Vaccine Strain Of Francisella Tularensis Research Articles

Improvement of Approaches to the Verification of the Vaccine Strain <i>Francisella tularensis</i> 15 NIIEG during Long-Term Storage

The aim of the study was to improve the methods for verifying the vaccine strain Francisella tularensis 15 NIIEG during long-term storage under current conditions.Materials and methods. The paper summarizes the results of studying the phenotypic and genetic properties of lyophilized cultures of the vaccine strain F. tularensis 15 NIIEG (1953, 1966, 1969, 1987, 1990, 2003, 2012 and 2013) stored at SCEMAP for a period of one to 60 years.Results and discussion. Previous studies have revealed that freeze-dried cultures of F. tularensis 15 NIIEG generally had the characteristics of the vaccine strain, with the exception of deviations from the regulatory requirements for residual virulence and specific safety. The stability of preservation of deletions in the pilA and pilE genes (the region of differentiation RD19) and the genes encoding lpp lipoprotein (RD18) in the vaccine strain, which was stored for various periods of time in a lyophilized state, has been confirmed. The vaccine-strain-specific mutation C178404T (by the genome of F. tularensis LVS strain, GenBank NCBI no. CP009694) has been identified, and an approach to determine it has been developed. The data obtained are promising as regards using the above deletions in the RD18/RD19 regions in combination with the C178404T mutation to assess the authenticity of the vaccine strain using molecular genetic methods. Thus, the conducted retrospective analysis of the data on the cultures of tularemia microbe vaccine strain from the 1940s to 2013 and the gathered experimental data, made it possible to supplement the uniform requirements for the manufacture, study, maintenance, storage and movement of F. tularensis 15 NIIEG vaccine strain with new evidence. Based on the results obtained, the authors have drawn a draft methodological recommendations of the federal level “Vaccinal strain Francisella tularensis 15 NIIEG: order of handling”.

Whole-Genome Sequencing and Phylogenetic Analysis of <i>Francisella tularensis</i> Vaccine Strain 15 NIIEG

Objective of the study was to conduct whole-genome sequencing of the vaccine strain Francisella tularensis 15 NIIEG and determine, based on the results, its phylogenetic relationships and the genetic organization features.Materials and methods. Whole-genome sequencing of F. tularensis 15 NIIEG strain was performed on Ion PGM (Ion Torrent, USA) and MinIon (Oxford Nanopore Technologies, UK) platforms. Alignment of readings obtained to the whole-genome of F. tularensis subsp. holarctica LVS (CP009694, USA, 2015) was performed using the software package DNASTAR Lasergene 15.3. Hybrid assembly of reads into contigs was performed by means of Unicycler v. 0.4.4, using data obtained by semiconductor sequencing technology (Ion PGM) and nanopore sequencing (MinIon). Phylogenetic analysis was performed on the basis of single nucleotide polymorphism (SNPs) data located in the core part of F. tularensis genome. Maximum parsimony algorithm was used to construct a dendrogram using the obtained data of common SNP-matrix.Results and discussion. The close relations of F. tularensis 15 NIIEG strain with F. tularensis LVS vaccine strain used in the countries of Western Europe and North America was confirmed. Searching for common single mutations characteristic of F. tularensis 15 vaccine strains of NIIEG and LVS, permitted to find 5 unique SNPs that distinguish them from all other 228 F. tularensis strains used in the comparison. Comparative genomic analysis ofF. tularensis 15 NIIEG vaccine strain and virulent strains revealed in its structure two extensive 526 bp deletions (genes pilA and pilE) and 1480 bp (genes encoding lipoprotein). Similar deletions are also present in the genome of the F. tularensis LVS vaccine strain.

