Evaluation of the long-term memory T cell in mice after immunization with a live tularemia vaccine

The vaccine strain F. tularensis 15 NIIEG induces long-lived cell-mediated immunity but exhibits a certain reactogenicity and genetic instability. Progress in development of a vaccine against tularemia has been limited by a lack of information regarding the mechanisms required to protect against this disease. The BALB/c mouse is the most commonly used animal to study tularemia due to its relatively low cost, well-characterized genetics, available immunological tools and mouse infection with virulent F. tularensis recapitulates human disease.CD4+ and CD8+T cells are known to be critical for the formation of protective immunity but the relative roles of memory T cell subpopulations in long lived protection against virulent strains of F. tularensis are not well established. We hypothesized that this immunity depends on central (TCM) and effector memory (TEM) T cells and their functional activity. In this study we have dissected the T cell immune response in BALB/c mice 30, 60 and 90 days after subcutaneous vaccination with 15 NIIEG.Multiparametric flow cytometry were used to characterize in vitro recall responses of splenocytes to F. tularensis antigen. TEM cells were identified as CD3+CD4+CD44+CD62L- and CD3+CD8+CD44+CD62L-, TCM cells as CD3+CD4+CD44+CD62L+ and CD3+CD8+CD44+CD62L+, respectively. The functional activity of memory T cells was assessed by the following parameters: the level of expression of the activation marker CD69 and cytokine-producing activity by staining with the intracellular cytokines IFNg and TNFa.Thus, development of a long-lived vaccine directed against F. tularensis is dependent on identifying not only the correlates of immunity present early after vaccination, but also those that persist in the host after the effector phase has ended. The maintenance of long-term protective immunity initiated by vaccination with F. tularensis strain 15 NIIEG has been shown to require the presence of antigen-specific CD4+ and CD8+ memory T cells producing IFNg and TNFa and expressing the activation marker CD69. A decrease in count and functional activity of CD8+TCM and CD8+TEM was detected in the long term after vaccination. The detected parameters of functional activity of memory T cells can be used as criteria for evaluation of protective immunity against virulent strains of F. tularensis

Construction and Investigation of the Vaccine Strain Francisella tularensis without iglC genes. Communication 1

Using site-specific mutagenesis constructed were the variants of the vaccine strain F. tularensis subsp. holarctica 15 with the deleted iglC genes. Identified was the fact that genome of the strain 15 contained two copies of iglC gene. Deletion of one of them as well as both had little effect on the cultural-morphological and growth properties of the microbe. At the same time F. tularensis 15 lacking one copy of the iglC gene propagated in mice macrophages several times slower, than the original strain. Inactivation of both of the copies in the chromosome leaded to the emergence of a variant incapable of intracellular reproduction. This capacity in F. tularensis 15/23-2 with two inactivated iglC gene copies was partially recovered after integration of a complementing plasmid. Therewith the data mentioned above testifies to the significance of iglC gene for the process of reproduction in macrophages

Construction and Investigation of the Variants of the Vaccine Strain <I>Francisella tularensis</I> Lacking <I>iglC </I>Genes. Communication 2

for BALB/c mice by an order while retaining protective properties. Strain Francisella tularensis 15 lacking two copies of the iglC gene cannot replicate in mice organism, induces weak humoral response, is fully avirulent, and has decreased protective activity. Treatment of mice with sera obtained from the animals immunized on day 49 post immunization both with the stock F. tularensis 15 strain and its variants with one or two inactivated iglC genes has provided for 100 % protection from challenge with 200 DCL of F. tularensis 15 strain. However in case of BALB/c mice exposure to virulent F. tularensis 503 and F. tularensis Schu strains (50 DCL and 25 DSL, respectively), treated with immune sera 24 hours before, registered has been only mean lifetime increase

Functional characterization and biological significance of Yersinia pestis lipopolysaccharide biosynthesis genes

