Novel Transcriptional and Translational Biomarkers of Tularemia Vaccine Efficacy in a Mouse Inhalation Model: Proof of Concept

Francisella tularensis subspecies tularensis (Ftt) is extremely virulent for humans when inhaled as a small particle aerosol (<5 µm). Inhalation of ≥20 viable bacteria is sufficient to initiate infection with a mortality rate ≥30%. Consequently, in the past, Ftt became a primary candidate for biological weapons development. To counter this threat, the USA developed a live vaccine strain (LVS), that showed efficacy in humans against inhalation of virulent Ftt. However, the breakthrough dose was fairly low, and protection waned with time. These weaknesses triggered extensive research for better vaccine candidates. Previously, we showed that deleting the clpB gene from virulent Ftt strain, SCHU S4, resulted in a mutant that was significantly less virulent than LVS for mice, yet better protected them from aerosol challenge with wild-type SCHU S4. To date, comprehensive searches for correlates of protection for SCHU S4 ΔclpB among molecules that are critical signatures of cell-mediated immunity, have yielded little reward. In this study we used transcriptomics analysis to expand the potential range of molecular correlates of protection induced by vaccination with SCHU S4 ΔclpB beyond the usual candidates. The results provide proof-of-concept that unusual host responses to vaccination can potentially serve as novel efficacy biomarkers for new tularemia vaccines.

Development, Phenotypic Characterization and Genomic Analysis of a Francisella tularensis Panel for Tularemia Vaccine Testing

Francisella tularensis is one of several biothreat agents for which a licensed vaccine is needed to protect against this pathogen. To aid in the development of a vaccine protective against pneumonic tularemia, we generated and characterized a panel of F. tularensis isolates that can be used as challenge strains to assess vaccine efficacy. Our panel consists of both historical and contemporary isolates derived from clinical and environmental sources, including human, tick, and rabbit isolates. Whole genome sequencing was performed to assess the genetic diversity in comparison to the reference genome F. tularensis Schu S4. Average nucleotide identity analysis showed &gt;99% genomic similarity across the strains in our panel, and pan-genome analysis revealed a core genome of 1,707 genes, and an accessory genome of 233 genes. Three of the strains in our panel, FRAN254 (tick-derived), FRAN255 (a type B strain), and FRAN256 (a human isolate) exhibited variation from the other strains. Moreover, we identified several unique mutations within the Francisella Pathogenicity Island across multiple strains in our panel, revealing unexpected diversity in this region. Notably, FRAN031 (Scherm) completely lacked the second pathogenicity island but retained virulence in mice. In contrast, FRAN037 (Coll) was attenuated in a murine pneumonic tularemia model and had mutations in pdpB and iglA which likely led to attenuation. All of the strains, except FRAN037, retained full virulence, indicating their effectiveness as challenge strains for future vaccine testing. Overall, we provide a well-characterized panel of virulent F. tularensis strains that can be utilized in ongoing efforts to develop an effective vaccine against pneumonic tularemia to ensure protection is achieved across a range F. tularensis strains.

Replicating bacterium-vectored vaccine expressing SARS-CoV-2 Membrane and Nucleocapsid proteins protects against severe COVID-19 disease in hamsters

An inexpensive readily manufactured COVID-19 vaccine that protects against severe disease is needed to combat the pandemic. We have employed the LVS ΔcapB vector platform, previously used successfully to generate potent vaccines against the Select Agents of tularemia, anthrax, plague, and melioidosis, to generate a COVID-19 vaccine. The LVS ΔcapB vector, a replicating intracellular bacterium, is a highly attenuated derivative of a tularemia vaccine (LVS) previously administered to millions of people. We generated vaccines expressing SARS-CoV-2 structural proteins and evaluated them for efficacy in the golden Syrian hamster, which develops severe COVID-19 disease. Hamsters immunized intradermally or intranasally with a vaccine co-expressing the Membrane (M) and Nucleocapsid (N) proteins, then challenged 5-weeks later with a high dose of SARS-CoV-2, were protected against severe weight loss and lung pathology and had reduced viral loads in the oropharynx and lungs. Protection by the vaccine, which induces murine N-specific interferon-gamma secreting T cells, was highly correlated with pre-challenge serum anti-N TH1-biased IgG. This potent vaccine against severe COVID-19 should be safe and easily manufactured, stored, and distributed, and given the high homology between MN proteins of SARS-CoV and SARS-CoV-2, has potential as a universal vaccine against the SARS subset of pandemic causing β-coronaviruses.

