Looking in the Rearview Mirror and to the Road Ahead on Dementia & Living Well

The number of Americans living with Alzheimer's and all other dementias continues to increase. Most of them will need long-term and community-based services as the disease progresses. While medical research is making advances, there is more work to be done to ensure that every person receives care that is person-centered and allows them to live with dignity and respect.

Tachykinins and the potential causal factors for post-COVID-19 condition.

The most prevalent symptoms of post-COVID-19 condition are pulmonary dysfunction, fatigue and muscle weakness, anxiety, anosmia, dysgeusia, headaches, difficulty in concentrating, sexual dysfunction, and digestive disturbances. Hence, neurological dysfunction and autonomic impairments predominate in post-COVID-19 condition. Tachykinins including the most studied substance P are neuropeptides expressed throughout the nervous and immune systems, and contribute to many physiopathological processes in the nervous, immune, gastrointestinal, respiratory, urogenital, and dermal systems and participate in inflammation, nociception, and cell proliferation. Substance P is a key molecule in neuroimmune crosstalk; immune cells near the peripheral nerve endings can send signals to the brain with cytokines, which highlights the important role of tachykinins in neuroimmune communication. We reviewed the evidence that relates the symptoms of post-COVID-19 condition to the functions of tachykinins and propose a putative pathogenic mechanism. The antagonism of tachykinins receptors can be a potential treatment target.

Plasma proteome of Long-COVID patients indicates HIF-mediated vasculo-proliferative disease with impact on brain and heart function.

AIMS: Long-COVID occurs after SARS-CoV-2 infection and results in diverse, prolonged symptoms. The present study aimed to unveil potential mechanisms, and to inform prognosis and treatment. METHODS: Plasma proteome from Long-COVID outpatients was analyzed in comparison to matched acutely ill COVID-19 (mild and severe) inpatients and healthy control subjects. The expression of 3072 protein biomarkers was determined with proximity extension assays and then deconvoluted with multiple bioinformatics tools into both cell types and signaling mechanisms, as well as organ specificity. RESULTS: Compared to age- and sex-matched acutely ill COVID-19 inpatients and healthy control subjects, Long-COVID outpatients showed natural killer cell redistribution with a dominant resting phenotype, as opposed to active, and neutrophils that formed extracellular traps. This potential resetting of cell phenotypes was reflected in prospective vascular events mediated by both angiopoietin-1 (ANGPT1) and vascular-endothelial growth factor-A (VEGFA). Several markers (ANGPT1, VEGFA, CCR7, CD56, citrullinated histone 3, elastase) were validated by serological methods in additional patient cohorts. Signaling of transforming growth factor-ß1 with probable connections to elevated EP/p300 suggested vascular inflammation and tumor necrosis factor-α driven pathways. In addition, a vascular proliferative state associated with hypoxia inducible factor 1 pathway suggested progression from acute COVID-19 to Long-COVID. The vasculo-proliferative process predicted in Long-COVID might contribute to changes in the organ-specific proteome reflective of neurologic and cardiometabolic dysfunction. CONCLUSIONS: Taken together, our findings point to a vasculo-proliferative process in Long-COVID that is likely initiated either prior hypoxia (localized or systemic) and/or stimulatory factors (i.e., cytokines, chemokines, growth factors, angiotensin, etc). Analyses of the plasma proteome, used as a surrogate for cellular signaling, unveiled potential organ-specific prognostic biomarkers and therapeutic targets.

Immune resilience despite inflammatory stress promotes longevity and favorable health outcomes including resistance to infection.

Some people remain healthier throughout life than others but the underlying reasons are poorly understood. Here we hypothesize this advantage is attributable in part to optimal immune resilience (IR), defined as the capacity to preserve and/or rapidly restore immune functions that promote disease resistance (immunocompetence) and control inflammation in infectious diseases as well as other causes of inflammatory stress. We gauge IR levels with two distinct peripheral blood metrics that quantify the balance between (i) CD8+ and CD4+ T-cell levels and (ii) gene expression signatures tracking longevity-associated immunocompetence and mortality-associated inflammation. Profiles of IR metrics in ~48,500 individuals collectively indicate that some persons resist degradation of IR both during aging and when challenged with varied inflammatory stressors. With this resistance, preservation of optimal IR tracked (i) a lower risk of HIV acquisition, AIDS development, symptomatic influenza infection, and recurrent skin cancer; (ii) survival during COVID-19 and sepsis; and (iii) longevity. IR degradation is potentially reversible by decreasing inflammatory stress. Overall, we show that optimal IR is a trait observed across the age spectrum, more common in females, and aligned with a specific immunocompetence-inflammation balance linked to favorable immunity-dependent health outcomes. IR metrics and mechanisms have utility both as biomarkers for measuring immune health and for improving health outcomes.

