezolid. Spectinamide 1599 (S) is also a protein synthesis inhibitor of Mycobacterium tuberculosis with an excellent safety profile but which lacks oral bioavailability. Here we hypothesize that inhaled spectinamide 1599, combined with BPa --BPaS regimen--has similar efficacy to that of BPaL regimen while simultaneously avoiding the L-associated AEs. The BPaL and BPaS regimens were compared in the Balb/c and C3HeB/FeJ murine chronic TB efficacy models. After 4-weeks of treatment, both regimens promoted equivalent bactericidal effect in both TB murine models. However, treatment with BPaL resulted in significant weight loss and the complete blood count suggested development of anemia. These effects were not similarly observed in mice treated with BPaS. BPaL treatment also decreased myeloid to erythroid ratio and increased concentration of proinflammatory cytokines in bone marrow compared to mice receiving BPaS regimen. During therapy both regimens improved the lung lesion burden, reduced neutrophil and cytotoxic T cells counts while increased the number of B and helper and regulatory T cells. These combined data suggest that inhaled spectinamide 1599 combined with BPa is an effective TB regimen that avoids L-associated AEs. IMPORTANCE: Tuberculosis (TB) is an airborne infectious disease that spreads via aerosols containing Mycobacterium tuberculosis (Mtb), the causative agent of TB. TB can be cured by administration of 3-4 drugs for 6-9 months but there are limited treatment options for patients infected with multidrug (MDR) and extensively resistant (XDR) strains of Mtb. BPaL is a new all-oral combination of drugs consisting of Bedaquiline (B), Pretomanid (Pa) and Linezolid (L). This regimen was able to cure ∼90% of MDR and XDR TB patients in clinical trials but many patients developed severe adverse effects (AEs) associated to the long-term administration of linezolid. We evaluated a new regimen in which Linezolid in the BPaL regimen was replaced with inhaled spectinamide 1599. In the current study, we demonstrate that 4-weeks of treatment with inhaled spectinamide 1599 in combination with Bedaquiline and Pretomanid has equivalent efficacy to the BPaL drug combination and avoids the L-associated-AEs.

Persistent CD8+ T cell proliferation and activation in COVID-19 adult survivors with post-acute sequelae: a longitudinal, observational cohort study of persistent symptoms and T cell markers.

Introduction: Post-acute sequelae of COVID-19 affects the quality of life of many COVID-19 survivors, yet the etiology of post-acute sequelae of COVID-19 remains unknown. We aimed to determine if persistent inflammation and ongoing T-cell activation during convalescence were a contributing factor to the pathogenesis of post-acute sequelae of COVID-19. Methods: We evaluated 67 individuals diagnosed with COVID-19 by nasopharyngeal polymerase chain reaction for persistent symptoms during convalescence at separate time points occurring up to 180 days post-diagnosis. Fifty-two of these individuals were evaluated longitudinally. We obtained whole blood samples at each study visit, isolated peripheral blood mononuclear cells, and stained for multiple T cell activation markers for flow cytometry analysis. The activation states of participants' CD4+ and CD8+ T-cells were next analyzed for each of the persistent symptoms. Results: Overall, we found that participants with persistent symptoms had significantly higher levels of inflammation at multiple time points during convalescence when compared to those who fully recovered from COVID-19. Participants with persistent dyspnea, forgetfulness, confusion, and chest pain had significantly higher levels of proliferating effector T-cells (CD8+Ki67+), and those with chest pain, joint pain, difficulty concentrating, and forgetfulness had higher levels of regulatory T-cells (CD4+CD25+). Additionally, those with dyspnea had significantly higher levels of CD8+CD38+, CD8+ Granzyme B+, and CD8+IL10+ cells. A retrospective comparison of acute phase inflammatory markers in adults with and without post-acute sequelae of COVID-19 showed that CD8+Ki67+ cells were significantly higher at the time of acute illness (up to 14 days post-diagnosis) in those who developed persistent dyspnea. Discussion: These findings suggest continued CD8+ T-cell activation following SARS-CoV-2 infection in adults experiencing post-acute sequelae of COVID-19 and that the increase in T regulatory cells for a subset of these patients represents the ongoing attempt by the host to reduce inflammation.