Комплекс Bfr-O-антиген внешних мембран Francisella tularensis: получение, характеристика, возможности использования

A method for the isolation of a complex antigen of protein-carbohydrate nature from outer membranes of the vaccine strain Francisella tularensis subsp. holarctica 15 NIIEG cells and its detailed characteristics have been described. A protein of bacterioferritin (Bfr) and O-antigen were identified in the antigenic complex. It was shown that the obtained complex antigen is non-toxic, harmless, has high immunogenic activity and protects white mice in the subcutaneous infection with a virulent strain of F. tularensis subsp. holarctica 503/840. This antigen was shown to occur in the tularemia microbe regardless of the subspecies and presence of a capsule. The high antigenic and immunobiological activity of the Bfr-O-antigen complex provides the prerequisites for its further use as a component in the development of preventive and diagnostic drugs against the tularemia infection. Francisella tularensis, antigens, outer membrane proteins, immunogenicity, lipopoly saccharide, O-antigen. Authors would like to thank to the staff of the Institute «Microbe»: laboratory of molecular diagnostics I.V. Terekhova for providing us with the monoclonal antibodies, laboratories for genomic and proteomic analysis N.V. Kotova for her excellent technical assistance.

Expansion and retention of pulmonary CD4+ T cells after prime boost vaccination correlates with improved longevity and strength of immunity against tularemia

Francisella tularensis subsp. tularensis strain SchuS4 (Ftt) is a highly virulent intracellular bacterium. Inhalation of 10 or fewer organisms results in an acute and potentially lethal disease called pneumonic tularemia. Ftt infections occur naturally in the U.S. and Ftt was developed as a bioweapon. Thus, there is a need for vaccines that protect against this deadly pathogen. Although a live vaccine strain of Francisella tularensis (LVS) exists, LVS fails to generate long-lived protective immunity against modest challenge doses of Ftt. We recently identified an important role for high avidity CD4+ T cells in short-term protection and hypothesized that expanding this pool of cells would improve overall vaccine efficacy with regard to longevity and challenge dose. In support of our hypothesis, application of a prime/boost vaccination strategy increased the pool of high avidity CD4+ T cells which correlated with improved survival following challenge with either increased doses of virulent Ftt or at late time points after vaccination. In summary, we demonstrate that both epitope selection and vaccination strategies that expand antigen-specific T cells correlate with superior immunity to Ftt as measured by survival.

Evaluation of a microfluidic chip system for preparation of bacterial DNA from swabs, air, and surface water samples

The detection of bacterial pathogens from complex sample matrices by PCR requires efficient DNA extraction. In this study, a protocol for extraction and purification of DNA from swabs, air, and water samples using a microfluidic chip system was established. The optimized protocol includes a combination of thermal, chemical and enzymatic lysis followed by chip-based DNA purification using magnetic particles. The procedure was tested using Gram-positive Bacillus thuringiensis Berliner var. kurstaki as a model organism for Bacillus anthracis and the attenuated live vaccine strain of Francisella tularensis subsp. holarctica as Gram-negative bacterium. The detection limits corresponded to 103 genome equivalents per milliliter (GE/ml) for surface water samples spiked with F. tularensis and 102 GE/ml for B. thuringiensis. In air, 10 GE of F. tularensis per 10 L and 1 GE of B. thuringiensis per 10 L were detectable. For swab samples obtained from artificially contaminated surfaces the detection limits were 4 × 103 GE/cm2 for F. tularensis and 4 × 102 GE/cm2 for B. thuringiensis. Suitability of the chip-assisted procedure for DNA preparation of real samples was demonstrated using livestock samples. The presence of thermophilic Campylobacter spp. DNA could be confirmed in air samples collected on pig and broiler farms.

The impact of Immunomodulators on Reactivity the immune System cells in Model Vaccinal process against Tularemia

Investigated is the effect of azoximer bromide (polioxidonium - PO) and dalargin (DA) on the immune system cell reactivity in the anti-tularemia vaccine prosess modeling in Balb/c mice. The animals were immunized subcutaneously with a dose of 10 4 live microbial cells of vaccine strain Francisella tularensis 15 NIIEG. PO or DA subcutaneously administered 60 min before vaccination, respectively, in doses of 4 mg and 2 mg. On 3rd and 21st days of immunogenesis the peripheral organs of the immune system and the contents of the abdominal cavity were investigated. Cell response to vaccination and investigated immunomodulators evaluated by flow cytometry (CyAn ADP) for two light scatter parameters and for changing the proliferating and apoptotic cell number. Additionally, for the characterization of changes in the functional status of lymphocytes nuclei the light microscopy was used and titers of specific antibodies were determined. Installed is the different response of intact mice lymphocytes and phagocytes to PO and DA introduction. As distinct from PO, DA stimulated the lymphocete proliferation and macrophage apoptosis by acting on their cytoplasmic granules. PO activated the migration of phagocytes with high cytoplasmic granules from mice abdominal cavity in the spleen. Only when using the PO as an immunomodulator in anti-tularemia vaccine process modeling recorded a significant increase in specific antibody titers against the background of a long-term functional splenocyte activation and macrophages reduced macrophage damage intensity in the spleen and abdomen.

