Obtaining and Characteristic of Antibodies to Vibrio cholerae Protective Antigens Conjugated with Gold Nanoparticles.

Gold nanoparticle conjugates with Vibrio cholerae antigens were synthesized. The animals were immunized with the obtained conjugates. Rabbit polyclonal antibodies to the antigens were obtained, which showed high specific activity. On the model of white laboratory mice, the protective activity of conjugates of cholera antigens with nanoparticles during infection of vaccinated animals was evaluated using a commercial vaccine as a control. It was shown that in terms of immunogenicity, the created prototypes of cholera vaccine using gold nanoparticles as a carrier and adjuvant complied with the production regulations for the Russian national cholera chemical vaccine.

[An avirulent vibrio cholerae strain--producer of the cholera toxin B subunit: obtaining and molecular genetic analysis].

The conjugative recombinant plasmid pIEM3 (KmR TcR) was constructed in order to introduce the cloned ctxB gene encoding the cholera toxin B subunit into the Vibrio cholerae cells. The plasmid was obtained as a result of co-integration of two plasmids: a conjugative plasmid, pIEM1(KmR), carrying mini-kan transposon and IS1 element, as well as the pCTdelta27(TcR) plasmid that is a derivative of the pBR322 which carries the cloned ctxB gene. The avirulent Vibrio cholerae strain eltor biovar deprived (according to the PCR analysis) of the key structural and regulatory pathogenicity genes and carrying a mutation in a single gene of the O1 antigen was chosen as the pIEM3 plasmid carrier strain. The cointegrate uncoupling was shown to take place in 5% the cholera vibrio cells followed by retention of only the multi-copy pCTdelta7 plasmid. This event leads to the formation of the TcRKmS clones characterized by high levels of the cholera toxin secreted B subunit production (10 to 14 microg/ml), one of these (KM93) being selected as a strain-producer of the protein. Molecular-genetic and biochemical assays were used to elucidate peculiar features of inheritance and expression of the cloned ctxAB gene within the KM93 cells. The expression of the cloned ctxB gene was shown to be independent of the presence of the toxR, tcpP, tcpH, toxT regulatory genes suggesting the existence of some other mechanisms that might exert their control over the transcriptional activity of the cholera toxin B subunit gene. Effective production of the cholera toxin B subunit would be also observed if the constructed producer strain was cultured under the conditions of industrial process. This indicates a possibility of its employment as a source of this protein involved in manufacturing cholera immunodiagnostic and prophylactic preparations.

[A study of the prevalence of regulatory genes controlling virulence gene expression among Vibrio choleraeeltor biovariant strains varying in their pandemic potential].

The evolution of the genome of the pathogenic agent of the seventh cholera pandemia Vibrio cholerae eltor biovariant was thought to occur by acquiring not only structural genes of virulence but also regulatory systems as a result of horizontal transfer events. The polymerase chain reaction revealed the presence of the following regulatory genes that control the virulence gene expression in the chromosome of pre-pandemic and pandemic strains of cholera vibrios eltor: toxR, toxT, tcpP, tcpH, luxS, luxO, crp, vicH, pepA. The avirulent V. cholerae strain ATCC14033 isolated in 1910 (hypothetical predecessor of the cholera eltor agent) was shown to be lacking the regulatory genes toxT, tcpP, tcpHlocalized in the pathogenicity island VPI-1, and to be capable of realizing positive control over the expression of the virulence genes involved in the ToxR regulon. The virulent strains isolated from cholera patients during the local cholera outbreak in Indonesia in 1937 did not differ from the strains that caused cholera eltor pandemic in 1961. The strains had identical content of the regulatory genes tested. Only one strain of the four isolates studied contained no tcpPgene. Two key regulatory genes, toxR and toxT, were sequenced in all the isolates. The toxR nucleotide sequence of three pre-pandemic strains was shown to be indistinguishable from that of the pandemic isolates. On the other hand, the clinical strain MAK757 isolated prior to the emergence of the epidemic demonstrated an altered nucleotide sequence in its toxR gene. Experiments with the intra-intestinal challenge of suckling rabbits were indicative of similar virulence levels for the pre-pandemic and pandemic clinical strains. These results may serve as the evidence of the in vivo activity of the pre-pandemic strains of the toxT, tcpH, and tcpP positive regulatory genes that acquired in V. cholerae during the evolutionary process.

