Construction of a recombinant live oral vaccine from a non-toxigenic strain of Vibrio cholerae O1 serotype inaba biotype E1 Tor and assessment of its reactogenicity and immunogenicity in the rabbit model.

The disease cholera is an important cause of mortality in many developing countries. Though it can be controlled through improved sanitation, this goal is not easily attainable in many countries. Development of an efficacious vaccine offers the best immediate solution. A new oral candidate vaccine has been constructed from a non-toxigenic strain of Vibrio cholerae E1 Tor, Inaba, which is not only devoid of the cholera toxin (CT) virulence cassette but also is completely non-reactogenic in rabbit ileal loop assay. The strain, however, had toxR and tcpA genes. Through a series of manipulations, the ctxB gene of V. cholerae, responsible for the production of the 'B' subunit of the cholera toxin (CTB) was introduced into the cryptic hemolysin locus of the strain. The resulting strain, named vaccine attempt 1.3 (VA1.3), was found to be able to produce copious amounts of CTB. In the RITARD model this strain was found to be non-reactogenic and provided full protection against the challenge doses of both V. cholerae O1, classical and E1 Tor. In the immunized rabbit it invoked significant levels of anti-bacterial and anti-toxin immunity.     

Randomized, controlled human challenge study of the safety, immunogenicity, and protective efficacy of a single dose of Peru-15, a live attenuated oral cholera vaccine

Peru-15 is a live attenuated oral vaccine derived from a Vibrio cholerae O1 El Tor Inaba strain by a series of deletions and modifications, including deletion of the entire CT genetic element. Peru-15 is also a stable, motility-defective strain and is unable to recombine with homologous DNA. We wished to determine whether a single oral dose of Peru-15 was safe and immunogenic and whether it would provide significant protection against moderate and severe diarrhea in a randomized, double-blind, placebo-controlled human volunteer cholera challenge model. A total of 59 volunteers were randomly allocated to groups to receive either 2 x 10(8) CFU of reconstituted, lyophilized Peru-15 vaccine diluted in CeraVacx buffer or placebo (CeraVacx buffer alone). Approximately 3 months after vaccination, 36 of these volunteers were challenged with approximately 10(5) CFU of virulent V. cholerae O1 El Tor Inaba strain N16961, prepared from a standardized frozen inoculum. Among vaccinees, 98% showed at least a fourfold increase in vibriocidal antibody titers. After challenge, 5 (42%) of the 12 placebo recipients and none (0%) of the 24 vaccinees had moderate or severe diarrhea (> or = 3,000 g of diarrheal stool) (P = 0.002; protective efficacy, 100%; lower one-sided 95% confidence limit, 75%). A total of 7 (58%) of the 12 placebo recipients and 1 (4%) of the 24 vaccinees had any diarrhea (P < 0.001; protective efficacy, 93%; lower one-sided 95% confidence limit, 62%). The total number of diarrheal stools, weight of diarrheal stools, incidence of fever, and peak stool V. cholerae excretion among vaccinees were all significantly lower than in placebo recipients. Peru-15 is a well-tolerated and immunogenic oral cholera vaccine that affords protective efficacy against life-threatening cholera diarrhea in a human volunteer challenge model. This vaccine may therefore be a safe and effective tool to prevent cholera in travelers and is a strong candidate for further evaluation to prevent cholera in an area where cholera is endemic.     

The vaccine candidate Vibrio cholerae 638 is protective against cholera in healthy volunteers