CONSTRUCTING AND STUDYING OF THE PROPERTIES OF Francisella tularensis 15 NIIEG VARIANTS WITH REDUCED EXPRESSION OF sodB GENE ENCODING Fe-SUPEROXIDE DISMUTASE

Within the frames of the investigation on constructing of an improved live tularemia vaccine with less reactogenicity by the method of allelic replacement using a suicidal plasmid pGM5, the mutant variants have been obtained from the vaccine strain Francisella tularensis 15 NIIEG with a reduced expression of the superoxide dismutase sodB gene. This reduction was a result of the substitution of rare for frequent natural codons in the F e-superoxide dismutase (FeSOD) gene. It was shown that the decrease in the FeSOD production up to 30% of the level in the original strain had no negative effect on the rate of the tularemia cause reproduction. It was shown that the mutant strains were more sensitive to hydrogen peroxide and paraquat as compared to the vacci- he bacteria. All the strains were shown (by Q-PCR) to have at the log phase the elevated content of gene groEL mRNA encoding GroEL chaperone.

T-bet Regulates Immunity to Francisella tularensis Live Vaccine Strain Infection, Particularly in Lungs

Upregulation of the transcription factor T-bet is correlated with the strength of protection against secondary challenge with the live vaccine strain (LVS) of Francisella tularensis. Thus, to determine if this mediator had direct consequences in immunity to LVS, we examined its role in infection. Despite substantial in vivo gamma interferon (IFN-γ) levels, T-bet-knockout (KO) mice infected intradermally (i.d.) or intranasally (i.n.) with LVS succumbed to infection with doses 2 log units less than those required for their wild-type (WT) counterparts, and exhibited significantly increased bacterial burdens in the lung and spleen. Lungs of LVS-infected T-bet-KO mice contained fewer lymphocytes and more neutrophils and interleukin-17 than WT mice. LVS-vaccinated T-bet-KO mice survived lethal LVS intraperitoneal secondary challenge but not high doses of LVS i.n. challenge, independently of the route of vaccination. Immune T lymphocytes from the spleens of i.d. LVS-vaccinated WT or KO mice controlled intracellular bacterial replication in an in vitro coculture system, but cultures with T-bet-KO splenocyte supernatants contained less IFN-γ and increased amounts of tumor necrosis factor alpha. In contrast, immune T-bet-KO lung lymphocytes were greatly impaired in controlling intramacrophage growth of LVS; this functional defect is the likely mechanism underpinning the lack of respiratory protection. Taken together, T-bet is important in host resistance to primary LVS infection and i.n. secondary challenge. Thus, T-bet represents a true, useful correlate for immunity to LVS.

A galU mutant of francisella tularensisis attenuated for virulence in a murine pulmonary model of tularemia

BackgroundA number of studies have revealed that Francisella tularensis (FT) suppresses innate immune responses such as chemokine/cytokine production and neutrophil recruitment in the lungs following pulmonary infection via an unidentified mechanism. The ability of FT to evade early innate immune responses could be a very important virulence mechanism for this highly infectious bacterial pathogen.ResultsHere we describe the characterization of a galU mutant strain of FT live vaccine strain (LVS). We show that the galU mutant was highly attenuated in a murine model of tularemia and elicited more robust innate immune responses than the wild-type (WT) strain. These studies document that the kinetics of chemokine expression and neutrophil recruitment into the lungs of mice challenged with the galU mutant strain are significantly more rapid than observed with WT FT, despite the fact that there were no observed differences in TLR2 or TLR4 signaling or replication/dissemination kinetics during the early stages of infection. We also show that the galU mutant had a hypercytotoxic phenotype and more rapidly induced the production of IL-1β following infection either in vitro or in vivo, indicating that attenuation of the galU mutant strain may be due (in part) to more rapid activation of the inflammasome and/or earlier death of FT infected cells. Furthermore, we show that infection of mice with the galU mutant strain elicits protective immunity to subsequent challenge with WT FT.ConclusionsDisruption of the galU gene of FTLVS has little (if any) effect on in vivo infectivity, replication, or dissemination characteristics, but is highly attenuating for virulence. The attenuated phenotype of this mutant strain of FT appears to be related to its increased ability to induce innate inflammatory responsiveness, resulting in more rapid recruitment of neutrophils to the lungs following pneumonic infection, and/or to its ability to kill infected cells in an accelerated fashion. These results have identified two potentially important virulence mechanisms used by FT. These findings could also have implications for design of a live attenuated vaccine strain of FT because sublethal infection of mice with the galU mutant strain of FTLVS promoted development of protective immunity to WT FTLVS.