The Gram negative bacterium Yersinia pestis is the etiological agent of flea transmitted fulminant systemic rodent zoonosis and the reason of the three devastating pandemics of plague Lipopolysaccharide (LPS, endotoxin) is an impor tant factor of pathogenicity of Gram negative bacteria. The full LPS molecule (S form LPS) consists of three well defined domains: i) lipid A composed of sugars, fatty acids, and phosphate; it represents the endotoxic princi ple of the LPS and anchors it in the outer membrane; ii) a core oligosaccharide containing charged groups; and iii) an O specific polysaccharide (O antigen), which carries ISSN 0006 2979, Biochemistry (Moscow), 2011, Vol. 76, No. 7, pp. 808 822. © Pleiades Publishing, Ltd., 2011. Published in Russian in Biokhimiya, 2011, Vol. 76, No. 7, pp. 989 1005 Abstract-In silico analysis of available bacterial genomes revealed the phylogenetic proximity levels of enzymes responsible for biosynthesis of lipopolysaccharide (LPS) of Yersinia pestis, the cause of plague, to homologous proteins of closely relat ed Yersinia spp. and some other bacteria (Serratia proteamaculans, Erwinia carotovora, Burkholderia dolosa, Photorhabdus luminescens and others). Isogenic Y. pestis mutants with single or double mutations in 14 genes of LPS biosynthetic path ways were constructed by site directed mutagenesis on the base of the virulent strain 231 and its attenuated derivative. Using high resolution electrospray ionization mass spectrometry, the full LPS structures were elucidated in each mutant, and the sequence of monosaccharide transfers in the assembly of the LPS core was inferred. Truncation of the core decreased sig nificantly the resistance of bacteria to normal human serum and polymyxin B, the latter probably as a result of a less effi cient incorporation of 4 amino 4 deoxyarabinose into lipid A. Impairing of LPS biosynthesis resulted also in reduction of LPS dependent enzymatic activities of plasminogen activator and elevation of LD 50 and average survival time in mice and guinea pigs infected with experimental plague. Unraveling correlations between biological properties of bacteria and partic ular LPS structures may help a better understanding of pathogenesis of plague and implication of appropriate genes as potential molecular targets for treatment of plague

BIOLOGICAL PROPERTIES AND MOLECULAR-GENETIC CHARACTERISTICS OF BACILLUS ANTHRACIS STRAINS, ISOLATED DURING THE OUTBREAK OF ANTHRAX IN THE YAMALO-NENETS AUTONOMOUS DISTRICT IN 2016

Objective of the study was to identify phenotypic properties and genetic peculiarities of Bacillus anthracis strains, isolated during the outbreak of anthrax in the territory of Yamal in 2016. Materials and methods. Investigated were the strains of anthrax agent, applying basic and subsequent identification tests and canSNP-, MLVA-genotyping methods and whole genome sequencing. Results and conclusions. The results showed the identity of the phenotypic properties, canSNPand MLVA25-genotypes, and profiles of whole genome-sequencing, regardless of the source of the strains isolation. Confirmed was a common source of human infection. Defined were phylogenetic interrelations of the tested strains and their position in global B. anthracis population. For the first time ever explored was variability of the gene pattern, associated with pathogenicity, and demonstrated – the efficiency of the proposed algorithm for genetic typing

Routes of Spread of Anthrax in Natural Ecosystems

Anthrax is a particularly dangerous zooanthroponosis caused by the Gram-positive spore-forming bacterium Bacillus anthracis. This disease mainly affects hoofed herbivores, including those used in agriculture, but can occur in other animals and in humans. That is why the majority of studies of this infection are focused on anthrax in humans and farm animals, as the most important issues from a practical point of view. At the same time, the issues of anthrax epidemiology in natural ecosystems are described in the literature in insufficient detail and often very fragmentary. This paper provides a review of the literature describing the main pathways, patterns and risks of the spread of various clinical forms of anthrax infection in nature, primarily among wild animals. Among other things, we cover some non-notable aspects of intestinal infection with anthrax which, for instance, explain the difference in sensitivity to infection in herbivorous and carnivorous mammals and even different sex and age groups within the same species

Construction of Candidate <i>Yersinia pestis</i> Vaccine Strains with Reduced Reactogenicity

