Efficacy trial of single-dose live oral cholera vaccine CVD 103-HgR in North Jakarta, Indonesia, a cholera-endemic area

A randomized, double-blind, placebo-controlled efficacy trial of one dose of CVD 103-HgR live oral cholera vaccine was performed in Indonesia from 1993 to 1997. 67,508 persons aged 2-41 years ingested vaccine or placebo and were followed for four years, detecting cholera cases using hospital-based surveillance. A nested reactogenicity study (538 vaccinees, 535 controls) revealed no vaccine-attributable side effects. A nested immunogenicity study (N=657) showed vibriocidal seroresponses in 64-70% of vaccinees vs 1-2% of controls. Cholera incidence was lower than expected. 103 cases of Vibrio cholerae O1 El Tor diarrhea were detected, 93 evaluable for vaccine efficacy (43 vaccine, 50 placebo; efficacy=14%). A suggestion of protection was observed among persons with blood group O [P=0.12]. Only seven cases occurred within six months of vaccination, precluding assessment of short-term efficacy. In Jakarta, single-dose CVD 103-HgR did not confer long-term protection. Short-term protection from a single-dose and long-term protection from two doses have yet to be studied.     

Randomized double-blind placebo controlled trial to evaluate the safety and immunogenicity of the live oral cholera vaccine strain CVD 103-HgR in Swiss adults

A randomized, double-blind, placebo controlled trial was conducted in 50 healthy Swiss adults to assess the safety and immunogenicity of the live oral attenuated cholera vaccine candidate strain Vibrio cholerae CVD 103-HgR (classical, Inaba). A single dose of 5 × 10⁸ viable CVD 103-HgR organisms, administered in a buffered liquid formulation, was well tolerated as compared with individuals who received an equivalent amount of heat-killed Escherichia coli K-12 placebo. Eighty-eight percent of subjects receiving CVD 103-HgR mounted a significant (>fourfold) rise in Inaba vibriocidal titre while 68% did so for the heterologous Ogawa serotype. The magnitude of the vibriocidal antibody response (as measured by peak geometric mean titre and by fold-rise in titre over baseline) was greater for the homologous Inaba serotype. Nineteen out of 25 volunteers (76%) responded with a significant (p < 0.05) rise in serum antitoxin levels. No vaccine who received the E. coli K-12 placebo mounted a significant rise in either vibriocidal or antitoxin antibody levels. These results corrobrate the safety and immunogenicity of CVD 103-HgR in healthy adult volunteers.     

Lipopolysaccharide-specific memory B cell responses to an attenuated live cholera vaccine are associated with protection against Vibrio cholerae infection

BackgroundThe single-dose live attenuated vaccine CVD 103-HgR protects against experimental Vibrio cholerae infection in cholera-naïve adults for at least 6 months after vaccination. While vaccine-induced vibriocidal seroconversion is associated with protection, vibriocidal titers decline rapidly from their peak 1–2 weeks after vaccination. Although vaccine-induced memory B cells (MBCs) might mediate sustained protection in individuals without detectable circulating antibodies, it is unknown whether oral cholera vaccination induces a MBC response.MethodsIn a study that enrolled North American adults, we measured lipopolysaccharide (LPS)- and cholera toxin (CtxB)-specific MBC responses to PXVX0200 (derived from the CVD 103-HgR strain) and assessed stool volumes following experimental Vibrio cholerae infection. We then evaluated the association between vaccine-induced MBC responses and protection against cholera.ResultsThere was a significant increase in % CT-specific IgG, % LPS-specific IgG, and % LPS-specific IgA MBCs which persisted 180 days after vaccination as well as a significant association between vaccine-induced increase in % LPS-specific IgA MBCs and lower post-challenge stool volume (r = −0.56, p < 0.001).DiscussionOral cholera vaccination induces antigen-specific MBC responses, and the anamnestic LPS-specific responses may contribute to long-term protection and provide correlates of the duration of vaccine-induced protection.Clinical trials registration: NCT01895855.     

Can oral cholera vaccination play a role in controlling a cholera outbreak?

Control measures to limit the spread of a cholera outbreak in Pohnpei Island (Micronesia), included mass vaccination with the single-dose live-attenuated oral cholera vaccine CVD 103-HgR as a potential adjunct measure. The outbreak provided a unique opportunity to evaluate the practicality of use and effectiveness of this vaccine. Under field conditions encountered in Pohnpei, crude vaccine efficacy was estimated at 79.2% (95% CI: 71.9-84.6%) in the target population. Retrospective analysis suggests that mass vaccination with oral cholera vaccines can be a useful adjunct tool for controlling outbreaks, particularly if implemented early in association with other standard control measures.     