Nursing

Nurses are advocates for their patients, and nurse directors are advocates for both patients and their nursing staff, as well as the eyes and ears of senior leadership. During the COVID-19 pandemic, the nursing staff at SBH Health System were fearful and anxious;CDC guidelines were changing frequently. Following the frequently changing instructions on the use and conservation of PPE and isolation precautions was not easy. The presence of nurse directors gave the nursing staff support and education. During this crisis, three staggered daily shifts were created in order to cover, 7 days a week. Along with immediately reducing nursing staff anxiety, nursing staff felt greater support and encouragement from management and leaders, especially on the evening and night shifts. The nursing education department was an indispensable resource at the core of retraining nursing staff from different departments and training nursing staff in new procedures and equipment. The nurse director is one of the most important leadership positions in any hospital, but the heroes of this COVID-19 pandemic are all hospital workers. © SBH Health System 2022.

Is France Once Again Looking for a Scapegoat?

On September 10, 2021, a special tribunal established by the French government launched an inquiry into the activities of former health minister Dr. Agnes Buzyn who was charged with "endangering the lives of others". It is surprising to learn of this accusation and inquiry into the actions of a public health official whose response to the epidemic was, to all appearances, exemplary.

Organ and cell-specific biomarkers of Long-COVID identified with targeted proteomics and machine learning.

BACKGROUND: Survivors of acute COVID-19 often suffer prolonged, diffuse symptoms post-infection, referred to as "Long-COVID". A lack of Long-COVID biomarkers and pathophysiological mechanisms limits effective diagnosis, treatment and disease surveillance. We performed targeted proteomics and machine learning analyses to identify novel blood biomarkers of Long-COVID. METHODS: A case-control study comparing the expression of 2925 unique blood proteins in Long-COVID outpatients versus COVID-19 inpatients and healthy control subjects. Targeted proteomics was accomplished with proximity extension assays, and machine learning was used to identify the most important proteins for identifying Long-COVID patients. Organ system and cell type expression patterns were identified with Natural Language Processing (NLP) of the UniProt Knowledgebase. RESULTS: Machine learning analysis identified 119 relevant proteins for differentiating Long-COVID outpatients (Bonferonni corrected P < 0.01). Protein combinations were narrowed down to two optimal models, with nine and five proteins each, and with both having excellent sensitivity and specificity for Long-COVID status (AUC = 1.00, F1 = 1.00). NLP expression analysis highlighted the diffuse organ system involvement in Long-COVID, as well as the involved cell types, including leukocytes and platelets, as key components associated with Long-COVID. CONCLUSIONS: Proteomic analysis of plasma from Long-COVID patients identified 119 highly relevant proteins and two optimal models with nine and five proteins, respectively. The identified proteins reflected widespread organ and cell type expression. Optimal protein models, as well as individual proteins, hold the potential for accurate diagnosis of Long-COVID and targeted therapeutics.

In silico analyses of diversity and dissemination of antimicrobial resistance genes and mobile genetics elements, for plasmids of enteric pathogens.