Vibrio cholerae 638 is a living candidate cholera vaccine strain attenuated by deletion of the CTXPhi prophage from C7258 (O1, El Tor Ogawa) and by insertion of the Clostridium thermocellum endoglucanase A gene into the hemagglutinin/protease coding sequence. This vaccine candidate was previously found to be well tolerated and immunogenic in volunteers. This article reports a randomized, double-blind, placebo-controlled trial conducted to test short-term protection conferred by 638 against subsequent V. cholerae infection and disease in volunteers in Cuba. A total of 45 subjects were enrolled and assigned to receive vaccine or placebo. The vaccine contained 10(9) CFU of freshly harvested 638 buffered with 1.3% NaHCO(3), while the placebo was buffer alone. After vaccine but not after placebo intake, 96% of volunteers had at least a fourfold increase in vibriocidal antibody titers, and 50% showed a doubling of at least the lipopolysaccharide-specific immunoglobulin A titers in serum. At 1 month after vaccination, five volunteers from the vaccine group and five from the placebo group underwent an exploratory challenge study with 10(9) CFU of DeltaCTXPhi attenuated mutant strain V. cholerae 81. Only two volunteers from the vaccine group shed strain 81 in their feces, but none of them experienced diarrhea; in the placebo group, all volunteers excreted the challenge strain, and three had reactogenic diarrhea. An additional 12 vaccinees and 9 placebo recipients underwent challenge with 7 x 10(5) CFU of virulent strain V. cholerae 3008 freshly harvested from a brain heart infusion agar plate and buffered with 1.3% NaHCO(3). Three volunteers (25%) from the vaccine group and all from the placebo group shed the challenge agent in their feces. None of the 12 vaccinees but 7 volunteers from the placebo group had diarrhea, and 2 of the latter exhibited severe cholera (>5,000 g of diarrheal stool). These results indicate that at 1 month after ingestion of a single oral dose (10(9) CFU) of strain 638, volunteers remained protected against cholera infection and disease provoked by the wild-type challenge agent V. cholerae 3008. We recommend that additional vaccine lots of 638 be prepared under good manufacturing practices for further evaluation.     

Alternative Mechanism of Cholera Toxin Acquisition by Vibrio cholerae: Generalized Transduction of CTXΦ by Bacteriophage CP-T1

Horizontal transfer of genes encoding virulence factors has played a central role in the evolution of many pathogenic bacteria. The unexpected discovery that the genes encoding cholera toxin (ctxAB), the main cause of the profuse secretory diarrhea characteristic of cholera, are encoded on a novel filamentous phage named CTXPhi, has resulted in a renewed interest in the potential mechanisms of transfer of virulence genes among Vibrio cholerae. We describe here an alternative mechanism of cholera toxin gene transfer into nontoxigenic V. cholerae isolates, including strains that lack both the CTXPhi receptor, the toxin coregulated pilus (TCP), and attRS, the chromosomal attachment site for CTXPhi integration. A temperature-sensitive mutant of the V. cholerae generalized transducing bacteriophage CP-T1 (CP-T1ts) was used to transfer a genetically marked derivative of the CTX prophage into four nontoxigenic V. cholerae strains, including two V. cholerae vaccine strains. We demonstrate that CTXPhi transduced by CP-T1ts can replicate and integrate into these nontoxigenic V. cholerae strains with high efficiency. In fact, CP-T1ts transduces the CTX prophage preferentially when compared with other chromosomal markers. These results reveal a potential mechanism by which CTXPhi(+) V. cholerae strains that lack the TCP receptor may have arisen. Finally, these findings indicate an additional pathway for reversion of live-attenuated V. cholerae vaccine strains.     

Lysogenic conversion of environmental Vibrio mimicus strains by CTXPhi.