Experimental evaluation of antitoxic protective effect of new cholera vaccines in mice.

Intraperitoneal immunization of mice and subsequent challenge with purified cholera toxin (CT) were employed to evaluate the anti-cholera toxin protective effect of two new oral cholera vaccines, live CVD 103-HgR and killed B subunit-whole cell (BS-WC). CVD 103-HgR vaccine demonstrated 100% protection of mice against 2.25 LD50 and 70% against 3 LD50 of CT. Mice immunized with BS-WC vaccine were protected against 2.25 and 3 LD50 of CT in 88 and 62% of cases, respectively. All three killed parenteral vaccines failed to protect against CT. We suggest this mouse system for preliminary evaluation of the antitoxic protective activity of cholera vaccines.     

Analysis of efficacy of CVD 103-HgR live oral cholera vaccine against all-cause travellers' diarrhoea in a randomised, double-blind, placebo-controlled study

Enterotoxigenic Escherichia coli (ETEC), which produces heat labile toxin (LT) and/or heat stable toxin (ST), is considered to be the most common known cause of travellers' diarrhoea (TD). Owing to the antigenic similarity between cholera toxin and LT, immunization with inactivated oral B-subunit/whole-cell cholera vaccine (BS-WC) offers short term (3 months) but significant (>67%) protection against TD caused by LT-related ETEC. Since it expresses the cholera toxin B (CTB) subunit, the live attenuated oral cholera vaccine strain CVD 103-HgR, may induce similar protection. A trial was performed to determine if CVD 103-HgR live oral cholera vaccine would provide a protective efficacy of at least 50% against TD. In addition, the protective efficacy of the vaccine against TD specifically due to LT-ETEC and LT/ST-ETEC was determined. Volunteers (n=134) travelling to Indonesia, India, Thailand or West-Africa were randomised to receive either a placebo (n=65) or the vaccine (n=69). In the placebo group, 46% reported an episode of diarrhoea, compared to 52% in the vaccine group. No significant group differences were found with regard to incidence, duration or severity of all caused TD or ETEC-associated TD. However, ETEC-associated TD occurred earlier in the placebo group (median 5 days), compared to the vaccine group (median 15 days). In conclusion, CVD 103-HgR live oral cholera vaccine failed to provide a 50% protection against TD. This study does not exclude that the vaccine may offer a short-lived protection against ETEC-associated TD. However, the power of the study was limited by the unexpected low incidence of LT-ETEC-associated diarrhoea (9% of all TD) compared to ST-associated TD (24% of all TD).     

Age-related immunogenicity and reactogenicity of live oral cholera vaccine CVD 103-HgR in a randomized,controlled clinical trial

Aging is accompanied by a decline in immune function which can lead to decreased responses to vaccines. Attenuated recombinant Vibrio cholerae O1 vaccine strain CVD 103-HgR elicits a rapid serum vibriocidal antibody (SVA) response and protects against cholera diarrhea in volunteer challenge studies but has not been studied in older adults. We evaluated CVD 103-HgR (PXVX0200) in adults age 46–64, compared them to previously studied adults age 18–45, and studied age-related immunogenicity across adults 18–64?years of age. Volunteers were randomized to receive a single dose of 1?×?10⁹?CFU of PXVX0200 or placebo. Immunogenicity endpoints included SVA and anti-cholera toxin (CT) antibody levels on days 1, 11, 29, 91 and 181 and lipopolysaccharide (LPS) and CT-specific IgA and IgG memory B cells on days 1, 91 and 181. Safety was assessed by comparing solicited signs and symptoms on days 1–8 and other adverse events through day 181. 2979 volunteers received vaccine, including 291 age 45–64. Day 11 seroconversion occurred in 90.4% of older adults vs 93.5%% of younger adults and met the endpoint of demonstrating non-inferiority between the two groups. Significant increases in LPS-specific IgG and IgA and CT-specific memory IgG memory B cells were seen at days 91 and 181. There appeared to be a continuous age-related decline in SVA seroconversion and geometric mean titers, but not memory B cell responses, across the 18–64?year age range. Most reactogenicity was mild and was more common in the placebo group. PXVX0200 appears safe and immunogenic in older adults.Clinical Trials Registration: clinicaltrials.gov NCT02100631.     

