ABSTRACT
Two formulations of pneumococcal vaccines are currently available to prevent invasive disease in adults and children. However, these vaccines will not protect against the majority of Streptococcus pneumoniae serotypes. The use of highly conserved cell-wall-associated proteins in vaccines may circumvent this problem. A proteomics approach was used to identify 270 S. pneumoniae cell-wall-associated proteins, which were then screened in a process that included in-silico, in-vitro and in-vivo validation criteria. Five potential candidates for inclusion in a vaccine were selected, expressed in Escherichia coli, and purified for use in immunisation experiments. These proteins were detected in at least 40 different serotypes of S. pneumoniae, and were expressed in S. pneumoniae isolates causing infection. Two of the five candidate proteins, the putative lipoate protein ligase (Lpl) and the ClpP protease, resulted in a reduced CFU titre and a trend towards reduced mortality in an animal sepsis model for investigating new S. pneumoniae protein vaccines.
Subject(s)
Bacterial Proteins/analysis , Membrane Proteins/analysis , Pneumococcal Vaccines/immunology , Proteome/analysis , Streptococcus pneumoniae/chemistry , Adult , Animals , Bacterial Proteins/isolation & purification , Cell Wall/chemistry , Child , Cloning, Molecular , Colony Count, Microbial , Escherichia coli/genetics , Gene Expression , Humans , Membrane Proteins/isolation & purification , Mice , Mice, Inbred BALB C , Pneumococcal Infections/immunology , Pneumococcal Infections/microbiology , Pneumococcal Infections/mortality , Sepsis/immunology , Sepsis/microbiology , Sepsis/mortalityABSTRACT
Campylobacter jejuni is one of the most common causes of traveller's diarrhoea and food poisoning, therefore development of a vaccine is important. Using biochemical fractionation and mass spectrometry analysis, we identified more than 110 surface polypeptides. Eight C. jejuni identified surface proteins were expressed in Escherichia coli and purified. Mice were immunized with different doses of these purified proteins and challenged orally with C. jejuni strains ML1 and ML53. The degree of protection of mice was tested by intestinal colonization. At least two groups of mice vaccinated with purified proteins clear the infection faster than control mice. Here, we present the use of a proteomics based approach for the identification of novel protein based C. jejuni vaccines for the first time.
Subject(s)
Bacterial Proteins/immunology , Bacterial Vaccines/immunology , Campylobacter jejuni/immunology , Diarrhea/prevention & control , Peptides/immunology , Proteomics , Travel , Animals , Antigens, Surface/immunology , Antigens, Surface/isolation & purification , Antigens, Surface/metabolism , Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Campylobacter jejuni/metabolism , Diarrhea/immunology , Mice , Peptides/isolation & purification , Peptides/metabolismABSTRACT
The synthesis of novel Boc/acyl protected monomers for the synthesis of peptide nucleic acid (PNA) is described. The oligomerization protocol using these new monomers has been optimized with regard to coupling reagents. The use of base-labile acyl protecting groups at the exocyclic amines of the heterocyclic bases (isobutyryl for guanine and benzoyl for adenine and cytosine) and a PAM-linked solid support offers an attractive alternative to the present procedures used in PNA synthesis. This strategy has been applied for the synthesis of a test 17mer PNA on both control pore glass (CPG) and a polystyrene MBHA support and was used in the preparation of PNA-DNA chimeras.
Subject(s)
Biochemistry/methods , Peptide Nucleic Acids/chemistry , Peptide Nucleic Acids/chemical synthesis , Chromatography, High Pressure Liquid , DNA/chemistry , Models, Chemical , Nucleic Acid ConformationABSTRACT
Deprotected compounds 1 and 9 were allowed to react with 4,4'-dimethoxytrityl chloride in pyridine to give 5'-O-DMT nucleosides 2 and 10. The 3'-phosphoramidites 4 and 11 were incorporated into oligodeoxynucleosides (ODNs). The hybridization properties of the modified ODNs with their complementary DNA strands were studied. Interesting results were obtained when 11 was inserted as a bulged nucleoside into TWAs, duplexes, and triplexes.