The filamentous bacteriophage CTXPhi, which encodes cholera toxin (CT) in toxigenic Vibrio cholerae, is known to propagate by infecting susceptible strains of V. cholerae by using the toxin coregulated pilus (TCP) as its receptor and thereby causing the origination of new strains of toxigenic V. cholerae from nontoxigenic progenitors. Besides V. cholerae, Vibrio mimicus strains which are normally TCP negative have also been shown to occasionally produce CT and cause diarrhea in humans. We analyzed nontoxigenic V. mimicus strains isolated from surface waters in Bangladesh for susceptibility and lysogenic conversion by CTXPhi and studied the expression of CT in the lysogens by using genetically marked derivatives of the phage. Of 27 V. mimicus strains analyzed, which were all negative for genes encoding TCP but positive for the regulatory gene toxR, 2 strains (7.4%) were infected by CTX-KmPhi, derived from strain SM44(P27459 ctx::km), and the phage genome integrated into the host chromosome, forming stable lysogens. The lysogens spontaneously produced infectious phage particles in the supernatant fluids of the culture, and high titers of the phage could be achieved when the lysogens were induced with mitomycin C. This is the first demonstration of lysogenic conversion of V. mimicus strains by CTXPhi. When a genetically marked derivative of the replicative form of the CTXPhi genome carrying a functional ctxAB operon, pMSF9.2, was introduced into nontoxigenic V. mimicus strains, the plasmid integrated into the host genome and the strains produced CT both in vitro and inside the intestines of adult rabbits and caused mild-to-severe diarrhea in rabbits. This suggested that in the natural habitat infection of nontoxigenic V. mimicus strains by wild-type CTXPhi may lead to the origination of toxigenic V. mimicus strains which are capable of producing biologically active CT. The results of this study also supported the existence of a TCP-independent mechanism for infection by CTXPhi and showed that at least one species of Vibrio other than V. cholerae may contribute to the propagation of the phage.     

Sunlight-induced propagation of the lysogenic phage encoding cholera toxin

In toxigenic Vibrio cholerae, the cholera enterotoxin (CT) is encoded by CTXPhi, a lysogenic bacteriophage. The propagation of this filamentous phage can result in the origination of new toxigenic strains. To understand the nature of possible environmental factors associated with the propagation of CTXPhi, we examined the effects of temperature, pH, salinity, and exposure to direct sunlight on the induction of the CTX prophage and studied the transmission of the phage to potential recipient strains. Exposure of cultures of CTXPhi lysogens to direct sunlight resulted in approximately 10,000-fold increases in phage titers. Variation in temperature, pH, or salinity of the culture did not have a substantial effect on the induction of the prophage, but these factors influenced the stability of CTXPhi particles. Exposure of mixed cultures of CTXPhi lysogens and potential recipient strains to sunlight significantly increased both the in vitro and in vivo (in rabbit ileal loops) transduction of the recipient strains by CTXPhi. Included in these transduction experiments were two environmental nontoxigenic (CTXPhi(-)) strains of V. cholerae O139. These two O139 strains were transduced at high efficiency by CTXPhi, and the phage genome integrated into the O139 host chromosome. The resulting CTXPhi lysogens produced biologically active CT both in vitro and in rabbit ileal loops. This finding suggests a possible mechanism explaining the origination of toxigenic V. cholerae O139 strains from nontoxigenic progenitors. This study indicates that sunlight is a significant inducer of the CTX prophage and suggests that sunlight-induced transmission of CTXPhi may constitute part of a natural mechanism for the origination of new toxigenic strains of V. cholerae.     

Live Oral Cholera Vaccine: Evaluation of the Clinical Effectiveness of Two Strains in Humans

El Tor Ogawa C14-S5 and EW-6, two live vaccine candidate strains, were given to volunteers in varying doses with and without bicarbonate. Vibrios were found in the stool of one of 32 men given the vaccine strain, and only three men developed a significant titer rise (fourfold or greater) at 2 weeks of vibriocidal or antitoxic antibody. Five men who had previously received 10⁹ organisms of the C14-S5 strain were challenged subsequently with virulent Ogawa 395 Vibrio cholerae. The rate of clinical infection in these men was no different than in unvaccinated controls. It was demonstrated that the live oral cholera vaccines did not remain viable in the intestine long enough to act antigenically.     