口服霍乱疫苗研究现状

[目的]了解目前国内外口服霍乱疫苗的研究现状.[方法]对近年来国内外医学期刊上发表的有关口服霍乱疫苗研究的论文进行综述.[结果]有3种疫苗具有良好的免疫原性、安全性,能有效预防霍乱流行.即灭活霍乱弧菌全菌体(WC)疫苗或霍乱毒素B亚单位-灭活霍乱弧菌全菌体(BS-WC)疫苗、重组霍乱毒素B亚单位-灭活霍乱弧菌全菌体(rBS-WC)疫苗、由活的基因修饰的O1群霍乱弧菌减毒株(CVD103-HgR)组成的减毒活菌苗.[结论]口服霍乱疫苗尚处在大规模的实验和论证中,仅在较小范围人群中被建议服用,未被作为大范围人群预防霍乱的公共卫生措施.     

Investigations into the safety and immunogenicity of a killed oral cholera vaccine developed in Viet Nam

OBJECTIVE: To evaluate a killed oral cholera vaccine produced in Viet Nam, and to compare the Vietnamese vaccine with one that is licensed internationally. METHOD: Two-dose regimens of a locally produced, bivalent, anti-O1, anti-O139 killed oral whole-cell cholera vaccine (biv-WC) and of a commercially available, monovalent (anti-O1) oral recombinant B subunit-killed whole-cell cholera vaccine (rBS-WC) were compared in two trials in Viet Nam. In the first trial, 144 adults were randomized to biv-WC with or without buffer, rBS-WC with buffer, or placebo without buffer. In the second, 103 children aged 1-12 years were randomized to biv-WC without buffer, rBS-WC with buffer, or placebo without buffer. FINDINGS: No regimen was associated with significant side-effects. In adults, ca 60% of recipients of either vaccine exhibited at least fourfold serum anti-O1 vibriocidal antibody responses and ca 40% of recipients of biv-WC demonstrated anti-O139 vibriocidal responses. Both anti-O1 (ca 90% in each vaccine groupand anti-O139 (68% in the biv-WC group) vibriocidal responses occurred more frequently in children. The responses to biv-WC were unaffected by the receipt of buffer. CONCLUSION: It was concluded that biv-WC was safe and immunogenic, that it could be administered without buffer, and that it could elicit robust immune responses even in children, for whom the risk of endemic cholera is highest.     

Safety and immunogenicity of single-dose live oral cholera vaccine strain CVD 103-HgR in healthy adults age 18–45

The attenuated recombinant Vibrio cholerae O1 vaccine strain CVD 103-HgR, re-developed as PXVX0200, elicits a rapid serum vibriocidal antibody (SVA) response and protects against cholera diarrhea in volunteer challenge studies. We performed a phase 3, placebo controlled, double blind, multi-center study to further assess the safety, immunogenicity, and lot-to-lot consistency of PXVX0200. Adult volunteers 18–45 years of age were randomized 8:1 to receive a single dose of 1 × 10⁹ CFU of PXVX0200 from three production lots or saline placebo. Immunogenicity endpoints included SVA and anti-cholera toxin (CT) antibody levels on days 1, 11, 29, 91 and 181. Safety was assessed by comparing solicited signs and symptoms on days 1–8, unsolicited adverse events through day 29 and serious adverse events through day 181. A total of 3146 participants were enrolled, including 2795 vaccine and 351 placebo recipients. The SVA seroconversion rates at day 11 were 94% and 4% in the PXVX0200 and placebo recipients, respectively (P < .0001). Cumulative SVA seroconversion occurred among 96% of vaccine recipients. PXVX0200 SVA GMTs peaked on day 11 and remained significantly higher than placebo through day 181 while the fold-rise over baseline in PXVX0200 anti-CT antibody was significantly greater than placebo at every post-vaccination time point. Most reactogenicity was mild and resolved within 1–3 days with headache and diarrhea more frequently reported in PXVX0200 recipients. There were no differences in unsolicited adverse events and no study-related serious adverse events. Immunogenicity and safety endpoints were equivalent between the three production lots. PXVX0200 is immunogenic and well tolerated across multiple production lots.Clinical Trials Registration: Clinicaltrials.gov NCT02094586.     

An oral B subunit: whole cell vaccine against cholera.

