Francisella Tularensis Vaccine Research Articles

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/β 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-1β, 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.

Evaluation of an outbred mouse model for Francisella tularensis vaccine development and testing.

Francisella tularensis (Ft) is a biothreat agent for which there is no FDA-approved human vaccine. Currently, there are substantial efforts underway to develop both vaccines and the tools to assess these vaccines. Tularemia laboratory research has historically relied primarily upon a small number of inbred mouse strains, but the utility of such findings to outbred animals may be limited. Specifically, C57BL/6 mice are more susceptible than BALB/c mice to Ft infection and less easily protected against challenge with highly virulent type A Ft. Thus, depending on the inbred mouse strain used, one could be misled as to which immunogen(s)/vaccine will ultimately be effective in an outbred human population. Accordingly, we evaluated an outbred Swiss Webster (SW) mouse model in direct comparison to a well-established, inbred C57BL/6 mouse model. Mucosal vaccination with the live, attenuated Ft LVS superoxide dismutase (sodB) mutant demonstrated significantly higher protection in outbred SW mice compared to inbred C57BL/6 mice against Ft SchuS4 respiratory challenge. The protection observed in vaccinated outbred mice correlated with lower bacterial density, reduced tissue inflammation, and reduced levels of pro-inflammatory cytokine production. This protection was CD4+ and CD8+ T cell-dependent and characterized by lower titers of serum antibody (Ab) that qualitatively differed from vaccinated inbred mice. Enhanced protection of vaccinated outbred mice correlated with early and robust production of IFN-γ and IL-17A. Neutralizing Ab administered at the time of challenge revealed that IFN-γ was central to this protection, while IL-17A neutralization did not alter bacterial burden or survival. The present study demonstrates the utility of the outbred mouse as an alternative vaccination model for testing tularemia vaccines. Given the limited MHC repertoire in inbred mice, this outbred model is more analogous to the human in terms of immunological diversity.

Humanized Mice for the Evaluation of Francisella tularensis Vaccine Candidates.

Francisella tularensis (FT), a highly infectious pathogen, is considered to be a potential biological weapon owing to the current lack of a human vaccine against it. Tul4 and FopA, both outer membrane proteins of FT, play an important role in the bacterium's immunogenicity. In the present study, we evaluated the immune response of mice-humanized with human CD34+ cells (hu-mice)-to a cocktail of recombinant Tul4 and FopA (rTul4 and rFopA), which were codon-optimized and expressed in Escherichia coli. Not only did the cocktail-immunized hu-mice produce a significant human immunoglobulin response, they also exhibited prolonged survival against an attenuated live vaccine strain as well as human T cells in the spleen. These results suggest that the cocktail of rTul4 and rFopA had successfully induced an immune response in the hu-mice, demonstrating the potential of this mouse model for use in the evaluation of FT vaccine candidates.

Activities of Murine Peripheral Blood Lymphocytes Provide Immune Correlates That Predict Francisella tularensis Vaccine Efficacy.

We previously identified potential correlates of vaccine-induced protection against Francisella tularensis using murine splenocytes and further demonstrated that the relative levels of gene expression varied significantly between tissues. In contrast to splenocytes, peripheral blood leukocytes (PBLs) represent a means to bridge vaccine efficacy in animal models to that in humans. Here we take advantage of this easily accessible source of immune cells to investigate cell-mediated immune responses against tularemia, whose sporadic incidence makes clinical trials of vaccines difficult. Using PBLs from mice vaccinated with F. tularensis Live Vaccine Strain (LVS) and related attenuated strains, we combined the control of in vitro Francisella replication within macrophages with gene expression analyses. The in vitro functions of PBLs, particularly the control of intramacrophage LVS replication, reflected the hierarchy of in vivo protection conferred by LVS-derived vaccines. Moreover, several genes previously identified by the evaluation of splenocytes were also found to be differentially expressed in immune PBLs. In addition, more extensive screening identified additional potential correlates of protection. Finally, expression of selected genes in mouse PBLs obtained shortly after vaccination, without ex vivo restimulation, was different among vaccine groups, suggesting a potential tool to monitor efficacious vaccine-induced immune responses against F. tularensis. Our studies demonstrate that murine PBLs can be used productively to identify potential correlates of protection against F. tularensis and to expand and refine a comprehensive set of protective correlates.