Mucosal exposure to non-tuberculous mycobacteria elicits B cell-mediated immunity against pulmonary tuberculosis.

Bacille Calmette-Guerin (BCG) is the only licensed vaccine against Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB) disease. However, BCG has limited efficacy, necessitating the development of better vaccines. Non-tuberculous mycobacteria (NTMs) are opportunistic pathogens present ubiquitously in the environment. TB endemic countries experience higher exposure to NTMs, but previous studies have not elucidated the relationship between NTM exposure and BCG efficacy against TB. Therefore, we develop a mouse model (BCG + NTM) to simulate human BCG immunization regime and continuous NTM exposure. BCG + NTM mice exhibit superior and prolonged protection against pulmonary TB, with increased B cell influx and anti-Mtb antibodies in serum and airways, compared with BCG alone. Notably, spatial transcriptomics and immunohistochemistry reveal that BCG + NTM mice formed B cell aggregates with features of germinal center development, which correlate with reduced Mtb burden. Our studies suggest a direct relationship between NTM exposure and TB protection, with B cells playing a crucial role.

Comprehensive Immune Profiling Reveals CD56+ Monocytes and CD31+ Endothelial Cells Are Increased in Severe COVID-19 Disease.

Immune response dysregulation plays a key role in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis. In this study, we evaluated immune and endothelial blood cell profiles of patients with coronavirus disease 2019 (COVID-19) to determine critical differences between those with mild, moderate, or severe COVID-19 using spectral flow cytometry. We examined a suite of immune phenotypes, including monocytes, T cells, NK cells, B cells, endothelial cells, and neutrophils, alongside surface and intracellular markers of activation. Our results showed progressive lymphopenia and depletion of T cell subsets (CD3+, CD4+, and CD8+) in patients with severe disease and a significant increase in the CD56+CD14+Ki67+IFN-γ+ monocyte population in patients with moderate and severe COVID-19 that has not been previously described. Enhanced circulating endothelial cells (CD45-CD31+CD34+CD146+), circulating endothelial progenitors (CD45-CD31+CD34+/-CD146-), and neutrophils (CD11b+CD66b+) were coevaluated for COVID-19 severity. Spearman correlation analysis demonstrated the synergism among age, obesity, and hypertension with upregulated CD56+ monocytes, endothelial cells, and decreased T cells that lead to severe outcomes of SARS-CoV-2 infection. Circulating monocytes and endothelial cells may represent important cellular markers for monitoring postacute sequelae and impacts of SARS-CoV-2 infection during convalescence and for their role in immune host defense in high-risk adults after vaccination.

Antigen Presentation of mRNA-Based and Virus-Vectored SARS-CoV-2 Vaccines.

Infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causes Coronavirus Disease 2019 (COVID-19), which has reached pandemic proportions. A number of effective vaccines have been produced, including mRNA vaccines and viral vector vaccines, which are now being implemented on a large scale in order to control the pandemic. The mRNA vaccines are composed of viral Spike S1 protein encoding mRNA incorporated in a lipid nanoparticle and stabilized by polyethylene glycol (PEG). The mRNA vaccines are novel in many respects, including cellular uptake and the intracellular routing, processing, and secretion of the viral protein. Viral vector vaccines have incorporated DNA sequences, encoding the SARS-CoV-2 Spike protein into (attenuated) adenoviruses. The antigen presentation routes in MHC class I and class II, in relation to the induction of virus-neutralizing antibodies and cytotoxic T-lymphocytes, will be reviewed. In rare cases, mRNA vaccines induce unwanted immune mediated side effects. The mRNA-based vaccines may lead to an anaphylactic reaction. This reaction may be triggered by PEG. The intracellular routing of PEG and potential presentation in the context of CD1 will be discussed. Adenovirus vector-based vaccines have been associated with thrombocytopenic thrombosis events. The anti-platelet factor 4 antibodies found in these patients could be generated due to conformational changes of relevant epitopes presented to the immune system.

A longitudinal SARS-CoV-2 biorepository for COVID-19 survivors with and without post-acute sequelae.