During the last decade there has been a rapid progress in the development of new, much improved vaccines against cholera. These vaccines, which are given orally to stimulate the gut mucosal immune system, are based on either a combination of purified cholera toxin B (binding) subunit and killed cholera vibrios of Inaba and Ogawa serotypes and El Tor and classical biotypes (B subunit-whole cell vaccine, B-WC) or on a live attenuated mutant strain of Vibrio cholerae producing the B subunit (CVD 103-HgR). The safety of the oral B-WC cholera vaccine and the immunogenicity and protective efficacy of this vaccine against both cholera and diarrhoea caused by enterotoxigenic Escherichia coli have been extensively documented, e.g. in a large randomized, placebo-controlled field trial in 90,000 persons living in a cholera endemic area. The potential for inexpensive large-scale manufacturing of the B-WC vaccine has recently been much facilitated by the introduction of recombinant DNA technology for production of the B subunit component. This now gives promise that this vaccine could become a useful, cost-effective tool in future strategies to control cholera both in endemic situations and in relation to acute epidemic outbreaks.     

A randomized,observer-blinded,equivalence trial comparing two variations of Euvichol®, a bivalent killed whole-cell oral cholera vaccine,in healthy adults and children in the Philippines

BackgroundTo contribute to the global demand for oral cholera vaccine (OCV), the production of Euvichol® was scaled up with elimination of thimerosal. To demonstrate the equivalence of the variations, a study was carried out in the Philippines.MethodsHealthy male and female adults and children in Manila were randomized to receive two doses of Euvichol® two weeks apart from either the 100L (Comparator) or the 600L (Test) variation. Primary and secondary immunogenicity endpoints were respectively geometric mean titer (GMT) of vibriocidal antibodies (two weeks post second dose) and seroconversion rate (two weeks after each dose) against O1 Inaba, Ogawa, and O139 serogroups. The GMT of vibriocidal antibodies against O1 Inaba, Ogawa, and O139 two weeks post first dose was also measured. To show the equivalence of two variations of Euvichol®, the ratio of GMT and the difference of seroconversion rate between Test and Comparator vaccines were tested with equivalence margin of [0.5, 2.0] for GMT ratio and of 15% for seroconversion rate, respectively. Safety assessment included solicited reactogenicity within 6 days after each dose and unsolicited and serious adverse events.ResultsA total of 442 participants were enrolled. For the overall population, equivalence between Test and Comparator was demonstrated for vibriocidal antibody response against O1 Inaba and Ogawa serotypes and O139 serogroup in both modified intention-to-treat (mITT) and per protocol analysis, since the 95% confidence intervals (CI) of GMT to any serotypes were within the lower and upper boundary [0.5, 2.0]. Seroconversion rates after two doses also showed equivalence for O1 Inaba, Ogawa, and O139. The vaccine was safe and well tolerated, similarly between the two groups.ConclusionThe study results support the equivalence of the 600L Euvichol® to the 100L formulation in healthy children and adults. The 600L Euvichol® is safe and immunogenic in adults and children.ClinicalTrials.gov registration number: NCT02502331.     

Local and systemic immune responses to combined vibrio cholerae CVD103-HgR and salmonella typhi ty21a live oral vaccines after primary immunization and reimmunization

The local and systemic antibody responses elicited following concomitant primary immunization and reimmunization with the live oral attenuated Vibrio cholerae CVD103-HgR and Salmonella typhi Ty21a vaccine strains were determined in healthy adult volunteers. A more pronounced serum vibriocidal antibody response was generated after primary immunization compared to reimmunization 2.5 or 3.5 yr later. The seroconversion rate (> or =4-fold rise over baseline) was 81% subsequent to primary immunization versus 57% (p=0.018) and 65% (p=0.639) upon reimmunization at 2.5 and 3.5 yr, respectively. A similar trend was observed for serum anti-S. typhi lipopolysaccharide (LPS) antibodies. After primary immunization, 48% of subjects manifested a significant rise in coproantibody levels to V. cholerae LPS while 60% did so for cholera toxin (CT). Upon reimmunization, the response rate for LPS ranged from 38% at 2.5 yr to 56% at 3.5 yr (p>0.05), while that for CT varied from 31% (p=0. 007) to 50% (p=0.541) at 2.5 and 3.5 yr, respectively. The anti-S. typhi IgA coproantibody response rate was 70% subsequent to primary immunization versus 47% at 2.5 yr (p=0.021) and 63% at 3.5 yr (p=0. 77).     