Pathogenic potential of environmental Vibrio cholerae strains carrying genetic variants of the toxin-coregulated pilus pathogenicity island

The major virulence factors of toxigenic Vibrio cholerae are cholera toxin (CT), which is encoded by a lysogenic bacteriophage (CTXPhi), and toxin-coregulated pilus (TCP), an essential colonization factor which is also the receptor for CTXPhi. The genes for the biosynthesis of TCP are part of a larger genetic element known as the TCP pathogenicity island. To assess their pathogenic potential, we analyzed environmental strains of V. cholerae carrying genetic variants of the TCP pathogenicity island for colonization of infant mice, susceptibility to CTXPhi, and diarrheagenicity in adult rabbits. Analysis of 14 environmental strains, including 3 strains carrying a new allele of the tcpA gene, 9 strains carrying a new allele of the toxT gene, and 2 strains carrying conventional tcpA and toxT genes, showed that all strains colonized infant mice with various efficiencies in competition with a control El Tor biotype strain of V. cholerae O1. Five of the 14 strains were susceptible to CTXPhi, and these transductants produced CT and caused diarrhea in adult rabbits. These results suggested that the new alleles of the tcpA and toxT genes found in environmental strains of V. cholerae encode biologically active gene products. Detection of functional homologs of the TCP island genes in environmental strains may have implications for understanding the origin and evolution of virulence genes of V. cholerae.     

Examination of diverse toxin-coregulated pilus-positive Vibrio cholerae strains fails to demonstrate evidence for Vibrio pathogenicity island phage

The major virulence factors of toxigenic Vibrio cholerae are cholera toxin, which is encoded by a lysogenic filamentous bacteriophage (CTXPhi), and toxin-coregulated pilus (TCP), an essential colonization factor that is also the receptor for CTXPhi. The genes involved in the biosynthesis of TCP reside in a pathogenicity island, which has been reported to correspond to the genome of another filamentous phage (designated VPIPhi) and to encode functions necessary for the production of infectious VPIPhi particles. We examined 46 V. cholerae strains having diverse origins and carrying different genetic variants of the TCP island for the production of the VPIPhi and CTXPhi in different culture conditions, including induction of prophages with mitomycin C and UV irradiation. Although 9 of 10 V. cholerae O139 strains and 12 of 15 toxigenic El Tor strains tested produced extracellular CTXPhi, none of the 46 TCP-positive strains produced detectable VPIPhi in repeated assays, which detected as few as 10 particles of a control CTX phage per ml. These results contradict the previous report regarding VPIPhi-mediated horizontal transfer of the TCP genes and suggest that the TCP island is unable to support the production of phage particles. Further studies are necessary to understand the mechanism of horizontal transfer of the TCP island.     

Strong biotype and serotype cross-protective antibacterial and antitoxic immunity in rabbits after cholera infection

We studied whether immunity evoked by infection with classical or El Tor V. cholerae 01 organisms in rabbits (the RITARD model) also gives protection against cholera caused by V. cholerae of heterologous biotype as well as serotype, and whether such protection is antibacterial and/or antitoxic. A primary infection with a classical Ogawa or El Tor Inaba strain resulted in intestinal colonization and diarrheal disease in a dose-related manner though the El Tor strain was more virulent. As few as 10(3) El Tor organisms gave disease in more than 90% of the rabbits as compared to 10(9) classical organisms (ED90); the El Tor strain also gave rise to diarrhea with earlier onset and of greater severity and longer duration. The primary infection induced strong protective immunity against later challenge with either the homologous or the heterologous strain in doses that corresponded to 1,000 x ED90. Protection was associated with marked inhibition of colonization, and when rabbits convalescing from cholera infection were challenged with graded doses of bacteria or purified toxin in ligated intestinal loops significant antibacterial as well as antitoxic immunity was evident. Titer rises in serum vibriocidal and anti-lipopolysaccharide antibodies were similar after infection with either strain, whilst antitoxin titer rises were more marked after El Tor infection. During infection V. cholerae 01 organisms seem to express protective antigens that stimulate immunity which extends across both biotype and serotype barriers.     

