Corrigendum: The Vacc-SeqQC project: Benchmarking RNA-Seq for clinical vaccine studies.

[This corrects the article DOI: 10.3389/fimmu.2022.1093242.].

The Vacc-SeqQC project: Benchmarking RNA-Seq for clinical vaccine studies.

Introduction: Over the last decade, the field of systems vaccinology has emerged, in which high throughput transcriptomics and other omics assays are used to probe changes of the innate and adaptive immune system in response to vaccination. The goal of this study was to benchmark key technical and analytical parameters of RNA sequencing (RNA-seq) in the context of a multi-site, double-blind randomized vaccine clinical trial. Methods: We collected longitudinal peripheral blood mononuclear cell (PBMC) samples from 10 subjects before and after vaccination with a live attenuated Francisella tularensis vaccine and performed RNA-Seq at two different sites using aliquots from the same sample to generate two replicate datasets (5 time points for 50 samples each). We evaluated the impact of (i) filtering lowly-expressed genes, (ii) using external RNA controls, (iii) fold change and false discovery rate (FDR) filtering, (iv) read length, and (v) sequencing depth on differential expressed genes (DEGs) concordance between replicate datasets. Using synthetic mRNA spike-ins, we developed a method for empirically establishing minimal read-count thresholds for maintaining fold change accuracy on a per-experiment basis. We defined a reference PBMC transcriptome by pooling sequence data and established the impact of sequencing depth and gene filtering on transcriptome representation. Lastly, we modeled statistical power to detect DEGs for a range of sample sizes, effect sizes, and sequencing depths. Results and Discussion: Our results showed that (i) filtering lowly-expressed genes is recommended to improve fold-change accuracy and inter-site agreement, if possible guided by mRNA spike-ins (ii) read length did not have a major impact on DEG detection, (iii) applying fold-change cutoffs for DEG detection reduced inter-set agreement and should be used with caution, if at all, (iv) reduction in sequencing depth had a minimal impact on statistical power but reduced the identifiable fraction of the PBMC transcriptome, (v) after sample size, effect size (i.e. the magnitude of fold change) was the most important driver of statistical power to detect DEG. The results from this study provide RNA sequencing benchmarks and guidelines for planning future similar vaccine studies.

Inhibition of the ubiquitin-proteasome system prevents vaccinia virus DNA replication and expression of intermediate and late genes.

The ubiquitin-proteasome system has a central role in the degradation of intracellular proteins and regulates a variety of functions. Viruses belonging to several different families utilize or modulate the system for their advantage. Here we showed that the proteasome inhibitors MG132 and epoxomicin blocked a postentry step in vaccinia virus (VACV) replication. When proteasome inhibitors were added after virus attachment, early gene expression was prolonged and the expression of intermediate and late genes was almost undetectable. By varying the time of the removal and addition of MG132, the adverse effect of the proteasome inhibitors was narrowly focused on events occurring 2 to 4 h after infection, the time of the onset of viral DNA synthesis. Further analyses confirmed that genome replication was inhibited by both MG132 and epoxomicin, which would account for the effect on intermediate and late gene expression. The virus-induced replication of a transfected plasmid was also inhibited, indicating that the block was not at the step of viral DNA uncoating. UBEI-41, an inhibitor of the ubiquitin-activating enzyme E1, also prevented late gene expression, supporting the role of the ubiquitin-proteasome system in VACV replication. Neither the overexpression of ubiquitin nor the addition of an autophagy inhibitor was able to counter the inhibitory effects of MG132. Further studies of the role of the ubiquitin-proteasome system for VACV replication may provide new insights into virus-host interactions and suggest potential antipoxviral drugs.

Transcriptomic and Metabolic Responses to a Live-Attenuated Francisella tularensis Vaccine.