Serum vibriocidal responses when second doses of oral cholera vaccine are delayed 6 months in Zambia

Two-dose killed oral cholera vaccines (OCV) are currently being used widely to control cholera. The standard dose-interval for OCV is 2 weeks; however, during emergency use of the vaccine, it may be more appropriate to use the available doses to quickly give a single dose to more people and give a delayed second dose when more vaccine becomes available.This study is an open label, randomized, phase 2 clinical trial of the vibriocidal response induced by OCV, comparing the responses when the second dose was given either 2 weeks (standard dose interval) or 6 months (extended dose interval) after the first dose. Vaccine was administered to healthy participants > 1 year of age living in the Lukanga Swamps area of Zambia. Three age cohorts (<5 years, 5–14 years, and ≥ 15 years) were randomized to the either dose-interval. The primary outcome was the vibriocidal GMT 14 days after the second dose.156 of 172 subjects enrolled in the study were included in this analysis. The Inaba vibriocidal titers were not significantly different 14 days post dose two for a standard dose-interval GMT: 45.6 (32–64.9), as compared to the GMT 47.6 (32.6–69.3), for the extended dose-interval, (p = 0.87). However, the Ogawa vibriocidal GMTs were significantly higher 14 days post dose two for the extended-dose interval at 87.6 (58.9–130.4) compared to the standard dose-interval group at 49.7 (34.1–72.3), p = 0.04. Vibriocidal seroconversion rates (a > 4-fold rise in vibriocidal titer) were not significantly different between dose-interval groups.This study demonstrated that vibriocidal titers 14 days after a second dose when given at an extended\ dose interval were similar to the standard dose-interval. The findings suggest that a flexible dosing schedule may be considered when epidemiologically appropriate.The trial was registered at Clinical Trials.gov (NCT03373669).     

Immune responses following one and two doses of the reformulated,bivalent, killed,whole-cell,oral cholera vaccine among adults and children in Kolkata,India: A randomized,placebo-controlled trial

Immune responses after one and two doses of the reformulated killed oral cholera vaccine were measured in a double-blind, randomized, placebo-controlled trial of 77 adults aged 18–40 years and 77 children aged 1–17 years residing in Kolkata, India. 65% of adults and 87% of children and 46% of adults and 82% of children exhibited a ≥4-fold rise in serum Vibrio cholerae O1 vibriocidal antibody titers from baseline following dose 1 and 2, respectively. Responses to V. cholerae O139 were less pronounced but followed a similar pattern. We demonstrate that in a cholera-endemic area, the vaccine elicited vibriocidal responses after a single-dose of the vaccine.     

A serological survey for cholera antibodies in rural east Pakistan. 2. A comparison of antibody titres in the innunized and control populationd of a cholera-vaccine field-trial area and the relation of antibody titre to cholera case rate

Controlled field trials of a highly antigenic cholera vaccine were held in Matlab Bazar in rural East Pakistan in 1963 and again in 1964. In July—September 1965, a serological survey for cholera antibodies was carried out on a random sample of the field-trial population. This survey revealed that it was possible to demonstrate the effect of a single injection of the cholera vaccine per head on the proportion of the population with detectable vibriocidal and agglutinating antibody 10 and 22 months after injection. More significantly, the reduction in cholera case rate caused by the vaccine could be correlated with the rise in vibriocidal antibody after vaccination, suggesting that the serological response to vaccine in man may be a useful measure of vaccine potency. The survey also indicated that in this endemic cholera area, with a high level of immunity in adults, a single injection of cholera vaccine was in fact a booster dose for the majority of the population. Thus, the results of cholera vaccine field trials in endemic areas cannot be directly extrapolated to predict the effects of the same vaccine in non-endemic areas.     

Biosafety aspects of the recombinant live oral Vibrio cholerae vaccine strain CVD 103-HgR

The development of live attenuated vaccines, allowing for the safe and effective immunisation at mucosal surfaces, is a strategy of great interest for vaccinologists. The main advantage of this approach over conventional parenteral vaccines is the induction of strong mucosal immune responses, allowing targeting of the pathogen at the initial point of contact with the host. Further advantages include the ease of administration, high acceptance by vaccines, and relatively low production costs. Finally, well-characterised, safe and immunogenic vaccine strains are well suited as vectors for the mucosal delivery of foreign vaccine antigens and of DNA vaccines. However, such vaccines, when based on or containing genetically modified organisms (GMOs), are facing new and specific regulatory hurdles, particularly regarding the potential risks for humans and the environment. In this contribution we address selected aspects of the risk assessment of live attenuated bacterial vaccines covered in the course of the registration of vaccine strain CVD 103-HgR as a recombinant live oral vaccine against cholera.     

