Construction and characterization of an auxotrophic ctxA mutant of O139 Vibrio cholerae

Chan, M.; Cheong, Tan Gim; Kurunathan, S.; Murugaiah, Chandrika; Ledón, Talena; Fando, Rafael; Lalitha, Pattabhiraman; Zafarina, Zainuddin; Ravichandran, Manickam

doi:10.1016/j.micpath.2010.06.001

Cited by 6 publications

(11 citation statements)

References 33 publications

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“…Similar to our results, a dose of 10 5 CFU of V. cholerae (El Tor strain C6706) was effective in colonization and biofilm development in infant mice, as reported in [50]. In contrast, the VCUSM2, VCUSM14 and VCUSM21P strains were recovered with 1.8 × 10 5 CFU, 2.0 × 10 4 CFU and 1.55 × 10 6 CFU respectively, from mouse intestines [44,51]. In comparison to the recovery of these VCUSM strains, a two-log higher recovery of VCUSM14P was recorded in the present study.…”

Section: Discussionsupporting

confidence: 90%

“…Our results indicate that both the LACV and unformulated VCUSM14P had no detectable diarrhoeagenic activity even at an inoculation dose of 10 4 -10 6 CFU/mL in a rabbit ileal loop model without signs of haemorrhage and reactogenicity. A similar observation with VCUSM14 at doses of 10 6 and 10 8 CFU was reported by [44]. A RITARD assay was performed to further validate the results of the rabbit ileal loops assay.…”

Section: Discussionsupporting

confidence: 77%

“…Hence, this strain was further improved by mutating the ctxA gene via substitution of 7th amino acid, arginine to lysine, R7K, and 112th amino acid, glutamate to glutamine, E112Q. And, two copies of ace and zot genes in ctx operon were also deleted to reduce the reactogenicity and resultant strain VCUSM14 (an aminolevulinic acid (ALA) auxotrophic and non-reactogenic) was characterised and evaluated in animal models [44]. Further, the hemA gene was reintroduced into VCUSM14 to enhance its colonization potential, and the strain was designated as VCUSM14P.…”

Section: Introductionmentioning

confidence: 99%

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Immunogenicity and protective efficacy of a live, oral cholera vaccine formulation stored outside-the-cold-chain for 140 days

et al. 2020

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Background: Cholera, an acute watery diarrhoeal disease caused by Vibrio cholerae serogroup O1 and O139 across the continents. Replacing the existing WHO licensed killed multiple-dose oral cholera vaccines that demand 'cold chain supply' at 2-8°C with a live, single-dose and cold chain-free vaccine would relieve the significant bottlenecks and cost determinants in cholera vaccination campaigns. In this direction, a prototype cold chain-free live attenuated cholera vaccine formulation (LACV) was developed against the toxigenic wild-type (WT) V. cholerae O139 serogroup. LACV was found stable and retained its viability (5 × 10 6 CFU/mL), purity and potency at room temperature (25°C ± 2°C, and 60% ± 5% relative humidity) for 140 days in contrast to all the existing WHO licensed cold-chain supply (2-8°C) dependent killed oral cholera vaccines. Results: The LACV was evaluated for its colonization potential, reactogenicity, immunogenicity and protective efficacy in animal models after its storage at room temperature for 140 days. In suckling mice colonization assay, the LACV recorded the highest recovery of (7.2 × 10 7 CFU/mL) compared to those of unformulated VCUSM14P (5.6 × 10 7 CFU/mL) and the WT O139 strain (3.5 × 10 7 CFU/mL). The LACV showed no reactogenicity even at an inoculation dose of 10 4-10 6 CFU/mL in a rabbit ileal loop model. The rabbits vaccinated with the LACV or unformulated VCUSM14P survived a challenge with WT O139 and showed no signs of diarrhoea or death in the reversible intestinal tie adult rabbit diarrhoea (RITARD) model. Vaccinated rabbits recorded a 275-fold increase in anti-CT IgG and a 15-fold increase in anti-CT IgA antibodies compared to those of rabbits vaccinated with unformulated VCUSM14P. Vibriocidal antibodies were increased by 31-fold with the LACV and 14-fold with unformulated VCUSM14P. Conclusion: The vaccine formulation mimics a natural infection, is non-reactogenic and highly immunogenic in vivo and protects animals from lethal wild-type V. cholerae O139 challenge. The single dose LACV formulation was found to be stable at room temperature (25 ± 2°C) for 140 days and it would result in significant cost savings during mass cholera vaccination campaigns.