The immune response to live-attenuated Francisella tularensis vaccine and its host evasion mechanisms are incompletely understood. Using RNA-Seq and LC-MS on samples collected pre-vaccination and at days 1, 2, 7, and 14 post-vaccination, we identified differentially expressed genes in PBMCs, metabolites in serum, enriched pathways, and metabolites that correlated with T cell and B cell responses, or gene expression modules. While an early activation of interferon α/ß signaling was observed, several innate immune signaling pathways including TLR, TNF, NF-κB, and NOD-like receptor signaling and key inflammatory cytokines such as Il-1α, Il-1ß, and TNF typically activated following infection were suppressed. The NF-κB pathway was the most impacted and the likely route of attack. Plasma cells, immunoglobulin, and B cell signatures were evident by day 7. MHC I antigen presentation was more actively up-regulated first followed by MHC II which coincided with the emergence of humoral immune signatures. Metabolomics analysis showed that glycolysis and TCA cycle-related metabolites were perturbed including a decline in pyruvate. Correlation networks that provide hypotheses on the interplay between changes in innate immune, T cell, and B cell gene expression signatures and metabolites are provided. Results demonstrate the utility of transcriptomics and metabolomics for better understanding molecular mechanisms of vaccine response and potential host-pathogen interactions.

Proteomic Analysis of Human Immune Responses to Live-Attenuated Tularemia Vaccine.

Francisella tularensis (F. tularensis) is an intracellular pathogen that causes a potentially debilitating febrile illness known as tularemia. F. tularensis can be spread by aerosol transmission and cause fatal pneumonic tularemia. If untreated, mortality rates can be as high as 30%. To study the host responses to a live-attenuated tularemia vaccine, peripheral blood mononuclear cell (PBMC) samples were assayed from 10 subjects collected pre- and post-vaccination, using both the 2D-DIGE/MALDI-MS/MS and LC-MS/MS approaches. Protein expression related to antigen processing and presentation, inflammation (PPARγ nuclear receptor), phagocytosis, and gram-negative bacterial infection was enriched at Day 7 and/or Day 14. Protein candidates that could be used to predict human immune responses were identified by evaluating the correlation between proteome changes and humoral and cellular immune responses. Consistent with the proteomics data, parallel transcriptomics data showed that MHC class I and class II-related signals important for protein processing and antigen presentation were up-regulated, further confirming the proteomic results. These findings provide new biological insights that can be built upon in future clinical studies, using live attenuated strains as immunogens, including their potential use as surrogates of protection.

Alterations in the Human Plasma Lipidome in Response to Tularemia Vaccination.

Tularemia is a highly infectious and contagious disease caused by the bacterium Francisella tularensis. To better understand human response to a live-attenuated tularemia vaccine and the biological pathways altered post-vaccination, healthy adults were vaccinated, and plasma was collected pre- and post-vaccination for longitudinal lipidomics studies. Using tandem mass spectrometry, we fully characterized individual lipid species within predominant lipid classes to identify changes in the plasma lipidome during the vaccine response. Separately, we targeted oxylipins, a subset of lipid mediators involved in inflammatory pathways. We identified 14 differentially abundant lipid species from eight lipid classes. These included 5-hydroxyeicosatetraenoic acid (5-HETE) which is indicative of lipoxygenase activity and, subsequently, inflammation. Results suggest that 5-HETE was metabolized to a dihydroxyeicosatrienoic acid (DHET) by day 7 post-vaccination, shedding light on the kinetics of the 5-HETE-mediated inflammatory response. In addition to 5-HETE and DHET, we observed pronounced changes in 34:1 phosphatidylinositol, anandamide, oleamide, ceramides, 16:1 cholesteryl ester, and other glycerophospholipids; several of these changes in abundance were correlated with serum cytokines and T cell activation. These data provide new insights into alterations in plasma lipidome post-tularemia vaccination, potentially identifying key mediators and pathways involved in vaccine response and efficacy.

Four superoxide dismutases contribute to Bacillus anthracis virulence and provide spores with redundant protection from oxidative stress.

The Bacillus anthracis genome encodes four superoxide dismutases (SODs), enzymes capable of detoxifying oxygen radicals. That two of these SODs, SOD15 and SODA1, are present in the outermost layers of the B. anthracis spore is indicated by previous proteomic analyses of the exosporium. Given the requirement that spores must survive interactions with reactive oxygen species generated by cells such as macrophages during infection, we hypothesized that SOD15 and SODA1 protect the spore from oxidative stress and contribute to the pathogenicity of B. anthracis. To test these theories, we constructed a double-knockout (Delta sod15 Delta sodA1) mutant of B. anthracis Sterne strain 34F2 and assessed its lethality in an A/J mouse intranasal infection model. The 50% lethal dose of the Delta sod15 Delta sodA1 strain was similar to that of the wild type (34F2), but surprisingly, measurable whole-spore SOD activity was greater than that in 34F2. A quadruple-knockout strain (Delta sod15 Delta sodA1 Delta sodC Delta sodA2) was then generated, and as anticipated, spore-associated SOD activity was diminished. Moreover, the quadruple-knockout strain, compared to the wild type, was attenuated more than 40-fold upon intranasal challenge of mice. Spore resistance to exogenously generated oxidative stress and to macrophage-mediated killing correlated with virulence in A/J mice. Allelic exchange that restored sod15 and sodA1 to their wild-type state restored wild-type characteristics. We conclude that SOD molecules within the spore afford B. anthracis protection against oxidative stress and enhance the pathogenicity of B. anthracis in the lung. We also surmise that the presence of four SOD alleles within the genome provides functional redundancy for this key enzyme.