BACKGROUND: SARS-CoV-2 has swept across the globe, causing millions of deaths worldwide. Though most survive, many experience symptoms of COVID-19 for months after acute infection. Successful prevention and treatment of acute COVID-19 infection and its associated sequelae is dependent on in-depth knowledge of viral pathology across the spectrum of patient phenotypes and physiologic responses. Longitudinal biobanking provides a valuable resource of clinically integrated, easily accessed, and quality-controlled samples for researchers to study differential multi-organ system responses to SARS-CoV-2 infection, post-acute sequelae of COVID-19 (PASC), and vaccination. METHODS: Adults with a history of a positive SARS-CoV-2 nasopharyngeal PCR are actively recruited from the community or hospital settings to enroll in the Northern Colorado SARS-CoV-2 Biorepository (NoCo-COBIO). Blood, saliva, stool, nasopharyngeal specimens, and extensive clinical and demographic data are collected at 4 time points over 6 months. Patients are assessed for PASC during longitudinal follow-up by physician led symptom questionnaires and physical exams. This clinical trial registration is NCT04603677 . RESULTS: We have enrolled and collected samples from 119 adults since July 2020, with 66% follow-up rate. Forty-nine percent of participants assessed with a symptom surveillance questionnaire (N = 37 of 75) had PASC at any time during follow-up (up to 8 months post infection). Ninety-three percent of hospitalized participants developed PASC, while 23% of those not requiring hospitalization developed PASC. At 90-174 days post SARS-CoV-2 diagnosis, 67% of all participants had persistent symptoms (N = 37 of 55), and 85% percent of participants who required hospitalization during initial infection (N = 20) still had symptoms. The most common symptoms reported after 15 days of infection were fatigue, loss of smell, loss of taste, exercise intolerance, and cognitive dysfunction. CONCLUSIONS: Patients who were hospitalized for COVID-19 were significantly more likely to have PASC than those not requiring hospitalization, however 23% of patients who were not hospitalized also developed PASC. This patient-matched, multi-matrix, longitudinal biorepository from COVID-19 survivors with and without PASC will allow for current and future research to better understand the pathophysiology of disease and to identify targeted interventions to reduce risk for PASC. Registered 27 October 2020 - Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT04603677 .

A Whole Virion Vaccine for COVID-19 Produced via a Novel Inactivation Method and Preliminary Demonstration of Efficacy in an Animal Challenge Model.

The COVID-19 pandemic has generated intense interest in the rapid development and evaluation of vaccine candidates for this disease and other emerging diseases. Several novel methods for preparing vaccine candidates are currently undergoing clinical evaluation in response to the urgent need to prevent the spread of COVID-19. In many cases, these methods rely on new approaches for vaccine production and immune stimulation. We report on the use of a novel method (SolaVAX) for production of an inactivated vaccine candidate and the testing of that candidate in a hamster animal model for its ability to prevent infection upon challenge with SARS-CoV-2 virus. The studies employed in this work included an evaluation of the levels of neutralizing antibody produced post-vaccination, levels of specific antibody sub-types to RBD and spike protein that were generated, evaluation of viral shedding post-challenge, flow cytometric and single cell sequencing data on cellular fractions and histopathological evaluation of tissues post-challenge. The results from this preliminary evaluation provide insight into the immunological responses occurring as a result of vaccination with the proposed vaccine candidate and the impact that adjuvant formulations, specifically developed to promote Th1 type immune responses, have on vaccine efficacy and protection against infection following challenge with live SARS-CoV-2. This data may have utility in the development of effective vaccine candidates broadly. Furthermore, the results of this preliminary evaluation suggest that preparation of a whole virion vaccine for COVID-19 using this specific photochemical method may have potential utility in the preparation of one such vaccine candidate.

Preservation of neutralizing antibody function in COVID-19 convalescent plasma treated using a riboflavin and ultraviolet light-based pathogen reduction technology.

