Title of Thesis
Preparation of Multiple Conjugates (LPS- Protein) for
Immunization Against Typhoidal Diseases
National Institute for Biotechnology and Genetic
Engineering (NIBGE), Faisalabad & Quaid-i-Azam University, Islamabad
Biotechnology and Genetic Engineering
|Number of Pages|
|Keywords (Extracted from title, table of contents and
abstract of thesis)|
Preparation, Multiple, Conjugates, LPS- Protein,
Immunization, Typhoidal, Diseases, pathogens, lipopolysaccharide
Salmonella enterica serovar Typhi (S. Typhi) is associated with
typhoid, a major health problem in developing countries. However, in
recent years Salmonella enterica serovar Paratyphi A (S. Paratyphi
A) and Vi negative variants of S. Typhi have emerged rapidly. For
development of multiple protein-polysaccharide conjugates with
potential to be used as vaccines, we needed to have a first hand
knowledge of local distribution of these pathogens. For this
purpose, we developed a nested multiplex PCR targeting 5 different
genes for differential diagnosis of typhoidal pathogens which has
been optimized to be directly applicable on clinical blood samples.
Out of 42 multiplex PCR positive blood samples, 26, 9, and 2 were
Vi-positive S. Typhi, Vi-negative S. Typhi and S. Paratyphi A,
respectively and 5 patients were found to have mixed infection.
Seventeen patients grew Salmonella from blood culture and the
remaining 25 were positive in the Salmonella specific PCR. Tests
with several common pathogens confirmed the specificity of the
assay. We conclude that the proposed multiplex PCR is rapid,
sensitive and specific for the diagnosis of typhoidal pathogens
directly from blood samples. Detection of Vi negative strains of S.
Typhi and S. Paratyphi A among typhoidal patients suggested the need
to work on vaccines which are not based on Vi polysaccharide only.
Therefore, O-specific polysaccharides (OSP) purified from
lipopolysaccharides (LPS) of S. Typhi and S. Paratyphi A were
conjugated with diphtheria toxoid (DT) using adipic acid dihydrazide
(ADH) as linker, and evaluated for immunogenicity in mice. Use of DT
as carrier protein for Salmonellae has not been reported before. S.
Typhi OSP-AH-DT conjugate 1 and 2 elicited significantly higher IgG
anti-LPS ELISA titer (P = 0.0241 and 0.0245 respectively) than
polysaccharide alone. The injection schedule-B (three injections
with 4-weeks interval) was found better than schedule-A (three
injections with 2-weeks interval). The conjugate of S. Paratyphi A
OSP with DT without linker molecule did not elicit sufficient immune
response to be used as conjugate vaccine candidate while antibody
response against S. Paratyphi A OSP-AH-DT onjugate was found
significantly higher (P = 0.0446) than polysaccharide alone.