Antimicrobial effects of interferon-inducible CXC chemokines against Bacillus anthracis spores and bacilli.

Based on previous studies showing that host chemokines exert antimicrobial activities against bacteria, we sought to determine whether the interferon-inducible Glu-Leu-Arg-negative CXC chemokines CXCL9, CXCL10, and CXCL11 exhibit antimicrobial activities against Bacillus anthracis. In vitro analysis demonstrated that all three CXC chemokines exerted direct antimicrobial effects against B. anthracis spores and bacilli including marked reductions in spore and bacillus viability as determined using a fluorometric assay of bacterial viability and CFU determinations. Electron microscopy studies revealed that CXCL10-treated spores failed to undergo germination as judged by an absence of cytological changes in spore structure that occur during the process of germination. Immunogold labeling of CXCL10-treated spores demonstrated that the chemokine was located internal to the exosporium in association primarily with the spore coat and its interface with the cortex. To begin examining the potential biological relevance of chemokine-mediated antimicrobial activity, we used a murine model of inhalational anthrax. Upon spore challenge, the lungs of C57BL/6 mice (resistant to inhalational B. anthracis infection) had significantly higher levels of CXCL9, CXCL10, and CXCL11 than did the lungs of A/J mice (highly susceptible to infection). Increased CXC chemokine levels were associated with significantly reduced levels of spore germination within the lungs as determined by in vivo imaging. Taken together, our data demonstrate a novel antimicrobial role for host chemokines against B. anthracis that provides unique insight into host defense against inhalational anthrax; these data also support the notion for an innovative approach in treating B. anthracis infection as well as infections caused by other spore-forming organisms.

Systems Vaccinology for a Live Attenuated Tularemia Vaccine Reveals Unique Transcriptional Signatures That Predict Humoral and Cellular Immune Responses.

Background: Tularemia is a potential biological weapon due to its high infectivity and ease of dissemination. This study aimed to characterize the innate and adaptive responses induced by two different lots of a live attenuated tularemia vaccine and compare them to other well-characterized viral vaccine immune responses. Methods: Microarray analyses were performed on human peripheral blood mononuclear cells (PBMCs) to determine changes in transcriptional activity that correlated with changes detected by cellular phenotyping, cytokine signaling, and serological assays. Transcriptional profiles after tularemia vaccination were compared with yellow fever [YF-17D], inactivated [TIV], and live attenuated [LAIV] influenza. Results: Tularemia vaccine lots produced strong innate immune responses by Day 2 after vaccination, with an increase in monocytes, NK cells, and cytokine signaling. T cell responses peaked at Day 14. Changes in gene expression, including upregulation of STAT1, GBP1, and IFIT2, predicted tularemia-specific antibody responses. Changes in CCL20 expression positively correlated with peak CD8+ T cell responses, but negatively correlated with peak CD4+ T cell activation. Tularemia vaccines elicited gene expression signatures similar to other replicating vaccines, inducing early upregulation of interferon-inducible genes. Conclusions: A systems vaccinology approach identified that tularemia vaccines induce a strong innate immune response early after vaccination, similar to the response seen after well-studied viral vaccines, and produce unique transcriptional signatures that are strongly correlated to the induction of T cell and antibody responses.

Detection of Bacillus anthracis spore germination in vivo by bioluminescence imaging.