Emergence and progression of Vibrio cholerae O1 El Tor variants and progenitor strains of Mozambique variants in Kolkata, India

Analysis of 75 Vibrio cholerae O1 strains isolated from hospitalized patients in Kolkata from 1989 to 1994 revealed the existence of true El Tor along with El Tor variants that possessed the classical allele of ctxB (ctxB(cl)) in strains having an El Tor backbone. Based on the existence of different combinations of ctxB and rstR alleles and their localization sites in the genome, these strains were classified into multiple genetic groups. Of 75 clinical strains, 11 were identified as non-toxigenic. These 8 strains were also devoid of pTLC, which is uncommon among the O1 strains. However, Mozambique variants isolated in 2004 were typically negative for pTLC, but these strains possessed tandemly arranged CTX prophages with ctxB(cl) in the small chromosome. Genetic manipulation studies with laboratory-generated kanamycin-tagged pCTX-Kan (derived from tandemly arranged small chromosome-localized ctxB(cl) bearing CTX prophages of 1992 VC53, a progenitor strain of the Mozambique variant) demonstrated that integration specificity of the pCTX-Kan was somewhat towards small chromosome. Such integration could be the prime step towards generation of the Mozambique variant. Based on the existence of multiple alleles of CTX? and their infections with non-toxigenic strains, we propose that the El Tor variant strains could have emerged following these genetic events. This study demonstrated existence of different 'intermediate strains' in a time frame that overlapped with a period of V. cholerae O139 emergence. Identification of these intermediate strains gave impetus to believe stepwise generation of the El Tor variants, and all these events profoundly influenced V. cholerae epidemiology in the following years.     

Immunogenicity of Vibrio cholerae O1 toxin-coregulated pili in experimental and clinical cholera.

A functional tcpA gene, encoding the major subunit of toxin-coregulated pili (TCP), is necessary for Vibrio cholerae O1 Ogawa strain 395 to colonize the human intestine and confer protective immunity to virulent challenge. The immunogenicity of TCP and other antigens in experimental and naturally acquired cholera was determined. Seroconversion to cholera toxin (CT), whole cell preparations, and to Ogawa lipopolysaccharide but not to purified native TCP or to a TcpA mimiotope was found in volunteers. Local intestinal secretory immunoglobulin A from volunteers showed conversions to cholera toxin and lipopolysaccharide but not to TCP. Cholera patients in Indonesia showed a seroconversion rate to TCP of 3 of 6 and a seroconversion to a TcpA mimiotope of 1 of 6. Volunteer and patient sera showed similar vibriocidal seroconversions when assayed against either TCP-positive and TCP-negative V. cholerae O1 strains, suggesting that TCP do not contribute demonstrably to the vibriocidal antigen. We conclude that although seroconversion to TCP can occur in naturally acquired cholera, solid long-term protection can be engendered in the absence of a detectable anti-TCP immune response.     

Randomized, double-blind, placebo-controlled, multicentered trial of the efficacy of a single dose of live oral cholera vaccine CVD 103-HgR in preventing cholera following challenge with Vibrio cholerae O1 El tor inaba three months after vaccination

CVD 103-HgR is a live oral cholera vaccine strain constructed by deleting 94% of the gene for the enzymatically active A subunit of cholera toxin from classical Inaba Vibrio cholerae O1 569B; the strain also contains a mercury resistance gene as an identifying marker. This vaccine was well tolerated and immunogenic in double-blind, controlled studies and was protective in open-label studies of volunteers challenged with V. cholerae O1. A randomized, double-blind, placebo-controlled, multicenter study of vaccine efficacy was designed to test longer-term protection of CVD 103-HgR against moderate and severe El Tor cholera in U.S. volunteers. A total of 85 volunteers (50 at the University of Maryland and 35 at Children's Hospital Medical Center/University of Cincinnati) were recruited for vaccination and challenge with wild-type V. cholerae El Tor Inaba. Volunteers were randomized in a double-blind manner to receive, with buffer, a single oral dose of either CVD 103-HgR (2 x 10(8) to 8 x 10(8) CFU) or placebo (killed E. coli K-12). About 3 months after immunization, 51 of these volunteers were orally challenged with 10(5) CFU of virulent V. cholerae O1 El Tor Inaba strain N16961, prepared from a standardized frozen inoculum. Ninety-one percent of the vaccinees had a >/=4-fold rise in serum vibriocidal antibodies after vaccination. After challenge, 9 (39%) of the 23 placebo recipients and 1 (4%) of the 28 vaccinees had moderate or severe diarrhea (>/=3-liter diarrheal stool) (P < 0.01; protective efficacy, 91%). A total of 21 (91%) of 23 placebo recipients and 5 (18%) of 28 vaccinees had any diarrhea (P < 0.001; protective efficacy, 80%). Peak stool V. cholerae excretion among placebo recipients was 1.1 x 10(7) CFU/g and among vaccinees was 4.9 x 10(2) CFU/g (P < 0.001). This vaccine could therefore be a safe and effective tool to prevent cholera in travelers.     