Construction and characterization of Francisella tularensis vaccine strain with a single copy of iglC gene and lacking recA gene

Live vaccine used in Russia against tularemia is based on Francisella tularensis subsp. holarctica NIIEG vaccine strain 15. This vaccine is highly effective, but fairly unstable. Therefore, the development of stable live tularemia vaccine with minimal side effects is rather pressing. F. tularensis iglC gene is required for intracellular production of the microbe, and recA gene is crucial for homologous recombination. Using methodology for allele removal, we deleted one copy of iglC gene and both copies of recA gene in F. tularensis vaccine strain. To achieve replacement of intact F. tularensis chromosome segments with modified segments, we constructed a novel pGM5 suicide vector based on pHV33 bireplicon plasmid. Modified chromosome segments contained a F. tularensis DNA fragment without 545 bp of iglC structural gene segment (in pGMΔiglC plasmid) and lacked a 1060-bp DNA fragment containing recA structural gene segment (pGMΔrecA plasmid). The constructed 15/23-lΔrecA mutant was capable to reproduce in macrophage-like J77A.l cell line with proliferation efficiency eight to ten times higher than parent vaccine strain 15. BALB/c mouse responded to immunization by 15/23-lΔrecA strain with a slight (about 2%) weight decrease compared to infection with strain 15 (about 14%). Bacteria of 15/23-lΔrecA strain were virtually incapable of maintaining germination in BALB/c murine spleen 14 days after invasion. In turn, bacteria of strain 15 were detected in murine spleen even after 21 days. Thus, F. tularensis 15/23-lΔrecA strain, which results in a lighter organismal reaction, can be successfully used as a basis for construction of stable live tularemia vaccines with minimal side effects.

Characterization of rationally attenuated Francisella tularensis vaccine strains that harbor deletions in the guaA and guaB genes

Francisella tularensis, the etiologic agent of tularemia, can cause severe and fatal infection after inhalation of as few as 10–100 CFU. F. tularensis is a potential bioterrorism agent and, therefore, a priority for countermeasure development. Vaccination with the live vaccine strain (LVS), developed from a Type B strain, confers partial protection against aerosal exposure to the more virulent Type A strains and provides proof of principle that a live attenuated vaccine strain may be efficacious. However LVS suffers from several notable drawbacks that have prevented its licensure and widespread use. To address the specific deficiencies that render LVS a sub-optimal tularemia vaccine, we engineered F. tularensis LVS strains with targeted deletions in the guaA or guaB genes that encode critical enzymes in the guanine nucleotide biosynthetic pathway. F. tularensis LVSΔ guaA and LVSΔ guaB mutants were guanine auxotrophs and were highly attenuated in a mouse model of infection. While the mutants failed to replicate in macrophages, a robust proinflammatory cytokine response, equivalent to that of the parental LVS, was elicited. Mice vaccinated with a single dose of the F. tularensis LVSΔ guaA or LVSΔ guaB mutant were fully protected against subsequent lethal challenge with the LVS parental strain. These findings suggest the specific deletion of these target genes could generate a safe and efficacious live attenuated vaccine.

Efficacy of the live attenuated Francisella tularensis vaccine (LVS) in a murine model of disease

A live attenuated vaccine Francisella tularensis live vaccine strain (LVS), that confers protection against tularemia infection in a number of animal models including man was developed during the 1960s in the US. In this study, we have established the median lethal dose (MLD) after intraperitoneal (i.p.) or intravenous (i.v.) delivery of NDBR Lot 4 F. tularensis LVS to be 4 cfu and 2.24 × 10 4 cfu, respectively, in BALB/c mice and less than 1 cfu and 1.29 × 10 4 cfu, respectively, in C57BL/6 mice. When delivered subcutaneously, the MLD for F. tularensis LVS was greater then 1 × 10 8 cfu in both strains of mouse. Using mouse models of systemic tularemia infection it was demonstrated that F. tularensis LVS immunised BALB/c mice were fully protected after challenge with approximately 1000 MLD of a strain of F. tularensis subsp. tularensis or a strain of F. tularensis subsp. holarctica. Under similar challenge conditions, protection in C57BL/6 mice was only evident against a subsp. holarctica strain. In BALB/c mice, protection against a subsp. holarctica strain was achieved 4 days after F. tularensis LVS immunisation whereas protection against a subsp. tularensis strain was only evident 14 days after F. tularensis LVS immunisation.

Live tularemia vaccine confers protection against lethal Legionella and Listeria infections in experimental animals.

The efficacy of a live Francisella tularensis vaccine strain to cause nonspecific immunity toward experimental legionellosis and listeriosis was studied. Immunisation with tularemia vaccine protected over 80% and 17% of experimental animals against subsequent lethal challenge with Legionella pneumophila and Listeria monocytogenes, respectively. The protection was maximal during the first month following immunisation and declined thereafter. In order to delineate the immunostimulatory moieties of the Francisella microbe, several cell wall proteins have been purified and characterized. However, isolated cell wall components failed to induce protection.

Live tularemia vaccine confers protection against lethal Legionella and Listeria infections in experimental animals

The efficacy of a live Francisella tularensis vaccine strain to cause nonspecific immunity toward experimental legionellosis and listeriosis was studied. Immunisation with tularemia vaccine protected over 80% and 17% of experimental animals against subsequent lethal challenge with Legionella pneumophila and Listeria monocytogenes, respectively. The protection was maximal during the first month following immunisation and declined thereafter. In order to delineate the immunostimulatory moieties of the Francisella microbe, several cell wall proteins have been purified and characterized. However, isolated cell wall components failed to induce protection.