We sought to visualize the site of Bacillus anthracis spore germination in vivo. For that purpose, we constructed a reporter plasmid with the lux operon under control of the spore small acid-soluble protein B (sspB) promoter. In B. subtilis, sspB-driven synthesis of luciferase during sporulation results in incorporation of the enzyme in spores. We observed that B. anthracis Sterne transformed with our sspBp::lux plasmid was only luminescent during germination. In contrast, Sterne transformed with a similarly constructed plasmid with lux expression under control of the protective antigen promoter displayed luminescence only during vegetative growth. We then infected A/J mice intranasally with spores that harbored the germination reporter. Mice were monitored for up to 14 days with the Xenogen In Vivo Imaging System. While luminescence only became evident in live animals at 18 h, dissection after sacrificing infected mice at earlier time points revealed luminescence in lung tissue at 30 min after intranasal infection. Microscopic histochemical and immunofluorescence studies on luminescent lung sections and imprints revealed that macrophages were the first cells in contact with the B. anthracis spores. By 6 h after infection, polymorphonuclear leukocytes with intracellular spores were evident in the alveolar spaces. After 24 h, few free spores were observed in the alveolar spaces; most of the spores detected by immunofluorescence were in the cytoplasm of interstitial macrophages. In contrast, mediastinal lymph nodes remained nonluminescent throughout the infection. We conclude that in this animal system, the primary site of B. anthracis spore germination is the lungs.

The Cynomolgus Macaque Natural History Model of Pneumonic Tularemia for Predicting Clinical Efficacy Under the Animal Rule.

Francisella tularensis is a highly infectious Gram-negative bacterium that is the etiologic agent of tularemia in animals and humans and a Tier 1 select agent. The natural incidence of pneumonic tularemia worldwide is very low; therefore, it is not feasible to conduct clinical efficacy testing of tularemia medical countermeasures (MCM) in human populations. Development and licensure of tularemia therapeutics and vaccines need to occur under the Food and Drug Administration's (FDA's) Animal Rule under which efficacy studies are conducted in well-characterized animal models that reflect the pathophysiology of human disease. The Tularemia Animal Model Qualification (AMQ) Working Group is seeking qualification of the cynomolgus macaque (Macaca fascicularis) model of pneumonic tularemia under Drug Development Tools Qualification Programs with the FDA based upon the results of studies described in this manuscript. Analysis of data on survival, average time to death, average time to fever onset, average interval between fever and death, and bacteremia; together with summaries of clinical signs, necropsy findings, and histopathology from the animals exposed to aerosolized F. tularensis Schu S4 in five natural history studies and one antibiotic efficacy study form the basis for the proposed cynomolgus macaque model. Results support the conclusion that signs of pneumonic tularemia in cynomolgus macaques exposed to 300-3,000 colony forming units (cfu) aerosolized F. tularensis Schu S4, under the conditions described herein, and human pneumonic tularemia cases are highly similar. Animal age, weight, and sex of animals challenged with 300-3,000 cfu Schu S4 did not impact fever onset in studies described herein. This study summarizes critical parameters and endpoints of a well-characterized cynomolgus macaque model of pneumonic tularemia and demonstrates this model is appropriate for qualification, and for testing efficacy of tularemia therapeutics under Animal Rule.

Anthrax vaccination strategies.

The biological attack conducted through the US postal system in 2001 broadened the threat posed by anthrax from one pertinent mainly to soldiers on the battlefield to one understood to exist throughout our society. The expansion of the threatened population placed greater emphasis on the reexamination of how we vaccinate against Bacillus anthracis. The currently-licensed Anthrax Vaccine, Adsorbed (AVA) and Anthrax Vaccine, Precipitated (AVP) are capable of generating a protective immune response but are hampered by shortcomings that make their widespread use undesirable or infeasible. Efforts to gain US Food and Drug Administration (FDA) approval for licensure of a second generation recombinant protective antigen (rPA)-based anthrax vaccine are ongoing. However, this vaccine's reliance on the generation of a humoral immune response against a single virulence factor has led a number of scientists to conclude that the vaccine is likely not the final solution to optimal anthrax vaccine design. Other vaccine approaches, which seek a more comprehensive immune response targeted at multiple components of the B. anthracis organism, are under active investigation. This review seeks to summarize work that has been done to build on the current PA-based vaccine methodology and to evaluate the search for future anthrax prophylaxis strategies.

Recombinant Bacillus anthracis spore proteins enhance protection of mice primed with suboptimal amounts of protective antigen.