[Prophage CTXphi genome variability and its role in alteration of Vibrio cholerae El Tor virulence characteristics].

Comparative analysis of CTXphi prophage genome of 366 V. cholerae El Tor strains isolated from infected people and water was carried out using the polymerase chain reaction. Four groups of vibrios, which carry different combinations of ctxA, zot, and ace genes from core region of CTXphi prophage coding key (cholera enterotoxin) and accessory (Zot and Ace toxins) pathogenicity factors, were determined: ctxA(+) zot(-) ace(+), ctxA(-) zot(+) ace(+), ctxA(-) zot(+) ace(-), ctxA(-) zot(-) ace(+). Vibrios that had lost all tested genes were also revealed. Genomic rearrangements occurring in water environment in virulent V. cholerae strains, which acquired foreign pathogenicity genes necessary for their existence in human organism, were proposed as one of the mechanisms of formation of clones with an incomplete or no prophage. Infection process in model animals challenged with wild and isogenic strains of V. cholerae differing in the set of the phage genes (ctxA, zot, and ace) was comparatively analyzed. It was shown that variability of CTXphi prophage genome was an important factor of modification of cholera vibrios virulent characteristics. Obtained data point to usefulness of ctxA, zot, and ace phage genes detection in wild V. cholerae isolates as it could permit evaluation of their virulent potential determining the severity of the infection.

Evaluation of the protective efficacy of Vibrio cholerae ghost (VCG) candidate vaccines in rabbits.

An effective Vibrio cholerae vaccine is needed to reduce the morbidity and mortality caused by this pathogen. Despite the availability of current oral vaccines with measurable efficacy, there is need for more effective vaccines with broad-spectrum efficacy in target populations. Recent studies have shown that bacterial ghosts, produced by the expression of cloned lysis gene E, possess adjuvant properties and are immunogenic. In this study, ghosts were prepared from V. cholerae O1 or O139 and evaluated as vaccines in the reversible intestinal tie adult rabbit diarrhea (RITARD) model. Rabbits were orally immunized with different doses of V. cholerae ghost (VCG) formulations. The vaccine formulations elicited high levels of serum vibriocidal titers against indicator strains. The magnitude of the response was measured as the geometric mean titer (GMT) increase for all rabbits in relation to prevaccination titers. The induction of cross protection was evidenced by the ability of serum from VCG-immunized rabbits to mediate complement-dependent killing of both the homologous and the heterologous strains. Immunized rabbits were protected against intraduodenal challenge 30 days after primary immunization. Protective immunity against challenge appeared to be dose dependent and was associated with marked inhibition of colonization. These results indicate that VCGs represent a novel approach to cholera vaccine development and constitute an effective vaccine delivery vehicle.

[Comparative study of the diagnostic value of new cholera eltor bacteriophages ctx+, ctx-, and KhDF-3, 4, and 5]. / Sravnitel'noe izuchenoe diagnosticheskoi tsennosti novykh bakteriofagov kholernykh él'tor ctx+, ctx-, i KhDF-3, 4, 5]

The comparative evaluation of the diagnostic value of new cholera eltor bacteriophages ctx+ and ctx-, as well as monophages X[symbol: see text]-3, 4, 5, demonstrated their high activity and specificity. Using of these bacteriophages epidemic potential of 95% Vibrio cholerae eltor strains ctx+ and 84.5% of V. cholerae eltor stains ctx- was determined. Commercial monophages X[symbol: see text]-3, 4, 5 were inferior to bacteriophages ctx+ and ctx- in their diagnostic value: only 55% of strains having gene ctxAB were found to be epidemically dangerous, i.e. 45% of strains capable of causing the disease were not detected. On the basis of the results obtained in this investigation cholera eltor bacteriophages ctx+ and ctx- were recommended for introduction into practical use, while further production of cholera diagnostic monophages X[symbol: see text]-3, 4, 5 was recommended to be stopped.