The state-of-the-art of approved and under-development cholera vaccines

Cholera remains a huge public health problem. Although in 1894, the first cholera vaccination was reported, an ideal vaccine that meets all the requirements of the WHO has not yet been produced. Among the different approaches used for cholera vaccination, attenuated vaccines represent a major category; these vaccines are beneficial in being able to induce a strong protective response after a single administration. However, they have possible negative effects on immunocompromised patient populations. Both the licensed CVD103-HgR and other vaccine approaches under development are detailed in this article, such as the Vibrio cholerae 638 vaccine candidate, Peru-15 or CholeraGarde^® and the VA1.3, VA1.4, IEM 108 VCUSM2 and CVD 112 vaccine candidates. In another strategy, killed V. cholerae vaccines have been developed, including Dukoral^®, mORCAX^® and Sanchol™. The killed vaccines are already sold, and they have successfully demonstrated their potential to protect populations in endemic areas or after natural disasters. However, these vaccines do not fulfill all the requirements of the WHO because they fail to confer long-term protection, are not suitable for children under two years, require more than a single dose and require a distribution chain with cold storage. Lastly, other vaccine strategies under development are summarized in this review. Among these strategies, vaccine candidates based on alternative drug delivery systems that have been reported lately in the literature are discussed, such as microparticles, proteoliposomes, LPS subunits, DNA vaccines and rice seeds containing toxin subunits. Preliminary results reported by many groups working on alternative delivery systems for cholera vaccines demonstrate the importance of new technologies in addressing old problems such as cholera. Although a fully ideal vaccine has not yet been designed, promising steps have been reported in the literature resulting in hope for the fight against cholera.     

Randomized,double-blind,placebo-controlled trial to evaluate the safety and immunogenicity of live oral cholera vaccine 638 in Cuban adults

A randomized, double-blind, placebo-controlled clinical trial was conducted to evaluate the safety, reactogenicity and the immunogenicity of a 2 × 10⁹ CFU dose of the 638 lyophilized live attenuated cholera vaccine for oral administration, formulated and produced at Finlay Institute, City of Havana, Cuba. Thirty-six healthy female and male adult volunteers from 18 to 40 years old were involved, clinically examined and laboratory tested after the informed consent signature. Adverse events were monitored and seroconversion rates and geometrical mean titer (GMT) of vibriocidal antibodies were tested in volunteer's sera samples. Neither serious adverse events nor other damages to the volunteers due to vaccine or placebo feeding were reported during the clinical follow-up period of this study; none of the adverse events registered within the first 72 h after inoculation were life-threatening for volunteers. Neither severe nor moderate adverse events were reported. Sixty-one percent of subjects showed mild expected adverse events in an interval lower than 24 h up to the first 72 h, 75% of these in the vaccinated group and 18% in the placebo group. Fourteen days after inoculation the GMT of vibriocidal antibodies in the vaccine group significantly increased in comparison to the placebo group. All subjects in the vaccine group (24) seroconverted (100%). Results show that this vaccine is safe, well tolerated and immunogenic in healthy female and male volunteers.     

Expression of enterotoxigenic Escherichia coli colonization factors in Vibrio cholerae

As a first step towards a vaccine against diarrhoeal disease caused by enterotoxigenic Escherichia coli (ETEC), we have studied the expression of several ETEC antigens in the live attenuated Vibrio cholerae vaccine strain CVD 103-HgR. Colonization factors (CF) CFA/I, CS3, and CS6 were expressed at the surface of V. cholerae CVD 103-HgR. Both CFA/I and CS3 required the co-expression of a positive regulator for expression, while CS6 was expressed without regulation. Up-regulation of CF expression in V. cholerae was very efficient, so that high amounts of CFA/I and CS3 similar to those in wild-type ETEC were synthesized from chromosomally integrated CF and positive regulator loci. Increasing either the operon and/or the positive regulator gene dosage resulted in only a small increase in CFA/I and CS3 expression. In contrast, the level of expression of the non-regulated CS6 fimbriae appeared to be more dependent on gene dosage. While CF expression in wild-type ETEC is known to be tightly thermoregulated and medium dependent, it seems to be less stringent in V. cholerae. Finally, co-expression of two or three CFs in the same strain was efficient even under the control of one single regulator gene.