Introduction: The antimicrobial resistance (AMR) mobilome plays a key role in the dissemination of resistance genes encoded by mobile genetics elements (MGEs) including plasmids, transposons (Tns), and insertion sequences (ISs). These MGEs contribute to the dissemination of multidrug resistance (MDR) in enteric bacterial pathogens which have been considered as a global public health risk. Methods: To further understand the diversity and distribution of AMR genes and MGEs across different plasmid types, we utilized multiple sequence-based computational approaches to evaluate AMR-associated plasmid genetics. A collection of 1,309 complete plasmid sequences from Gammaproteobacterial species, including 100 plasmids from each of the following 14 incompatibility (Inc) types: A/C, BO, FIA, FIB, FIC, FIIA, HI1, HI2, I1, K, M, N, P except W, where only 9 sequences were available, was extracted from the National Center for Biotechnology Information (NCBI) GenBank database using BLAST tools. The extracted FASTA files were analyzed using the AMRFinderPlus web-based tools to detect antimicrobial, disinfectant, biocide, and heavy metal resistance genes and ISFinder to identify IS/Tn MGEs within the plasmid sequences. Results and Discussion: In silico prediction based on plasmid replicon types showed that the resistance genes were diverse among plasmids, yet multiple genes were widely distributed across the plasmids from enteric bacterial species. These findings provide insights into the diversity of resistance genes and that MGEs mediate potential transmission of these genes across multiple plasmid replicon types. This notion was supported by the observation that many IS/Tn MGEs and resistance genes known to be associated with them were common across multiple different plasmid types. Our results provide critical insights about how the diverse population of resistance genes that are carried by the different plasmid types can allow for the dissemination of AMR across enteric bacteria. The results also highlight the value of computational-based approaches and in silico analyses for the assessment of AMR and MGEs, which are important elements of molecular epidemiology and public health outcomes.

COVID-19 plasma proteome reveals novel temporal and cell-specific signatures for disease severity and high-precision disease management.

Coronavirus disease 2019 (COVID-19) is a systemic inflammatory condition with high mortality that may benefit from personalized medicine and high-precision approaches. COVID-19 patient plasma was analysed with targeted proteomics of 1161 proteins. Patients were monitored from Days 1 to 10 of their intensive care unit (ICU) stay. Age- and gender-matched COVID-19-negative sepsis ICU patients and healthy subjects were examined as controls. Proteomic data were resolved using both cell-specific annotation and deep-analysis for functional enrichment. COVID-19 caused extensive remodelling of the plasma microenvironment associated with a relative immunosuppressive milieu between ICU Days 3-7, and characterized by extensive organ damage. COVID-19 resulted in (1) reduced antigen presentation and B/T-cell function, (2) increased repurposed neutrophils and M1-type macrophages, (3) relatively immature or disrupted endothelia and fibroblasts with a defined secretome, and (4) reactive myeloid lines. Extracellular matrix changes identified in COVID-19 plasma could represent impaired immune cell homing and programmed cell death. The major functional modules disrupted in COVID-19 were exaggerated in patients with fatal outcome. Taken together, these findings provide systems-level insight into the mechanisms of COVID-19 inflammation and identify potential prognostic biomarkers. Therapeutic strategies could be tailored to the immune response of severely ill patients.

Food Insecurity Trajectories in the US During the First Year of the COVID-19 Pandemic.

INTRODUCTION: The objective of this study was to characterize population-level trajectories in the probability of food insecurity in the US during the first year of the COVID-19 pandemic and to examine sociodemographic correlates associated with identified trajectories. METHODS: We analyzed data from the Understanding America Study survey, a nationally representative panel (N = 7,944) that assessed food insecurity every 2 weeks from April 1, 2020, through March 16, 2021. We used latent class growth analysis to determine patterns (or classes) of pandemic-related food insecurity during a 1-year period. RESULTS: We found 10 classes of trajectories of food insecurity, including 1 class of consistent food security (64.7%), 1 class of consistent food insecurity (3.4%), 5 classes of decreasing food insecurity (15.8%), 2 classes of increasing food insecurity (4.6%), and 1 class of stable but elevated food insecurity (11.6%). Relative to the class that remained food secure, other classes were younger, had a greater proportion of women, and tended to identify with a racial or ethnic minority group. CONCLUSION: We found heterogeneous longitudinal patterns in the development, resolution, or persistence of food insecurity during the first year of the COVID-19 pandemic. Experiences of food insecurity were highly variable across the US population, with one-third experiencing some form of food insecurity risk. Findings have implications for identifying population groups who are at increased risk of food insecurity and related health disparities beyond the first year of the pandemic.

COVID-19 in Assisted Living: Protecting a Critical Long-Term Care Resource.