Analysis of a live attenuated bacterial vaccine for tularemia in the MIMIC® (Modular Immune In vitro Construct) System (52.6)

Abstract Sanofi Pasteur has optimized a series of unique, sensitive, automatable, and cost-effective assay systems, termed the Modular IMmune In vitro Construct (MIMIC®), to permit the evaluation of human innate and adaptive immunity in a laboratory setting. The lymphoid tissue equivalent (LTE) module of the MIMIC® platform reproduces the correct cellular interactions and kinetics to initiate primary (naïve) human B and T cell responses in an in vitro setting. We are currently investigating LVS, a live attenuated bacterial vaccine strain of Francisella tularensis, in the MIMIC® System. LVS generates a strong, naïve, TH1-skewed CD4 T cell response demonstrated by the percentage of responding cells producing intracellular IFNγ and TNFα. This data is consistent with human in vivo data showing that protection elicited by LVS vaccine includes a strong cellular immunity component. Previous studies have also shown that LVS drives production of protective antibodies in humans. We have currently demonstrated that the MIMIC® System can drive the generation of LVS-specific antibody responses in vitro using a fully autologous system. These studies indicate that the in vitro MIMIC® system offers a platform in which to conduct experiments in a fully human system prior to the involvement of human subjects. Therefore, the MIMIC® system provides a platform for analysis of new vaccine candidates and can be utilized for greater understanding of how efficient vaccines function.

Biological properties and structure of the lipopolysaccharide of a vaccine strain of Francisella tularensis generated by inactivation of a quorum sensing system gene qseC

A knockout mutant with a deletion in a quorum sensing system gene qseC was generated from the vaccine strain Francisella tularensis 15 by site-directed mutagenesis. The variant with the inactivated gene qseC differed from the parental strain in growth rate on solid nutrient medium but had the same growth dynamics in liquid nutrient medium. The mutation abolished almost completely the resistance of the vaccine strain to normal rabbit serum and its ability to survive in macrophages; in addition, the strain lost the residual virulence. A significant phenotypic alteration was observed in the lipopolysaccharide of F. tularensis. Particularly, the mutant strain synthesized no noticeable amount of the lipopolysaccharide with the high-molecular-mass O-polysaccharide, presumably as a result of impairing biosynthesis of the repeating unit, namely, a loss of the ability to incorporate a formyl group, an N-acyl substituent of 4-amino-4,6-dideoxy-D-glucose.

Live Vaccine Strain Francisella tularensis Is Detectable at the Inoculation Site but Not in Blood after Vaccination against Tularemia

Live vaccine strain (LVS) Francisella tularensis is a live, attenuated investigational tularemia vaccine that has been used by the US Army for decades to protect laboratory workers. Postvaccination bacterial kinetic characteristics of LVS at the inoculation site and in the blood are unknown and, therefore, were assessed in a prospective study. LVS vaccination of laboratory workers provided the opportunity to compare culture with polymerase chain reaction (PCR) for the detection of F. tularensis in human clinical samples. Blood and skin swab samples were prospectively collected from volunteers who received the LVS tularemia vaccine at baseline (negative controls) and at 5 specified time points (days 1, 2, 7 or 8, 14 or 15, and 35 after vaccination). Bacterial culture and PCR of whole blood samples (17 volunteers) and inoculation site swabs (41 volunteers) were performed. The culture and PCR results of all blood samples were negative. Results of real-time PCR from the inoculation site samples were positive for 41 (100%) of 41 volunteers on day 1, for 40 (97.6%) of 41 volunteers on day 2, for 24 (58.5%) of 41 on day 7 or 8, for 6 (16.7%) of 36 on day 14 or 15, and for 0 (0%) of 9 on day 35. Positive results of bacterial cultures of the inoculation site samples occurred significantly less frequently, compared with PCR testing, with 4 (9.8%) of 41 volunteers having positive results on day 1 (P<.001) and 4 (9.8%) of 41 on day 2 (P<.001); all results from subsequent days were negative. F. tularensis LVS genomic DNA was detected in the majority of samples from the inoculation site up to 1 week after LVS vaccination, with real-time PCR being more sensitive than culture. Our data suggest that bacteremia does not occur after LVS vaccination in normal, healthy human volunteers.