Inactivated Bacillus anthracis spores given with protective antigen (PA) contribute to immunity against anthrax in several animal models. Antiserum raised against whole irradiated B. anthracis spores has been shown to have anti-germination and opsonic activities in vitro. Based on these observations, we hypothesized that surface-exposed spore proteins might serve as supplemental components of a PA-based anthrax vaccine. The protective anti-spore serum was tested for reactivity with recombinant forms of 30 proteins known, or believed to be, present within the B. anthracis exosporium. Eleven of those proteins were reactive with this antiserum, and, subsequently a subset of this group was used to generate rabbit polyclonal antibodies. These sera were evaluated for recognition of the immunogens on intact spores generated from Sterne strain, as well as from an isogenic mutant lacking the spore surface protein Bacillus collagen-like antigen (BclA). The data were consistent with the notion that the antigens in question were located beneath BclA on the basal surface of the exosporium. A/J mice immunized with either the here-to-for hypothetical protein p5303 or the structural protein BxpB, each in combination with subprotective levels of PA, showed enhanced protection against subcutaneous spore challenge. While neither anti-BxpB or anti-p5303 antibodies reduced the rate of spore germination in vitro, both caused increased uptake and lead to a higher rate of destruction by phagocytic cells. We conclude that by facilitating more efficient phagocytic clearance of spores, antibodies against individual exosporium components can contribute to protection against B. anthracis infection.

Bacillus anthracis exosporium protein BclA affects spore germination, interaction with extracellular matrix proteins, and hydrophobicity.

Bacillus collagen-like protein of anthracis (BclA) is the immunodominant glycoprotein on the exosporium of Bacillus anthracis spores. Here, we sought to assess the impact of BclA on spore germination in vitro and in vivo, surface charge, and interaction with host matrix proteins. For that purpose, we constructed a markerless bclA null mutant in B. anthracis Sterne strain 34F2. The growth and sporulation rates of the DeltabclA and parent strains were nearly indistinguishable, but germination of mutant spores occurred more rapidly than that of wild-type spores in vitro and was more complete by 60 min. Additionally, the mean time to death of A/J mice inoculated subcutaneously or intranasally with mutant spores was lower than that for the wild-type spores even though the 50% lethal doses of the two strains were similar. We speculated that these in vitro and in vivo differences between mutant and wild-type spores might reflect the ease of access of germinants to their receptors in the absence of BclA. We also compared the hydrophobic and adhesive properties of DeltabclA and wild-type spores. The DeltabclA spores were markedly less water repellent than wild-type spores, and, probably as a consequence, the extracellular matrix proteins laminin and fibronectin bound significantly better to mutant than to wild-type spores. These studies suggest that BclA acts as a shield to not only reduce the ease with which spores germinate but also change the surface properties of the spore, which, in turn, may impede the interaction of the spore with host matrix substances.

Recombinant exosporium protein BclA of Bacillus anthracis is effective as a booster for mice primed with suboptimal amounts of protective antigen.

Bacillus collagen-like protein of anthracis (BclA) is an immunodominant glycoprotein located on the exosporium of Bacillus anthracis. We hypothesized that antibodies to this spore surface antigen are largely responsible for the augmented immunity to anthrax that has been reported for animals vaccinated with inactivated spores and protective antigen (PA) compared to vaccination with PA alone. To test this theory, we first evaluated the capacity of recombinant, histidine-tagged, nonglycosylated BclA (rBclA) given with adjuvant to protect A/J mice against 10 times the 50% lethal dose of Sterne strain spores introduced subcutaneously. Although the animals elicited anti-rBclA antibodies and showed a slight but statistically significant prolongation in the mean time to death (MTD), none of the mice survived. Similarly, rabbit anti-rBclA immunoglobulin G (IgG) administered intraperitoneally to mice before spore inoculation increased the MTD statistically significantly but afforded protection to only 1 of 10 animals. However, all mice that received suboptimal amounts of recombinant PA and that then received rBclA 2 weeks later survived spore challenge. Additionally, anti-rBclA IgG, compared to anti-PA IgG, promoted a sevenfold-greater uptake of opsonized spores by mouse macrophages and markedly decreased intramacrophage spore germination. Since BclA has some sequence similarity to human collagen, we also tested the extent of binding of anti-rBclA antibodies to human collagen types I, III, and V and found no discernible cross-reactivity. Taken together, these results support the concept of rBclA as being a safe and effective boost for a PA-primed individual against anthrax and further suggest that such rBclA-enhanced protection occurs by the induction of spore-opsonizing and germination-inhibiting antibodies.