The COVID-19 pandemic had a big impact on assisted living (AL), a vital setting in long-term care (LTC). Understanding the strengths and opportunities for improvement through practice, policy, and research are essential for AL to be prepared for the next pandemic and other challenges. AL communities experienced the pandemic in unique ways, because of varying regulatory environments, differences in familiarity with using and procuring personal protective equipment not typically used in AL (such as N95 masks), loss of family involvement, the homelike environment, and lower levels of licensed clinical staff. Being state rather than federally regulated, much less national data are available about the COVID-19 experience in AL. This article reviews what is known about cases and deaths, infection control, and the impact on residents and staff. For each, we suggest actions that could be taken and link them to the Assisted Living Workgroup Report (ALW) recommendations. Using the Center for Excellence in Assisted Living (CEAL) 15-year ALW report, we also review which of these recommendations have and have not been implemented by states in the preceding decade and half, and how their presence or absence may have affected AL pandemic preparedness. Finally, we provide suggestions for policy, practice, and research moving forward, including improving state-level reporting, staff vaccine requirements, staff training and work-life, levels of research-provider partnerships, dissemination of research, and uptake of a holistic model of care for AL.

Cross-immunity against SARS-COV-2 variants of concern in naturally infected critically ill COVID-19 patients.

Critically ill patients infected with SARS-CoV-2 display adaptive immunity, but it is unknown if they develop cross-reactivity to variants of concern (VOCs). We profiled cross-immunity against SARS-CoV-2 VOCs in naturally infected, non-vaccinated, critically ill COVID-19 patients. Wave-1 patients (wild-type infection) were similar in demographics to Wave-3 patients (wild-type/alpha infection), but Wave-3 patients had higher illness severity. Wave-1 patients developed increasing neutralizing antibodies to all variants, as did patients during Wave-3. Wave-3 patients, when compared to Wave-1, developed more robust antibody responses, particularly for wild-type, alpha, beta and delta variants. Within Wave-3, neutralizing antibodies were significantly less to beta and gamma VOCs, as compared to wild-type, alpha and delta. Patients previously diagnosed with cancer or chronic obstructive pulmonary disease had significantly fewer neutralizing antibodies. Naturally infected ICU patients developed adaptive responses to all VOCs, with greater responses in those patients more likely to be infected with the alpha variant, versus wild-type.

Plasma Proteome of Long-covid Patients Indicates Hypoxia-mediated Vasculo-proliferative Disease With Impact on Brain and Heart Function (preprint)

Aims Long-COVID occurs after SARS-CoV-2 infection and results in diverse, prolonged symptoms. The present study aims to determine the underlying mechanisms, and to inform prognosis and treatment.Methods Plasma proteome from Long-COVID outpatients was analyzed in comparison to acutely ill COVID-19 (mild and severe) inpatients and healthy control subjects. The expression of approximately 3000 protein biomarkers was determined with proximity extension assays and then deconvoluted with multiple bioinformatics tools into both cell types and signaling mechanisms, as well as organ specificity.Results Compared to age- and sex-matched acutely ill COVID-19 inpatients and healthy control subjects, Long-COVID outpatients showed natural killer cells with a resting phenotype, as opposed to active, and neutrophils that formed extracellular traps. This resetting of cell phenotypes was reflected in vascular events mediated by both angiopoietin-1 (ANGPT1) and vascular-endothelial growth factor-A (VEGFA). Levels of ANGPT1 and VEGFA were validated by serological methods in different patient cohorts. Silent signaling of transforming growth factor-β1 with elevated EP300 favored not only vascular inflammation, but also tumor necrosis factor-α driven pathways. In addition, a vascular proliferative state associated with hypoxia inducible factor 1 pathway was predicted that progressed from COVID-19 to Long-COVID. The vasculo-proliferative process identified in Long-COVID was associated with significant changes in the organ-specific proteome reflective of neurological and cardiometabolic dysfunction.Conclusions Taken together, our study uncovered a vasculo-proliferative process in Long-COVID initiated by prior hypoxia, and identified potential organ-specific prognostic biomarkers and therapeutic targets.

Initial evaluation of extracorporeal immunomodulatory therapy for the treatment of critically ill COVID-19 infected patients.