The technology of directed constructing of non-reverting Y. pestis ∆lpxM mutants, lacking antibiotic resistance markers was developed. It included direct site-specific mutagenesis, cloning of mutant ∆lpxM::cat allele in pCVD442 suicide vector, homologous recombination in vivo and deletion of the antibiotic resistance cassette. lpxM gene knockout mutant created on the basis of vaccine Y. pestis EV NIIEG strain lacking antibiotic resistance markers synthesized a lipopolysaccharide (LPS) with reduced ability to stimulate TNF-α production. The level of reduction of anti-inflammatory activity of the LPS synthesized by the mutant Y. pestis EV∆ lpxM strain was significantly higher in the test with human target cells in comparison with mouse model

Truncation of Lipopolysaccharide Core Decreases <i>Yersinia pestis</i> Virulence

The isogenic set of Yersinia pestis strains including wild-type strain 231 and its mutants with deletions of the structural genes, wabD, waaL, waaQ, waaE, and hldE, responsible for core oligosaccharide synthesis was generated using site directed mutagenesis. It was shown that gradual decrease of the core-oligosaccharide length was accompanied by reduction of mutants' virulence in subcutaneously infected mice and guinea pigs

FEATURES OF BETA-LACTAMASE ACTIVITY IN FRANCISELLA TULARENSIS subsp. MEDIASIATICA

Small Gram-negative bacteria Francisella tularensis is the tularemia causative agent. This species subdivides on four subspecies — ssp. tularensis, holarctica, mediasiatica and novicida, which have some differences in their distribution areas, pathogenicity and epidemical potencial. Until recently only subspecies holarctica was found on the territory of the Russian Federation, but in 2013 a natural focus of tularemia in which circulates F. tularensis subsp. mediasiatica was found on the Altai. Till now this subspecies was found only in Central Asia. The data of laboratory studies indicate the ability of strains of this subspecies to cause infection in rabbits and mice which is comparable in severity to infection caused by subsp. holarctica strains. However, the virulence of F. tularensis subsp. mediasiatica for humans and its epidemical potential are still unclear, since no cases of human infection caused by the strains of this subspecies have been recorded, probably due to the geographical aspects — mountainous Altai and Central Asia are extremely sparsely populated regions. The main phenotypic feature of this subspecies is the lack of activity of β-lactamase, which is responsible for the natural resistance to β-lactam antibiotics (penicillins, cephalosporins and carbapenems). Despite the absence of detectable enzymatic activity, subsp. mediasiatica strains are resistant to these antibiotics. In this article we report that subsp. mediasiatica strains have β-lactamase activity despite to current opinion, but the of β-lactams hydrolysis rate is much more lower in comparison with reaction rate of subs. holarctica strains. In addition, in case of a decrease of the microbial cells number in the nutrient medium, antibiotic susceptibility appears. We identified a single specific for subsp. mediasiatica nucleotide substitution G/A at the 290 position of the blaB gene, which encodes the active serine β-lactamase. This substitution leads to the amino acid substitution Gly/Arg at the 97 position of the protein BlaB. We assume, that enzymatic activity decreasing is the most likely caused by this substitution), for example it may cause some conformational changes leading either to enzyme — substrate affinity decreasing or to in the lifetime of the enzyme-substrate complex increasing. On the basis of the found nucleotide substitution, we developed an allele-specific PCR test that makes it possible to determine whether the studied strain F. tularensis belongs to the subspecies mediasiatica

Determination of the Expression of CD69 Marker of Early Activation in the Immune Mice Lymphocytes after their Stimulation with Plague Agent Antigens

Evaluated was the ability of plague microbe antigens F1 and V to activate specifically in the in vitro system subpopulations of T-lymphocytes of BALB/c mice immunized against plague. The level of CD69 expression at the lymphocytes surface was proposed to be evaluated as the marker of lymphocytes activation. Expression of CD69 early marker of activation in subpopulations of T-helpers and cytotoxic lymphocytes, reciprocated by the in vitro stimulation with Y. pestis F1, correlated with the intensity of the anti-plague postvaccinal immunity induced by immunization with F1 or F1and V antigens mixtures