[Strains of Vibrio cholerae serogroups O1 and O139 that produce the basic protective antigens]. / Shtammy Vibrio cholerae serogrupp O1 i O139--produtsenty osnovnykh protektivnykh antigenov.

To find out stable and effective producers of major protective antigens intended for use as components of cholera chemical vaccine against V. cholerae strains of serogroups O and O139, the comparative analysis of the production of cholera toxin, toxin-coregulated pili (TCP), antigens O1 and O139, polysaccharide capsule and outer membrane protein OmpU in different V. cholerae strains groups O1 and O139 has been made. V. cholerae strain KM68, serogroup O1, has been found capable of the production of antigen O1, serovar Ogawa, protein OmpU at a sufficiently high level and the hyperproduction of cholera toxin and TCP, and thus suitable for use in the manufacture of cholera bivalent vaccine as the source of these antigens. Specially selected alysogenic noncapsular strain KM137 of serogroup O139, characterized by a high and stable level of the biosynthesis of this somatic antigen when grown in both laboratory and production conditions, may serve as the produces of antigen O139.

[Vibrio cholerae temperate phage O139: characteristics and role in changing expression of chromosomal virulence genes]. / Umerennyi fag Vibrio cholerae O139: kharakteristika i rol' v izmenenii ékspressii khromosomnykh genov virulentnosti.

Restriction analysis of temperate cholera phage 139 isolated from Vibrio cholerae P16064, serogroup 0139, showed its DNA to be double-stranded linear with cohesive terminals. DNA-DNA hybridization on nylon membranes revealed that many V. cholerae strains of serogroup 0139 isolated in different regions contained a temperate cholera phage 139 in their genomes. Southern blot hybridization of chromosomal DNA PST-fragments with phage probe showed that the temperate phage 139 was identical to the temperate phage of serogroup II V. eltor. The phage integrated in the chromosome near genes encoding motility (mot) and production of the capsule (cap) and purine (pur). Phage genome is apparently responsible for instability of cap, pur, and mot genes whose products are important for the development of an infectious process in cholera.

[Some immunochemical and immunobiological properties of Vibrio cholerae serovar 0139 capsule antigen]. / Nekotorye immunokhimicheskie i immunobiologicheskie svoistva kapsul'nogo antigena Vibrio cholerae 0139 serovara.

Capsular antigen (C-antigen) was isolated from culture fluid of V. cholerae serovar 0139 strain P16064 4-fold fractionation by (NH4)2SO4 after Weinblatt (1980). Electrophoretic separation in PAAG-SDS revealed two protein subunits with molecular weights 38 +/- 2 and 61 +/- 2 KD in the C-antigen and whole-cell lysates of two strains (MO45 and P16064) of V. cholerae serovar 0139. Murine polyclonal anticapsular antibodies reacted with these protein subunits in immunoblotting, as well as with two rapidly migrating carbohydrate components. After proteinase treatment of the C-antigen and cell lysates from opaque colonies these antibodies helped detect two high-molecular zones in addition to rapidly migrating ones. C-antigen protects from death 75% of mice infected with V. cholerae 0.139 and 43% (P < 0.05) of those infected with V. cholerae eltor 230. The protective properties of C-antigen are due to lipopolysaccharide (LPS) but not to the protein component. The immunogenicity of LPS contained in the C-antigen is selective, whereas LPS isolated from biomass of V. cholerae 0139 cells possesses similar protective activity towards the homologous strain (49.5%) and the agent of Eltor cholera (50.0%).

[Emergence of toxigenic Vibrio cholerae strains on non-O1 serotype as a result of the exchange of genetic information]. / Vozniknovenie toksigennykh shtammov Vibrio cholerae ne-O1 serogruppy v rezul'tate obmena geneticheskoi informatsiei.

To study the possibilities of genetic exchange between Vibrio cholerae of O1 and non-O1 serogroups, donor and recipient strains were developed. It was shown that toxicogenic strains of V. cholerae non-O1 appeared in vitro and in vivo as the result of conjugative transfer of rfb-NAG genes from avirulent V. cholerae non-O1 strains to toxicogenic strains belonging to V. cholerae O1 classical and eltor biovars. These genes are responsible for synthesis of O antigen of non-O1 serotype. It was established that foreign rfb-NAG genes have no effect on virulence properties of a causative agent of cholera. Apparently, pathogenic V. cholerae non-O1 strains with cholera toxin genes are generated due to transfer of rfb-NAG genes under natural conditions.