Severe COVID-19 infection results in significant immune dysregulation resulting from excessive recruitment and activation of neutrophils. The aim of this study was to confirm feasibility, initial safety and detect signal of efficacy of a non-propriety device delivered using an intermittent extra-corporeal system (LMOD) allowing leucocytes modulation in the setting of Severe COVID-19 infection. Twelve patients were recruited. Inclusion criteria were > 18 years age, confirmed COVID-19, acute respiratory distress syndrome requiring mechanical support and hypotension requiring vasopressor support. Primary end point was vasopressor requirements (expressed as epinephrine dose equivalents) and principle secondary endpoints related to safety, ability to deliver the therapy and markers of inflammation assessed over five days after treatment initiation. LMOD treatment appeared safe, defined by hemodynamic stability and no evidence of white cell number depletion from blood. We demonstrated a significant decrease in vasopressor doses (-37%, p = 0.02) in patients receiving LMOD therapy (despite these patients having to tolerate an additional extracorporeal intermittent therapy). Vasopressor requirements unchanged/increasing in control group (+ 10%, p = 0.48). Although much about the use of this therapy in the setting of severe COVID-19 infection remains to be defined (e.g. optimal dose and duration), this preliminary study supports the further evaluation of this novel extracorporeal approach.

Endothelial Glycocalyx Degradation in Critical Illness and Injury.

The endothelial glycocalyx is a gel-like layer on the luminal side of blood vessels that is composed of glycosaminoglycans and the proteins that tether them to the plasma membrane. Interest in its properties and function has grown, particularly in the last decade, as its importance to endothelial barrier function has come to light. Endothelial glycocalyx studies have revealed that many critical illnesses result in its degradation or removal, contributing to endothelial dysfunction and barrier break-down. Loss of the endothelial glycocalyx facilitates the direct access of immune cells and deleterious agents (e.g., proteases and reactive oxygen species) to the endothelium, that can then further endothelial cell injury and dysfunction leading to complications such as edema, and thrombosis. Here, we briefly describe the endothelial glycocalyx and the primary components thought to be directly responsible for its degradation. We review recent literature relevant to glycocalyx damage in several critical illnesses (sepsis, COVID-19, trauma and diabetes) that share inflammation as a common denominator with actions by several common agents (hyaluronidases, proteases, reactive oxygen species, etc.). Finally, we briefly cover strategies and therapies that show promise in protecting or helping to rebuild the endothelial glycocalyx such as steroids, protease inhibitors, anticoagulants and resuscitation strategies.

Elevated vascular transformation blood biomarkers in Long-COVID indicate angiogenesis as a key pathophysiological mechanism.

BACKGROUND: Long-COVID is characterized by prolonged, diffuse symptoms months after acute COVID-19. Accurate diagnosis and targeted therapies for Long-COVID are lacking. We investigated vascular transformation biomarkers in Long-COVID patients. METHODS: A case-control study utilizing Long-COVID patients, one to six months (median 98.5 days) post-infection, with multiplex immunoassay measurement of sixteen blood biomarkers of vascular transformation, including ANG-1, P-SEL, MMP-1, VE-Cad, Syn-1, Endoglin, PECAM-1, VEGF-A, ICAM-1, VLA-4, E-SEL, thrombomodulin, VEGF-R2, VEGF-R3, VCAM-1 and VEGF-D. RESULTS: Fourteen vasculature transformation blood biomarkers were significantly elevated in Long-COVID outpatients, versus acutely ill COVID-19 inpatients and healthy controls subjects (P < 0.05). A unique two biomarker profile consisting of ANG-1/P-SEL was developed with machine learning, providing a classification accuracy for Long-COVID status of 96%. Individually, ANG-1 and P-SEL had excellent sensitivity and specificity for Long-COVID status (AUC = 1.00, P < 0.0001; validated in a secondary cohort). Specific to Long-COVID, ANG-1 levels were associated with female sex and a lack of disease interventions at follow-up (P < 0.05). CONCLUSIONS: Long-COVID patients suffer prolonged, diffuse symptoms and poorer health. Vascular transformation blood biomarkers were significantly elevated in Long-COVID, with angiogenesis markers (ANG-1/P-SEL) providing classification accuracy of 96%. Vascular transformation blood biomarkers hold potential for diagnostics, and modulators of angiogenesis may have therapeutic efficacy.

Cohort-Specific Serological Recognition of SARS-CoV-2 Variant RBD Antigens.