BACKGROUND AND OBJECTIVES: Convalescent plasma (CP) has been embraced as a safe therapeutic option for coronavirus disease 2019 (COVID-19), while other treatments are developed. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is not transmissible by transfusion, but bloodborne pathogens remain a risk in regions with high endemic prevalence of disease. Pathogen reduction can mitigate this risk; thus, the objective of this study was to evaluate the effect of riboflavin and ultraviolet light (R + UV) pathogen reduction technology on the functional properties of COVID-19 CP (CCP). MATERIALS AND METHODS: COVID-19 convalescent plasma units (n = 6) from recovered COVID-19 research donors were treated with R + UV. Pre- and post-treatment samples were tested for coagulation factor and immunoglobulin retention. Antibody binding to spike protein receptor-binding domain (RBD), S1 and S2 epitopes of SARS-CoV-2 was assessed by ELISA. Neutralizing antibody (nAb) function was assessed by pseudovirus reporter viral particle neutralization (RVPN) assay and plaque reduction neutralization test (PRNT). RESULTS: Mean retention of coagulation factors was ≥70%, while retention of immunoglobulins was 100%. Starting nAb titres were low, but PRNT50 titres did not differ between pre- and post-treatment samples. No statistically significant differences were detected in levels of IgG (P ≥ 0·3665) and IgM (P ≥ 0·1208) antibodies to RBD, S1 and S2 proteins before and after treatment. CONCLUSION: R + UV PRT effects on coagulation factors were similar to previous reports, but no significant effects were observed on immunoglobulin concentration and antibody function. SARS-CoV-2 nAb function in CCP is conserved following R + UV PRT treatment.

BCG-Prime and boost with Esx-5 secretion system deletion mutant leads to better protection against clinical strains of Mycobacterium tuberculosis.

Although vaccination with BCG prevents disseminated forms of childhood tuberculosis (TB), it does not protect against pulmonary infection or Mycobacterium tuberculosis (Mtb) transmission. In this study, we generated a complete deletion mutant of the Mtb Esx-5 type VII secretion system (Mtb Δesx-5). Mtb Δesx-5 was highly attenuated and safe in immunocompromised mice. When tested as a vaccine candidate to boost BCG-primed immunity, Mtb Δesx-5 improved protection against highly virulent Mtb strains in the murine and guinea pig models of TB. Enhanced protection provided by heterologous BCG-prime plus Mtb Δesx-5 boost regimen was associated with increased pulmonary influx of central memory T cells (TCM), follicular helper T cells (TFH) and activated monocytes. Conversely, lower numbers of T cells expressing exhaustion markers were observed in vaccinated animals. Our results suggest that boosting BCG-primed immunity with Mtb Δesx-5 is a potential approach to improve protective immunity against Mtb. Further insight into the mechanism of action of this novel prime-boost approach is warranted.

Cyto-Feature Engineering: A Pipeline for Flow Cytometry Analysis to Uncover Immune Populations and Associations with Disease.

Flow cytometers can now analyze up to 50 parameters per cell and millions of cells per sample; however, conventional methods to analyze data are subjective and time-consuming. To address these issues, we have developed a novel flow cytometry analysis pipeline to identify a plethora of cell populations efficiently. Coupled with feature engineering and immunological context, researchers can immediately extrapolate novel discoveries through easy-to-understand plots. The R-based pipeline uses Fluorescence Minus One (FMO) controls or distinct population differences to develop thresholds for positive/negative marker expression. The continuous data is transformed into binary data, capturing a positive/negative biological dichotomy often of interest in characterizing cells. Next, a filtering step refines the data from all identified cell phenotypes to populations of interest. The data can be partitioned by immune lineages and statistically correlated to other experimental measurements. The pipeline's modularity allows customization of statistical testing, adoption of alternative initial gating steps, and incorporation of other datasets. Validation of this pipeline through manual gating of two datasets (murine splenocytes and human whole blood) confirmed its accuracy in identifying even rare subsets. Lastly, this pipeline can be applied in all disciplines utilizing flow cytometry regardless of cytometer or panel design. The code is available at https://github.com/aef1004/cyto-feature_engineering.

Acquisition of High-Quality Spectral Flow Cytometry Data.