[Creating a system of conjugated chromosome transfer and genetic mapping of Vibrio cholerae serogroup O139 chromosomes]. / Sozdanie sistemy kon''iugatsionnogo perenosa khromosomy i geneticheskoekartirovanie khromosomy Vibrio cholerae serogruppy O139.

A conjugational gene transfer system consisting of donor and recipient strains has been developed for genetic analysis of Vibrio cholerae 0139 serogroup, a new cholera agent. Donor strains constructed using the Tn5-Mob carrying the origin of transfer (ori T) of plasmid RP4 and helper plasmid pRP4-4 were able to perform a directed transfer of chromosomal markers. Recipient strains carried mutations in auxotrophic genes as well as in virulence genes. Based on this gene transfer system, a genetic map of V. cholerae chromosome has been created showing the order of 17 gene markers. Relationship between the genetic structure of V. cholerae 0139 and 01 is discussed.

Virulence-associated characteristics and phage lysogenicity of two morphologically distinct colonies of Vibrio cholerae O139 serogroup.

The presence of a temperate phage was demonstrated in a strain of Vibrio cholerae O139 isolated from a patient. Spontaneous variants with translucent colonies had lost this phage. The loss of the phage was associated with increased hydrophobicity, indicating the loss of the capsule. These clones were sensitive to serum bactericidal activity, showed decreased expression of such presumed virulence factors as proteases, motility and mannose-sensitive pili. Furthermore, excision of the phage made the strain dependent on purines for growth.

Construction and characterization of recombinant Vibrio cholerae strains carrying heterologous genes encoding non-01 antigen or cholera enterotoxin.

In an attempt to study the effect of heterologous genes on the virulence of Vibrio cholerae 01 and non-01, rfb genes encoding biosynthesis of non-01 antigens were introduced by homologous recombination into the chromosome of V. cholerae 01 strain 569B (serotype Inaba, biotype classical). Recombinant strains were obtained which were not agglutinated with the diagnostic cholera 01 antiserum and were not sensitive to the cholera diagnostic bacteriophage, but produced as much cholera toxin as 569B and were highly virulent in the infant rabbit intraintestinal injection model. These data indicate that the rfb genes from the studied V. cholerae non-01 did not alter the virulence phenotype of V. cholerae 01. In contrast, cloned ctxAB genes from V. cholerae 01 encoding cholera toxin introduced into a non-pathogenic strain lead to efficient secretion of cholera toxin but to only low virulence in the infant rabbit model.

[Phenotypic analysis of two morphologically different types of Vibrio cholerae 0139 colonies]. / Fenotipicheskii analiz dvukh morfologicheski raznykh tipov kolonii Vibrio cholerae 0139.

A clinical isolate Vibrio cholerae P16064 serogroup 0139 was shown to produce two different kinds of colonies: opaque encapsulated and translucent devoid of capsule. Low virulence of translucent colonies seems to be due to not only loss of capsule which determines serum resistance, but also to decreased expression of genes controlling the motility, and production of protease and mannose-sensitive adhesion pili. Analysis of the lysogenic properties of the two types of colonies permitted us to propose that simultaneous spontaneous alteration of capsule production and the above-mentioned virulence factors in translucent colonies may be caused by a temperate phage in turbid clones of strain P16064.

[Mapping a genetic determinant determining the increased synthesis of cholera toxin by the Dakka 35 strain of Vibrio cholerae]. / Kartirovanie geneticheskogo determinanta, opredeliaiushchego povyshennyi sintez kholernogo toksina shtammom Vibrio cholerae Dakka 35.

A new mutation tox-2 defining the increased level of cholera exotoxin production by the strain Vibrio cholerae Dakka 35 isolated from nature has been mapped by conjugational crosses of donor and recipient strains differing by toxin production and serovar. The mutation has been localized on the chromosomal fragment containing the ilv, pur, ura, rfb genes adjacent to ura-94 locus. The linkage of the tox-2 mutation with the rfb locus coding for the synthesis of somatic Ol-antigen has been also established.