Characterization of Vibrio cholerae O1 El Tor biotype variant clinical isolates from Bangladesh and Haiti, including a molecular genetic analysis of virulence genes

Vibrio cholerae serogroup O1, the causative agent of the diarrheal disease cholera, is divided into two biotypes: classical and El Tor. Both biotypes produce the major virulence factors toxin-coregulated pilus (TCP) and cholera toxin (CT). Although possessing genotypic and phenotypic differences, El Tor biotype strains displaying classical biotype traits have been reported and subsequently were dubbed El Tor variants. Of particular interest are reports of El Tor variants that produce various levels of CT, including levels typical of classical biotype strains. Here, we report the characterization of 10 clinical isolates from the International Centre for Diarrhoeal Disease Research, Bangladesh, and a representative strain from the 2010 Haiti cholera outbreak. We observed that all 11 strains produced increased CT (2- to 10-fold) compared to that of wild-type El Tor strains under in vitro inducing conditions, but they possessed various TcpA and ToxT expression profiles. Particularly, El Tor variant MQ1795, which produced the highest level of CT and very high levels of TcpA and ToxT, demonstrated hypervirulence compared to the virulence of El Tor wild-type strains in the infant mouse cholera model. Additional genotypic and phenotypic tests were conducted to characterize the variants, including an assessment of biotype-distinguishing characteristics. Notably, the sequencing of ctxB in some El Tor variants revealed two copies of classical ctxB, one per chromosome, contrary to previous reports that located ctxAB only on the large chromosome of El Tor biotype strains.     

RS1 element of Vibrio cholerae can propagate horizontally as a filamentous phage exploiting the morphogenesis genes of CTXphi.

In toxigenic Vibrio cholerae, cholera toxin is encoded by the CTX prophage, which consists of a core region carrying ctxAB genes and genes required for CTXPhi morphogenesis, and an RS2 region encoding regulation, replication, and integration functions. Integrated CTXPhi is often flanked by another genetic element known as RS1 which carries all open reading frames (ORFs) found in RS2 and an additional ORF designated rstC. We identified a single-stranded circularized form of the RS1 element, in addition to the CTXPhi genome, in nucleic acids extracted from phage preparations of 32 out of 83 (38.5%) RS1-positive toxigenic V. cholerae strains analyzed. Subsequently, the corresponding double-stranded replicative form (RF) of the RS1 element was isolated from a representative strain and marked with a kanamycin resistance (Km(r)) marker in an intergenic site to construct pRS1-Km. Restriction and PCR analysis of pRS1-Km and sequencing of a 300-bp region confirmed that this RF DNA was the excised RS1 element which formed a novel junction between ig1 and rstC. Introduction of pRS1-Km into a V. cholerae O1 classical biotype strain, O395, led to the production of extracellular Km(r) transducing particles, which carried a single-stranded form of pRS1-Km, thus resembling the genome of a filamentous phage (RS1-KmPhi). Analysis of V. cholerae strains for susceptibility to RS1-KmPhi showed that classical biotype strains were more susceptible to the phage compared to El Tor and O139 strains. Nontoxigenic (CTX(-)) O1 and O139 strains which carried genes encoding the CTXPhi receptor toxin-coregulated pilus (TCP) were also more susceptible (>1,000-fold) to the phage compared to toxigenic El Tor or O139 strains. Like CTXPhi, the RS1Phi genome also integrated into the host chromosomes by using the attRS sequence. However, only transductants of RS1-KmPhi which also harbored the CTXPhi genome produced a detectable level of extracellular RS1-KmPhi. This suggested that the core genes of CTXPhi are also required for the morphogenesis of RS1Phi. The results of this study showed for the first time that RS1 element, which encodes a site-specific recombination system in V. cholerae, can propagate horizontally as a filamentous phage, exploiting the morphogenesis genes of CTXPhi.     