Flow cytometry allows the visualization of physical, functional, and/or biological properties of cells including antigens, cytokines, size, and complexity. With increasingly large flow cytometry panels able to analyze up to 50 parameters, there is a need to standardize flow cytometry protocols to achieve high-quality data that can be input into analysis algorithms. Without this clean data, algorithms may incorrectly categorize the cell populations present in the samples. In this protocol, we outline a comprehensive methodology to prepare samples for polychromatic flow cytometry. The use of multiple washing steps and rigorous controls creates high-quality data with good separation between cell populations. Experimental data acquired using this protocol can be analyzed via computational algorithms that perform end-to-end analysis. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Preparation of single-cell suspension for flow cytometry Support Protocol 1: Lung preparation Support Protocol 2: Counting cells on a flow cytometer Basic Protocol 2: Surface and intracellular flow cytometry staining Support Protocol 3: Single-color bead controls.

Enhanced antibody response to ovalbumin coupled to poly-dispersed acid functionalized single walled carbon nanotubes.

Activated mouse B cells as compared to the resting B cells are known to internalize substantially more acid-functionalized single walled carbon nanotubes (AF-SWCNTs). It was hypothesized that the antigens coupled to AF-SWCNTs would also be taken up more efficiently by B cells. Further the enhanced uptake of the antigen by B cells may facilitate antigen presentation by B cells resulting in a better antibody response. Aim of this study was to test this hypothesis. Ovalbumin was chemically coupled to AF-SWCNTs that yielded a coupled product that had 0.08% of all carbon atoms in AF-SWCNTs occupied by ovalbumin. Coupling of ovalbumin to AF-SWCNTs was confirmed by staining the product with anti-ovalbumin antibodies. B cells incubated with ovalbumin-AF-SWCNT internalized more ovalbumin than the B cells incubated with free ovalbumin. Groups of mice were immunized subcutaneously with (a) free ovalbumin, (b) free ovalbumin and AF-SWCNTs at two different subcutaneous sites respectively on mice, and (c) ovalbumin-AF-SWCNT coupled product. In each case a primary immunization was followed by three weekly booster doses. It was found that the anti-ovalbumin antibody response assessed by ELISA, was highest in the group where ovalbumin coupled to AF-SWCNTs was used for immunization (p < 0.001). Antibody response in ovalbumin-AF-SWCNT group was comparable to the group where ovalbumin was used for immunization using complete and incomplete Freund's adjuvant (primary and secondary immunizations respectively). We propose that AF-SWCNTs could be explored as an adjuvant to improve the antibody response especially in vaccine development.

Elevated internalization and cytotoxicity of polydispersed single-walled carbon nanotubes in activated B cells can be basis for preferential depletion of activated B cells in vivo.

Uptake of polydispersed acid-functionalized single-walled carbon nanotubes (AF-SWCNTs) in resting and LPS-activated B cells was studied using fluorescence-tagged AF-SWCNTs (FAF-SWCNTs). Activated B cells internalized substantially higher amounts of FAF-SWCNTs [76.5% AF-SWCNT+ B cells, mean fluorescence intensity (MFI) 720.6] as compared to the resting B cells [39.5% AF-SWCNT+ B cells, MFI 198.5]. B cells in S and G2/M phases were found to have significantly higher uptake of FAF-SWCNTs as compared to cells in G0/G1 phase. Confocal microscopy indicated that AF-SWCNTs were essentially localized on cell membrane in resting B cells, whereas in activated B cells, AF-SWCNTs were distributed throughout the cytoplasm. Targeting of AF-SWCNTs specifically to activated B cells in vivo was examined by first administering intravenously LPS-activated B cells tagged with fluorescence tracer (CFSE) in mice, followed by FAF-SWCNTs through the same route. It was found that FAF-SWCNTs were specifically taken up by CFSE+CD19+-activated B cells (95% FAF-SWCNT+ B cells, MFI 3725) as compared to CFSE- CD19+ resting B cells (31.1% FAF-SWCNT+ B cells, MFI 428). Administration (i.v.) of LPS resulted in a significant increase in the proportion of B cell in mouse spleen that was reduced by 68% by administering AF-SWCNTs. In control mice, the corresponding decrease in B cell proportion was 49%, which was significantly lower (p < 0.005) than the decline in LPS-treated mice. These results indicate that AF-SWCNTs may have the potential as an agent for depleting activated B cells in vivo.