[The isolation of Vibrio cholerae strains that produce cholera toxin and its B-subunit]. / Poluchenie shtammov Vibrio cholerae--produtsentov kholernogo toksina i B-sub''edinitsy kholernogo toksina.

The creation of two V. cholerae strains, belonging to the classical biovar, serovars Ogawa and Inaba, and serving as hyperproducers of cholera toxin, is described. The strains have been created by means of recombinant plasmid pCO107-2, carrying the cloned gene of cholera toxin vctAB. The use of recombinant plasmid pIEMZ3 carrying the cloned gene of the B-subunit of cholera toxin vctB has made it possible to obtain a strain, capable of producing the B-subunit of cholera toxin, on the basis of V. cholerae strain non 01. Besides, V. cholerae isogenic toxigenic strains, serovars Inaba and Ogawa, with the increased production of the B-subunit of cholera toxin, have been obtained.

[The capacity to produce cholera exotoxin in natural strains of Vibrio cholerae]. / Izuchenie sposobnosti k produktsii kholernogo ékzotoksina u prirodnykh shtammov Vibrio cholerae.

The study of 27 V. cholerae strains, isolated from cholera patients and found to be hemolytically inactive, with a view to establish their capacity for the production of cholera toxin has revealed that 4 strains (V. cholerae cholerae Dacca 35, V. cholerae cholerae Dacca 3, V. cholerae cholerae B1307, V. cholerae cholerae J89) produce this protein. The quantitative determination of enterotoxin has been made with the use of GM1 ELISA technique. Strain Dacca 35 has been found to be highly toxigenic and, as regards the amount of exotoxin it produces, no different from V. cholerae cholerae strain 569B, a well-known producer of cholera toxin. In strain Dacca 35 correlation between the capacity of the cells for toxin production and the morphology of colonies has been established. The study has revealed that the chromosome of strain Dacca 35 contains two copies of gene vctAB responsible for the synthesis of cholera toxin.

[Inheritance and expression of cholera toxin genes introduced into Vibrio cholerae el tor cells in a hybrid plasmid]. / Osobennosti nasledovaniia i ékspressii genov kholernogo toksina, vvedennykh v kletki Vibrio cholerae eltor v sostave gibridnoi plazmidy.

Two replicons, pOX38 (in F-factor derivative lacking all IS elements) and pCT105 (containing cholera toxin operon cloned in pBR322) have been fused to produce recombinant plasmid, pCO109, which was then introduced into Vibrio cholerae eltor by conjugation. Restriction analysis showed pCO109 to dissociate in V. cholerae cells at a higher frequency than in Escherichia coli strains, its pOX38 component being lost, while the pCT105 component demonstrated relative stability. V. cholerae eltor RV79 (pCT105) produced 4-5 micrograms/ml of cholera toxin. Occasional insertion of cloned vctA, B operon into RV79 chromosome was also observed.

[Genetic mapping of the regulatory gene determining the synthesis of enterotoxin in Vibrio cholerae]. / Geneticheskoe kartirovanie reguliatornogo gena, opredeliaiushchego sintez énterotoksina u Vibrio cholerae.

The data of genetic mapping of the cholera toxin regulatory gene by conjugation mating of Vibrio cholerae eltor donor strain with V. cholerae classica recipients are presented. The close genetic linkage of tox locus to pur-63 is shown. The gene order asp - cys - nal - pur-61 - trp - his - pur-63 - tox - ile of the chromosomal region examined is established.

[Genetic analysis of mutations affecting the synthesis of somatic O-antigen of Vibrio cholerae]. / Geneticheskii analiz mutatsii, vliiaiushchikh na sintez somaticheskogo O-antigena u kholernogo vibriona.

The mutations oagM and oagR affecting the synthesis of somatic O-antigen have been localized on the chromosome of Vibrio cholerae El Jor and classic biotypes by conjugational crosses between different donor and recipient strains. The mutations are localized in the vicinity of the arg marker in both classic and El Tor biotypes of Vibrio cholerae.