霍乱弧菌毒力表达调控基因toxR缺失株的构建及其功能的研究

目的　通过构建霍乱弧菌toxR基因缺失株来研究toxR基因对霍乱弧菌减毒菌株IEM101和高产毒株569B毒力表达的调控作用。方法　采用自杀性质粒和接合转移技术，将2个中间含有四环素基因的toxR基因分别与霍乱弧菌减毒株IEM101和高产毒株569B染色体toxR基因重组，从而获得toxR基因缺失株IEM101-4和569B-43，并对2个toxR基因缺失株和其原出发菌株的霍乱肠毒素的产率和主要外膜蛋白图谱进行比较。结果　采用GM1-ELISA检测受测菌CT基因表达，toxR基因缺失株569B-43的P/N值为1.82，而其原出发菌株569B的P/N为4.52，而IEM101和其toxR基因缺失株的P/N值均低于2。采用SDS-PAGE对受试菌外膜蛋白进行分析，toxR基因缺失株569B和IEM101的外膜蛋白图谱相比，均多出2条相对分子质量（Mr）为40×103和43×103外膜蛋白区带。结论　toxR蛋白是霍乱肠毒素基因ctx表达的正调控因子，是霍乱弧菌主要外膜蛋白（Mr为40×103和43×103)编码基因的负调控因子。     

Mucosal and Systemic Immune Responses in Humans after Primary and Booster Immunizations with Orally Administered Invasive and Noninvasive Live Attenuated Bacteria

The mucosal and systemic immune responses after primary and booster immunizations with two attenuated live oral vaccine strains derived from a noninvasive (Vibrio cholerae) and an invasive (Salmonella typhi) enteric pathogen were comparatively evaluated. Vaccination with S. typhi Ty21a elicited antibody-secreting cell (ASC) responses specific for S. typhi O9, 12 lipopolysaccharide (LPS), as well as significant increases in levels of immunoglobulin G (IgG) and IgA antibodies to the same antigen in serum. A strong systemic CD4(+) T-helper type 1 cell-mediated immune (CMI) response was also induced. In contrast to results with Ty21a, no evidence of a CMI response was obtained after primary immunization with V. cholerae CVD 103-HgR in spite of the good immunogenicity of the vaccine. Volunteers who received a single dose of CVD 103-HgR primarily developed an IgM ASC response against whole vaccine cells and purified V. cholerae Inaba LPS, and seroconversion of serum vibriocidal antibodies occurred in four of five subjects. Serum IgG anti-cholera toxin antibody titers were of lower magnitude. For both live vaccines, the volunteers still presented significant local immunity 14 months after primary immunization, as revealed by the elevated baseline antibody titers at the time of the booster immunization and the lower ASC, serum IgG, and vibriocidal antibody responses after the booster immunization. These results suggest that local immunity may interfere with colonization of the gut by both vaccine strains at least up to 14 months after basis immunization. Interestingly, despite a low secondary ASC response, Ty21a was able to boost both humoral (anti-LPS systemic IgG and IgA) and CMI responses. Evidence of a CMI response was also observed for one of three volunteers given a cholera vaccine booster dose. The direct comparison of results with two attenuated live oral vaccine strains in human volunteers clearly showed that the capacity of the vaccine strain to colonize specific body compartments conditions the pattern of vaccine-induced immune responses.     