Live tularemia vaccine but not proteins purified from Francisella tularensis can confer protection against lethal Listeria infection in mice

Immunization of Balb/c mice with Francisella tularensis vaccine strain 15/10 conferred significant protection against subsequent listerial infection. Since immunostimulatory activities could apparently be relevant to surface components of the bacterium, a technique for purification of cell wall proteins was developed. The scheme designed consisted of Triton X-100 extraction with subsequent FPLC chromatography steps, and resulted in the isolation of homogeneous proteins with molecular masses of 54 kDa (pI = 6.7) and 82 kDa (pI = 5.3), and partially purified 50, 85 and 100 kDa components. It was shown that immunization with isolated proteins failed to protect mice against lethal Listeria monocytogenes infection. Possible reasons for failure are discussed.

Long-lasting cell-mediated immunity induced by a live Francisella tularensis vaccine.

The lymphocyte stimulation test was used to estimate specific cell-mediated immunity after vaccination with the live vaccine strain of Francisella tularensis. Nonvaccinated individuals and individuals vaccinated 1, 5 to 6, 7 to 8, and 9 years previously were tested. Lymphocytes from most vaccinees responded to an antigen preparation of the vaccine strain, and those vaccinated 9 years before testing responded to a similar extent as did vaccines in the other groups. A new technique was developed to study the presence of T lymphocytes among the stimulated cells. Stimulated cells were allowed to incorporate [14C]thymidine and were then fractionated into T and non-T lymphocytes. Most of the incorporation appeared in the fraction containing T lymphocytes. The data indicate that cell-mediated immunity endures for at least 9 years after vaccination with the live F. tularensis vaccine.

Cell-mediated and humoral immunity induced by a live Francisella tularensis vaccine.

Live attenuated Francisella tularensis vaccine induced long-lasting humoral and cell-mediated immune responses in all 13 subjects studied. Lymphocyte blast transformation reactivity to F. tularensis appeared 2 weeks after vaccination in most subjects and remained unchanged for up to 1.5 years. Similarly, in most recipients, antibodies against F. tularensis were detectable by both the enzyme-linked immunosorbent assay (ELISA) and the agglutination method from 2 weeks after vaccination, although diagnostically significant agglutination titers (greater than or equal to 80) were not detectable until 4 weeks after vaccination. Maximal agglutination titers of 80 to 2,560 appeared at 4 to 8 weeks, and in spite of decreasing tendency, titers as high as 320 were still present 1.5 years after vaccination. ELISA showed the simultaneous, but not parallel, appearance of different immunoglobulin classes, immunoglobulin M (IgM) reaching individual maximal values 1.8 months after vaccination on average, at the same time as agglutinating antibodies, 1 week earlier than IgA, and about 1 month earlier than IgG. All of these immunoglobulin classes persisted in significant amounts up to 1.5 years, with IgG generally dominant. Long-lasting IgA and IgM responses after vaccination, as also after infection, suggested that the serodiagnosis of tularemia generally requires two consecutive serum samples with a significant increase in the titer.

Immunization against tularemia: analysis of the effectiveness of live Francisella tularensis vaccine in prevention of laboratory-acquired tularemia.

A retrospective analysis was made of cases of laboratory-acquired infections with Francisella tularensis among civilian employees at Fort Detrick, Maryland. The incidence and clinical presentation of tularemia during the decade 1950-1959, when the phenol-killed Foshay vaccine was used routinely for immunization of employees, were compared with similar data from the first decade (1960-1969) after the live tularemia vaccine had come into use. The incidence of typhoidal tularemia fell (from 5.70 to 0.27 cases per 1,000 at-risk employee-years; P less than 0.001), whereas the incidence of ulceroglandular tularemia remained unchanged (from 0.76 to 0.54 cases per 1,000 at-risk employee-years). Ulceroglandular tularemia in employees immunized with live vaccine was characterized by clinical signs and symptoms that were milder than those in employees vaccinated with the Foshay vaccine.

The relationship between immune response and hydrolase activity in rat mononuclear leucocytes.

Studies of differences in hydrolase activity of peritoneal mononuclear leucocytes from normal rats and rats given three injections of killed Francisella tularensis vaccine demonstrated increases of 37–69% in ribonuclease, deoxyribonuclease, and acid phosphatase in lysates of cells obtained from vaccinated animals. No deviation from normal was observed for B-glucuronidase, alkaline phosphatase, or cathepsin. Following a single injection of vaccine the activity of ribonuclease, deoxyribonuclease, and acid phosphatase decreased slightly and then increased, reaching a maximum of 96 h and returning to normal levels after 30 d. Stimulation of the animals with a second injection of vaccine at 96 h resulted in an immediate and pronounced depression in activity of deoxyribonuclease and acid phosphatase which also was followed by an increase similar to that observed initially. Analysis of the various leucocytic fractions revealed 52.2–56.4% of the total enzymatic activity to be associated with the cytoplasmic granules.