Electro-Optical Analysis of Cell Viability of Tularemia Microbe Vaccine Strain

Objective: to study the possibility of applying electro-optical analysis for the assessment of cell viability of tularemia microbe vaccine strain at different stages of experimental live tularemia vaccine production.Materials and methods. The research object was a cell culture of Francisella tularensis 15 NIIEG. Investigations were carried out at all stages of experimental live tularemia vaccine (ELTV) manufacturing according to an advanced technology: cultivation, concentrating, diafiltration, mixing with drying media, stabilization, and storage (two-year period of observation). Electro-optical analysis by the parameter “polarizability anisotropy” of bacterial cell was conducted with the help of EloTrace (EloSystems, Germany). Total concentration of cells was evaluated using density metering at 590 nm and spectrometry – at 650 nm. Viability was assessed through inoculation of plates with FT-agar.Results and discussion. The experiment has demonstrated that the change in polarizability anisotropy of the cell at the frequencies of 900 kHz and 2100 kHz, reflecting the state of cytoplasm and cytoplasmic membrane, respectively, is the earliest response to changes in vital indicators of bacterial culture in the process of cultivation. Thereby, the decrease in viability of F. tularensis cells occurrs well before the decrease in cell concentration. We have shown the preservation of viability of F. tularensis 15 NIIEG cells at all stages of experimental live tularemia vaccine production. Electro-optical analysis allows for registering the changes in vital parameters of microorganism cells in real-time mode, while the assessment of viability applying bacteriological method takes up to 5 days. Different stages of tularemia vaccine manufacturing have impact on the vital indicators of F. tularensis cells, and electro-optical analysis is a prospect method of control of such parameter as “Specific activity (the number of live microbial cells)”.

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.

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 rare but highly contagious and potentially fatal disease caused by bacteria Francisella tularensis where as few as ten inhaled organisms can lead to an infection, making it one of the most infectious microorganisms known and a potential bioweapon. To better understand the response to a live, attenuated tularemia vaccine and the biological pathways altered post-vaccination, healthy adults were vaccinated by scarification and plasma was collected pre- and post-vaccination for longitudinal lipidomics studies. Using tandem mass spectrometry, we identified and quantified individual lipid molecular species within representative lipid classes in plasma to characterize alterations 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), an eicosanoid produced following arachidonic acid liberation and epoxygenation, 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 several 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.

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.

Features of humoral answer in experimental animal tularemia with different sensitivity to infection

Tularemia is an anthropozoonotic infection caused by Francisella tularensis. In clinical and sanitary-epidemiological practice, traditional diagnostics methods in tularemia are based on serological assays for detecting specific antibodies, allowing to diagnose it and estimate durability of patients’ immunity after vaccination. Previously, it was shown that specific serum antibodies in patients recovered after tularemia, unlike to those vaccinated with the live tularemia vaccine F. tularensis 15 NIIEG, can interact with specific epitopes on lipopolysaccharides isolated from strains of various subspecies — F. tularensis (Ft) and F. novicida (LPS Fn), while LPS Fn-specific immunoglobulins are lacked in the blood of vaccinated individuals. A set of experiments on identifying antibodies with similar specificity in laboratory animals of various species — mice, guinea pigs and rats with differed sensitivity to tularemia, administered with live tularemia vaccine strain as well as virulent F. tularensis strains to simulate vaccine-mediated and infectious processes, respectively was conducted. A methodical approach has been developed that allows to analyze humoral response in modelled infectious process in animals highly sensitive to tularemia such as BALB/c mice and guinea pigs that consisted of preliminary immunization with live tularemia vaccine followed by infection with virulent F. tularensis strains. It was shown that induction of specific anti-LPS Ft antibodies occured in these animal species, both after vaccination and infection with virulent strains. It was noted that, unlike guinea pigs and rats, mice both during vaccination and infection were characterized by significantly lower titers of LPS Ft-specific antibodies. However, no specific interaction between mouse serum and LPS Fn might be detected. Moreover, two types of immunoglobulins with different antigen specificities to the LPS Ft and LPS Fn epitopes were detected by dot-blot analysis in guinea pigs immunized with live tularemia vaccine, followed by infection with a virulent strain. In addition, antibodies to LPS Fn were also detected in the serum of rats infected with virulent, but not vaccine-based, F. tularensis strains. Thus, previous experimental data on the production of immunoglobulins with different antigenic specificity were confirmed in an experimental tularemia modelled in rats and guinea pigs that demonstrated a diagnostic significance and feasibility of using LPS Fn to confirm tularemia infection in humans.