El Tor and Calcutta CTXPhi precursors coexisting with intact CTXPhi copies in Vibrio cholerae O139 isolates

Restriction fragment length polymorphism analyses of the array of CTXPhi prophages in strains CRC262 and CRC266 of Vibrio cholerae O139 revealed the presence of copies of complete CTXPhi and pre-CTXPhi prophages coexisting at a single chromosomal locus in each strain. Restriction pattern and comparative nucleotide sequence analysis revealed pre-CTXPhi precursors of both the El Tor and Calcutta lineages. Thus, we hypothesize that two precursor variants independently acquired cholera toxin genes and gave rise to the current El Tor and Calcutta CTXPhi prophages. We discuss the implications of these results in terms of the evolution and origin of the current diversity of CTXPhi prophages.     

Construction and analysis of a Vibrio cholerae delta-aminolevulinic acid auxotroph which confers protective immunity in a rabbit model.

Vibrio cholerae CVD101 is a very effective live vaccine. Although this strain does not produce active cholera toxin because of a mutation in the gene for the cholera toxin A subunit, it still shows residual pathogenicity. To attenuate CVD101 further, we set out to isolate derivatives of CVD101 which were limited in their ability to proliferate in vivo. Two delta-aminolevulinic acid auxotrophs of CVD101, designated V286 and V287, were isolated by transposon mutagenesis and penicillin enrichment. Southern blotting revealed that the mutants differed with respect to the location of the transposon insertion. Under aerobic conditions, in the absence of delta-aminolevulinic acid, both mutants showed diminished growth compared with CVD101. The growth of V286 was most severely affected. Microaerophilic growth of both mutants was less affected. Competition experiments with a rabbit model showed that strain V286 was found in numbers 10(3)- to 10(4)-fold lower than its parental strain. This observation indicates that strain V286 is impaired in its ability to colonize the rabbit intestine. It also supports an important role for aerobic growth in the colonization of the intestine by V. cholerae. Vaccination of rabbits with a single dose of strain V286 resulted in full protection against challenge with a virulent strain. Strain V286 was not shed from rabbits in a cultivatable form. Our results suggest that delta-aminolevulinic acid auxotrophy can attenuate V. cholerae by limiting its ability to colonize without affecting its capacity to induce protective immunity. Furthermore, this type of mutation may prevent the spread of V. cholerae vaccine strains in the environment.     

New variants of Vibrio cholerae O1 biotype El Tor with attributes of the classical biotype from hospitalized patients with acute diarrhea in Bangladesh

The sixth pandemic of cholera and, presumably, the earlier pandemics were caused by the classical biotype of Vibrio cholerae O1, which was progressively replaced by the El Tor biotype representing the seventh cholera pandemic. Although the classical biotype of V. cholerae O1 is extinct, even in southern Bangladesh, the last of the niches where this biotype prevailed, we have identified new varieties of V. cholerae O1, of the El Tor biotype with attributes of the classical biotype, from hospitalized patients with acute diarrhea in Bangladesh. Twenty-four strains of V. cholerae O1 isolated between 1991 and 1994 from hospitalized patients with acute diarrhea in Matlab, a rural area of Bangladesh, were examined for the phenotypic and genotypic traits that distinguish the two biotypes of V. cholerae O1. Standard reference strains of V. cholerae O1 belonging to the classical and El Tor biotypes were used as controls in all of the tests. The phenotypic traits commonly used to distinguish between the El Tor and classical biotypes, including polymyxin B sensitivity, chicken cell agglutination, type of tcpA and rstR genes, and restriction patterns of conserved rRNA genes (ribotypes), differentiated the 24 strains of toxigenic V. cholerae O1 into three types designated the Matlab types. Although all of the strains belonged to ribotypes that have been previously found among El Tor vibrios, type I strains had more traits of the classical biotype while type II and III strains appeared to be more like the El Tor biotype but had some classical biotype properties. These results suggest that, although the classical and El Tor biotypes have different lineages, there are possible naturally occurring genetic hybrids between the classical and El Tor biotypes that can cause cholera and thus provide new insight into the epidemiology of cholera in Bangladesh. Furthermore, the existence of such novel strains may have implications for the development of a cholera vaccine.