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Section: Discussionsupporting

confidence: 90%

Section: Discussionsupporting

confidence: 77%

Section: Introductionmentioning

confidence: 99%

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Immunogenicity and protective efficacy of a live, oral cholera vaccine formulation stored outside-the-cold-chain for 140 days

et al. 2020

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“…Recently, we have constructed a live VCUSM14 auxotroph vaccine candidate, shown to be a potentially efficient attenuated cholera vaccine that secretes enzymatically inactive ctx A subunit which is immunogenic yet safe [33]. The 7 th amino acid of the ctx A which is the arginine has been substituted with lysine (R7K), and the 112 th glutamate was substituted with glutamine (E112Q).…”

Section: Introductionmentioning

confidence: 99%

Construction and Evaluation of V. cholerae O139 Mutant, VCUSM21P, as a Safe Live Attenuated Cholera Vaccine

et al. 2014

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Cholera is a major infectious disease, affecting millions of lives annually. In endemic areas, implementation of vaccination strategy against cholera is vital. As the use of safer live vaccine that can induce protective immunity against Vibrio cholerae O139 infection is a promising approach for immunization, we have designed VCUSM21P, an oral cholera vaccine candidate, which has ctxA that encodes A subunit of ctx and mutated rtxA/C, ace and zot mutations. VCUSM21P was found not to disassemble the actin of HEp2 cells. It colonized the mice intestine approximately 1 log lower than that of the Wild Type (WT) strain obtained from Hospital Universiti Sains Malaysia. In the ileal loop assay, unlike WT challenge, 1×106 and 1×108 colony forming unit (CFU) of VCUSM21P was not reactogenic in non-immunized rabbits. Whereas, the reactogenicity caused by the WT in rabbits immunized with 1×1010 CFU of VCUSM21P was found to be reduced as evidenced by absence of fluid in loops administered with 1×102–1×107 CFU of WT. Oral immunization using 1×1010 CFU of VCUSM21P induced both IgA and IgG against Cholera Toxin (CT) and O139 lipopolysaccharides (LPS). The serum vibriocidal antibody titer had a peak rise of 2560 fold on week 4. Following Removable Intestinal Tie Adult Rabbit Diarrhoea (RITARD) experiment, the non-immunized rabbits were found not to be protected against lethal challenge with 1×109 CFU WT, but 100% of immunized rabbits survived the challenge. In the past eleven years, V. cholerae O139 induced cholera has not been observed. However, attenuated VCUSM21P vaccine could be used for vaccination program against potentially fatal endemic or emerging cholera caused by V. cholerae O139.

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“…They have been licensed and manufactured in Viet Nam and India [5,21]. Nevertheless, up to date there is no live attenuated vaccine available for O139 organisms, although a number of such vaccines candidates has been developed and some of them evaluated in small pilot studies [22,23].…”

Section: Introductionmentioning

confidence: 99%

TLP01, an mshA mutant of Vibrio cholerae O139 as vaccine candidate against cholera

Ledón

Ferrán

Pérez

et al. 2012

Microbes and Infection

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Construction and characterization of an auxotrophic ctxA mutant of O139 Vibrio cholerae

Cited by 6 publications

References 33 publications

Immunogenicity and protective efficacy of a live, oral cholera vaccine formulation stored outside-the-cold-chain for 140 days

Immunogenicity and protective efficacy of a live, oral cholera vaccine formulation stored outside-the-cold-chain for 140 days

Construction and Evaluation of V. cholerae O139 Mutant, VCUSM21P, as a Safe Live Attenuated Cholera Vaccine

TLP01, an mshA mutant of Vibrio cholerae O139 as vaccine candidate against cholera

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