Grey variants of the live vaccine strain of Francisella tularensis lack lipopolysaccharide O-antigen, show reduced ability to survive in macrophages and do not induce protective immunity in mice

Francisella tularensis live vaccine strain (LVS) produces two colony types when grown on solid media, often referred to as blue variants (BV) and grey variants (GV). Whereas blue variant bacteria possessed a lipopolysaccharide O-side chain, grey variant bacteria lacked O-side chains. Grey variant bacteria appeared in stationary phase bacterial cultures and could be identified using a novel FACS-based assay. Compared to blue variant bacteria, grey variants showed a reduced ability to infect and survive in macrophages. The immunisation of mice with blue variant bacteria, but not grey variant bacteria, induced protective immunity towards fully virulent F. tularensis.

Aerosol-, but not intradermal-immunization with the live vaccine strain of Francisella tularensis protects mice against subsequent aerosol challenge with a highly virulent type A strain of the pathogen by an αβ T cell- and interferon gamma- dependent mechanism

Francisella tularensis is an extremely virulent facultative intracellular bacterial pathogen of many mammalian species including mice and humans in which it causes a spectrum of disease collectively called tularemia. In humans, intradermal or inhaled inocula of 10 cfu or less of the most virulent strains of the pathogen are sufficient to cause severe infection and possible death; in mice similar inocula are routinely lethal. An attenuated live vaccine strain, F. tularensis LVS, was developed almost 50 years ago, and remains the sole prophylactic against virulent strains of the pathogen. Using F. tularensis LVS as a model vaccine, we recently showed that it was possible to systemically immunize various mouse strains and protect them against subsequent massive (2000 cfu) intradermal (i.d.) challenge, but not against low dose (∼10 cfu) aerosol challenge, with virulent strains of the pathogen. This is troubling because the latter route is considered an important means of deliberately disseminating F. tularensis in a bioterrorist attack. Others have previously shown that administering LVS to humans, guinea pigs and monkeys as an aerosol enhanced protection against subsequent aerosol challenge with virulent F. tularensis. In the present study, we show the same phenomenon in BALB/c and C3H/HeN mice. In this model, interferon gamma (IFNγ) and CD4 + and CD8 + T cells are essential for the expression of anti- Francisella immunity in the lungs. Combined this immune response operates by limiting dissemination of the pathogen to susceptible internal organs. Further, understanding of how inhaled LVS elicits local cell-mediated protective immunity will be critical for devising improved vaccines against pulmonary tularemia.

Tularemia in BALB/c and C57BL/6 mice vaccinated with Francisella tularensis LVS and challenged intradermally, or by aerosol with virulent isolates of the pathogen: protection varies depending on pathogen virulence, route of exposure, and host genetic background

In order to begin understanding the immunological basis for immunity to tularemia, and to establish a baseline for judging the efficacy of potential novel vaccines, the present study examined the ability of the live vaccine strain of Francisella tularensis (F. tularensis) LVS, to elicit immunity in mice against subsequent systemic and aerosol challenge with highly virulent strains of the pathogen. The results show, that infection with LVS protects BALB/c mice against systemic challenge with virulent Types A and B F. tularensis. In contrast, C57BL/6 mice vaccinated with LVS were only rendered immune to systemic challenge with Type B F. tularensis. Neither mouse strain immunized with LVS was able to resist aerosol challenge with Type A F. tularensis, and only immunized BALB/c mice withstood exposure to aerosols of Type B F. tularensis.

