S.Typhi derived OmpC peptide conjugated with Vi-polysaccharide evokes better immune response than free Vi-polysaccharide in mice
Introduction
Typhoid fever is a serious systemic common disease of developing countries. Salmonella enterica serovar Typhi (S. Typhi) is a causative agent of typhoid fever in humans only. Recent data from World Health Organization (WHO) shows, annually there are 16–33 million cases and 500,000 to 600,000 deaths due to typhoid fever worldwide [1]. Tourism from the UK and the USA to India, resulted in approximately 400 imported cases each year [2,3]. It is common notion that typhoid fever is a disease of school going children [4,5], while most vulnerable age group for S. typhi infection is 1–2 years [6]. Free Vi capsular polysaccharide (Vi) vaccine was shown to be 60–70% protective [7,8], and licensed for ≥2 years of age against typhoid fever [9]. It gives protection for almost 3 years [10] but re-vaccination does not induce booster response. There are three typhoid conjugate vaccine, all are Vi polysaccharide conjugated with tetanus toxoid as carrier peptide, are now licensed and on the market in India [39] Vi polysaccharide vaccine has certain limitations such as poor immunogenicity, lack of memory and booster response, and most importantly not immunogenic in infants.
Outer Membrane Proteins (OMPs) of S. typhi that can be porin or non-porin have been found to be protective against typhoid fever [11]. S. Typhi OMPs have been proven to be protective in mice and this phenomenon is evident in a wide range of other micro-organisms [Niesseria gonorrhea, Niesseria meningitides,] [[12], [13], [14], [15]]. Porins are excellent antigens and elicit IgG specific anti-porin antibodies for diagnostics [16]. In addition, these immunologically potential surface antigens are important for vaccine design [17].
The outer membrane protein C (OmpC) (a 39 kD protein) is a highly conserved porin [18], and is regarded as one the major immunodominant OMPs. Generally, porin contains five or more surface epitopes i.e. 6 to 25 residues in length [18,19] that are partially obscured by the lipopolysaccharide (LPS) and completely blocked by O-antigen sugars [20]. The OmpC peptide-resin conjugate (S.typhi peptide was deduced from exposed loops of OmpC) evoked OmpC peptide specific antibodies with Freund's complete adjuvant in mice [21]. Despite these epitopes could have potentially been blocked by LPS, monoclonal antibodies induced by immunization with OmpC peptides reacted with native and recombinant OmpC.
We selected the outer loops of OmpC that are rich in T cell epitopes and conjugated with Vi polysaccharide. Conjugation was mediated by carbodiimide chemistry using ADH as linker and immune response of this Vi-conjugate was tested in mice. We have shown that the full length recombinant OmpC conjugated to Vi proved to be an effective carrier protein as mentioned in US patent application [22]. So, we extended our work and hypothesized that T site rich OmpC peptide carrier with Vi may be a promising partner. Indeed the Vi-peptide conjugate overcame the limitations of the plain Vi vaccine: immunisations induced a 20 fold IgG response and a booster response after two booster injections.
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Materials and methods
Vi polysaccharide of S. Typhi (Ty2 strain) and Citrobacter freundii were obtained from Bharat Biotech International Limited (Table 1). OmpCp of S. Typhi was synthesized chemically at Department of Biochemistry, All India Institute of Medical Sciences (AIIMS), New Delhi, India.
General purpose white Swiss mice were used for the immunologic studies approved by institutional animal ethics committee. All the animals were kept under the rules and regulations of AIIMS animal facility. Vi of S. Typhi
Selection of T-cell epitope rich OmpCp
A schematic diagram of S. Typhi OmpC loops as shown (Fig. 1A) and the T-cell epitopes within the entire OmpC loops are shown (Fig. 1B). Major sites rich in T-cell epitopes were observed in loop number 3a. These were confirmed by running the program in selected loops 3a and 7 (Fig. 1C).
Characterization of Vi-conjugate and yield
Vi polysaccharide is soluble in aqueous medium while OmpC peptide is insoluble in aqueous medium without urea. Urea had to be added to the aqueous solution for the conjugation of OmpCp to Vi polysaccharide to
Discussion
Bacterial surface polysaccharides alone are widely used as successful vaccines. However, polysaccharides are T-independent antigens, poorly immunogenic, no memory response and not immunogenic in infants [27]. Conjugation of polysaccharides with carrier protein or peptide is the only way to convert polysaccharides into T-dependent antigens thereby providing immune memory and extended the longevity of protection. Indeed, repeated immunizations of glycoconjugates induce a protective booster in
Conclusions
We conclude that selected OmpCp is an immunodominant peptide. Since, this peptide evoked T-helper response. We suggest that selected OmpCp as a carrier with Vi polysaccharide is assumed to be a promising molecule for candidate vaccine for typhoid fever.
Declaration of competing interest
Nothing to declare.
Acknowledgments
This work was supported by the grant from Department of Biotechnology, New Delhi, India. Authors are grateful to Dr. Arif Azam Khan, AIIMS (Lab: Dr. Rao), Department of Biochemistry, New Delhi for peptide synthesis. Authors are thankful to Savita Saini for constant support in the lab.
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