OBJECTIVE: Estimating the response of different population cohorts to new SARS-CoV-2 variants is important to customize measures of control. Our goal was to evaluate how antibodies from sera of infected and vaccinated people recognize antigens expressed by different SARS-CoV-2 variants. METHODS: We compared sera from vaccinated donors and four patient/donor cohorts: Sera from critically ill patients collected 2-7 days and more than 10 days after admission to an intensive care unit, a NIBSC/WHO reference panel of SARS-CoV-2 positive individuals, and ambulatory or hospitalized (but not critically ill) positive donors. Samples were tested with an anti-SARS-CoV-2 ELISA kit coated with SARS-CoV-2 RBD recombinant antigens including mutations present in eleven of the most widespread variants. RESULTS: Sera from vaccinated individuals exhibited higher antibody binding (P<0.001) than sera from infected (but not critically ill) individuals when tested against the wild type (WT) and each of 11 variants' receptor binding domain (RBD). Antibodies' binding to the SARS-CoV-2 antigens of at least 6 variants, including Variants of Concern (VOCs), was reduced in comparison to the WT in vaccinated and non-critically ill convalescence individuals. CONCLUSION: Understanding differences between population cohorts in the antibody titers against WT vs variant RBD antigens can help design variant-specific immunoassays for surveillance and evaluation of the epidemiology of new variants.

Community Health Resources, Globalization, Trust in Science, and Voting as Predictors of COVID-19 Vaccination Rates: A Global Study with Implications for Vaccine Adherence.

The COVID-19 global pandemic requires, not only an adequate supply of, but public adherence to safe and effective vaccinations. This study analyzes the human and economic resources and political and public attitudinal factors that influence widely varying country-level coronavirus vaccination rates. Using data on up to 95 countries, we found that countries' strength of community health training and research (CHTR), education index, globalization, and vaccine supply are associated with a greater COVID-19 vaccination rate. In a separate analysis, certain political factors, and public attitudes (perceived government effectiveness, government fiscal decentralization, trust in science, and parliamentary voter turnout) predicted vaccination rates. Perceived corruption and actual freedoms (political rights and civil liberties) related to vaccination rates in prior studies were not significantly predictive when controlling for the above factors. The results confirm our prior findings on the importance of CHTR resources for increasing COVID-19 vaccination rates. They also suggest that to motivate vaccine adherence countries need, not only an adequate vaccine supply (which depends on a country having either its own resources or effective global political, social, and economic connections) and community health workforce training and research, but also a population that trusts in science, and is actively engaged in the political process.

Preclinical immunogenicity and efficacy of a candidate COVID-19 vaccine based on a vesicular stomatitis virus-SARS-CoV-2 chimera.

BACKGROUND: To investigate a vaccine technology with potential to protect against coronavirus disease 2019 (COVID-19) and reduce transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with a single vaccine dose, we developed a SARS-CoV-2 candidate vaccine using the live vesicular stomatitis virus (VSV) chimeric virus approach previously used to develop a licensed Ebola virus vaccine. METHODS: We generated a replication-competent chimeric VSV-SARS-CoV-2 vaccine candidate by replacing the VSV glycoprotein (G) gene with coding sequence for the SARS-CoV-2 Spike glycoprotein (S). Immunogenicity of the lead vaccine candidate (VSV∆G-SARS-CoV-2) was evaluated in cotton rats and golden Syrian hamsters, and protection from SARS-CoV-2 infection also was assessed in hamsters. FINDINGS: VSV∆G-SARS-CoV-2 delivered with a single intramuscular (IM) injection was immunogenic in cotton rats and hamsters and protected hamsters from weight loss following SARS-CoV-2 challenge. When mucosal vaccination was evaluated, cotton rats did not respond to the vaccine, whereas mucosal administration of VSV∆G-SARS-CoV-2 was found to be more immunogenic than IM injection in hamsters and induced immunity that significantly reduced SARS-CoV-2 challenge virus loads in both lung and nasal tissues. INTERPRETATION: VSV∆G-SARS-CoV-2 delivered by IM injection or mucosal administration was immunogenic in golden Syrian hamsters, and both vaccination methods effectively protected the lung from SARS-CoV-2 infection. Hamsters vaccinated by mucosal application of VSV∆G-SARS-CoV-2 also developed immunity that controlled SARS-CoV-2 replication in nasal tissue. FUNDING: The study was funded by Merck Sharp & Dohme, Corp., a subsidiary of Merck & Co., Inc., Rahway, NJ, USA, and The International AIDS Vaccine Initiative, Inc. (IAVI), New York, USA. Parts of this research was supported by the Biomedical Advanced Research and Development Authority (BARDA) and the Defense Threat Reduction Agency (DTRA) of the US Department of Defense.