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

Chandrika Murugaiah,Ravichandran Manickam,Nik Zuraina Nik Mohd Noor,Shyamoli Mustafa,Lalitha Pattabhiraman,Odir A Dellagostin

doi:10.1371/journal.pone.0081817

Abstract

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.

Highlights

V. cholerae is the causative agent of cholera which is a lifethreatening, acute secretory diarrhoeal disease, a major public health problem that affects indigenous populations [1]
Cholera is associated with expression of Cholera Toxin (CT) encoded in the ctxAB gene, which is acquired by V. cholerae from the filamentous phage CTX [8]
Construction of vaccine strains The vaccine candidate VCUSM20 was successfully obtained by introducing the rtxC::aphA gene into the chromosome of VCUSM14 via suicide plasmid integration into the chromosome and subsequent sucrose counterselection (Figure 1)

Summary

Introduction

V. cholerae is the causative agent of cholera which is a lifethreatening, acute secretory diarrhoeal disease, a major public health problem that affects indigenous populations [1]. Despite efforts taken to control V. cholerae infection [2,3], the global burden of cholera is high, mostly affecting young children [4]. Cholera is associated with expression of CT encoded in the ctxAB gene, which is acquired by V. cholerae from the filamentous phage CTX [8]. Apart from the ctxAB toxin, the zonula occludens toxin (zot) and accessary cholera enterotoxin (ace), located on a 4.5-kb central core region of the CTX genetic element [10,11,12,13], were suggested to cause probable diarrhoea symptom [11,14,15]

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