Inability of the Francisella tularensis lipopolysaccharide to mimic or to antagonize the induction of cell activation by endotoxins.

We studied the ability of the lipopolysaccharide (LPS) extracted from a vaccine strain of Francisella tularensis (LPS-Ft) to mimic LPSs from other gram-negative bacteria for activation of various murine cell types or to antagonize the effects of other LPSs. We found that activation of macrophages for the production of tumor necrosis factor alpha and NO, of pre-B lymphocytes for the expression of surface immunoglobulins, and of bone marrow cells for the expression of LPS-binding sites was either undetectable with LPS-Ft or required concentrations 100 to 1,000 times higher than for standard LPSs. Preexposure of macrophages to LPS-Ft also failed to trigger down-regulation of tumor necrosis factor alpha (desensitization) or up-regulation of NO responses to an endotoxin challenge. In contrast to other atypical LPSs, LPS-Ft was also unable to antagonize any of the endotoxin-induced cellular responses mentioned above, suggesting that this LPS does not interact with LPS receptors.

The new vaccine strains (or variants) of Francisella tularensis.

A colony (N83) of the vaccine strain of Francisella tularensis (15/10) and a strain (N268) isolated from a water sample in nature were revealed for susceptibility to cultivation at 42 degrees C. Both strains had low virulence for white mice and were avirulent for guinea pigs but possessed high immunogenicity in these animals. The spontaneous mutant of vaccine strain 15/10 showed resistance to doxicycline and rifampicine (15/10 Dox (r)40 Rif (r)40). The obtained mutant had biological characteristics similar to the parent vaccine strain. It provided immunity in experimental animals when vaccination and antimicrobial agents were used in combination.

The new vaccine strains (or variants) of Francisella tularensis

A colony (N83) of the vaccine strain of Francisella tularensis ( 15 10 ) and a strain (N268) isolated from a water sample in nature were revealed for susceptibility to cultivation at 42°C. Both strains had low virulence for white mice and were avirulent for guinea pigs but possessed high immunogenicity in these animals. The spontaneous mutant of vaccine strain 15 10 showed resistance to doxicycline and rifmanpicine ( 15 10 Dox r40 Rif r40). The obtained mutant had biological characteristics similar to the parent vaccine strain. It provided immunity in experimental animals when vaccination and antimicrobial agents were used in combination.

Levels of interleukin 6 and tumor necrosis factor in serum from humans vaccinated with live, attenuated Francisella tularensis

The levels of interleukin 6 (IL-6) and tumor necrosis factor alpha in serum did not change significantly during the course of immunization with the live vaccine strain of Francisella tularensis. Higher levels of circulating IL-6 were found in the sera of vaccinees with good antibody responses than in the sera from nonresponders. Preimmunization levels of IL-6 in serum were also higher in responders than in nonresponders.

Cell-mediated and humoral immune responses after vaccination of human volunteers with the live vaccine strain of Francisella tularensis

The specific humoral and cell-mediated immune responses of human volunteers vaccinated with the Francisella tularensis live vaccine strain (LVS) were evaluated. In the search for an optimal antigen to measure the immunogenicity of the vaccine in an enzyme-linked immunosorbent assay, we tested irradiation-killed LVS, an aqueous ether extract of the LVS (EEx), lipopolysaccharide (LPS) from LVS, and a virulent strain (SCHU4). Volunteers were immunized with LVS by scarification. Immunoglobulin G (IgG) responses to LVS and LPS gave the highest background titers when tested with sera from unimmunized volunteers, whereas IgA, IgG, and IgM background titers to EEx and SCHU4 were low. Vaccination caused a significant rise (P < 0.01) in IgA, IgG, and IgM titers to all antigens tested, except for the IgG response to LPS. Eighty percent of vaccinated volunteers developed a positive IgG response to EEx 14 days postvaccination, while 50% were positive to LVS. By day 14 after vaccination, 70% of immunized volunteers exhibited a positive response to EEx in an in vitro peripheral blood lymphocyte proliferation assay. EEx, a specific and sensitive antigen for evaluating immune responses of vaccinated volunteers, may be a superior antigen for the diagnosis of tularemia.