Immune responses of selected phagotopes from monoclonal antibodies of Burkholderia pseudomallei.

Random peptide libraries displayed by bacteriophage T7 and M13 were employed to identify mimotopes from 4 monoclonal antibodies (MAbs) specific to Burkholderia pseudomallei. Insert DNA sequences of bound phages selected from four rounds of panning with each MAb revealed peptide sequences corresponding to B. pseudomallei K96243 hypothetical protein BPSL2046, hypothetical protein BpseP_02000035, B. pseudomallei K96243 hypothetical protein BPSS0784, B. pseudomallei 1710b hypothetical protein BURPS1710b_1104, and B. cenocepacia H12424 TonB-dependent siderophore receptor, all located at the outer membrane. The immune responses from all selected phagotopes were significantly higher than that of lipopolysaccharide. The study demonstrates the feasibility of identifying mimotopes through screening of phage-displayed random peptide libraries with B. pseudomallei MAbs.

Antimicrobial susceptibility patterns and phage types of Salmonella typhi from Vietnam.

A retrospective study of the patterns of antimicrobial susceptibility and phage types of 111 Salmonella typhi strains isolated in 1996 from Vietnam was carried out. The strains were tested for susceptibility to chloramphenicol, ampicillin, tetracycline, trimethoprim-sulfamethoxazole, nalidixic acid, ceftazidime, ceftriaxone and ciprofloxacin. Simultaneous resistance to chloramphenicol, ampicillin, tetracycline and trimethoprim-sulfamethoxazole were present in 84 strains (75.7%). Nalidixic acid resistance was only observed in 2 multidrug-resistant strains (1.8%). Twenty-one strains (18.9%) were completely susceptible to all drugs tested. All 111 strains were susceptible to ceftazidime, ceftriaxone and cipropfloxacin. The MIC values for chloramphenicol, ampicillin and trimethoprim-sulfamethoxazole corresponded with the results by disk diffusion method. On Vi phage-typing, 5 different phage types (28, A, D1, E1 and M1) were found in 12 strains (10.8%). However, most S. typhi strains were indistinguishable by this typing technique because they were degraded Vi-positive or untypeable Vi-positive strains (35.1% and 54.1%, respectively). There were no correlations between antimicrobial resistance patterns and phage types in the tested S. typhi strains in this study.

Patient and sample-related factors that effect the success of in vitro isolation of Orientia tsutsugamushi.

Orientia tsutsugamushi is the causative agent of scrub typhus infection, a major cause of human disease in rural areas of Southeast Asia. Twenty-six blood samples collected from patients with serologically proven scrub typhus during a six month period were sent to Bangkok (535 km from the clinical site) by road at ambient temperature (average daily temperature range: 27.1-29.1 degrees C) for attempted in vitro isolation in Vero cells. O. tsutsugamushi was isolated from 12 samples (sensitivity 46.7%) with the time to isolation ranging from 16 to 37 days [median 27 days, inter-quartile range (IQR) 22.5-33.5 days]. Patient factors such as days of fever and O. tsutsugamushi IgM antibody titer, transport factors such as transit time, and isolate genotype (Karp and Gilliam/Kawasaki) were assessed to determine their influence on the outcome of in vitro isolation. None of the factors significantly influenced the isolation outcome. This study demonstrates that O. tsutsugamushi can often be isolated in vitro from the blood of scrub typhus patients when transported at ambient tropical temperatures for many days.

CpG DNA, liposome and refined antigen oral cholera vaccine.

An oral cholera vaccine made up of three Vibrio cholerae antigens, i.e. lipopolysaccharide (LPS), recombinant toxin co-regulated pili (rTcpA) and heat-treated cholera toxin (H-CT) has been developed in six different formulations. Eight-week-old Wistar rats were divided into nine groups and immunized as follows: the first group received the oral vaccine 1 consisting of the three antigens (LPS, rTcpA and H-CT) associated with a liposome (L) and bacterial CpG-DNA (ODN#1826). The rats of groups 2 and 3 received oral vaccines 2 and 3 consisting of the liposome-associated three antigens with and without non-bacterial CpG-DNA (ODN#1982), respectively. Rats of groups 4 received oral vaccine 4 consisting of the three antigens mixed with the ODN#1826, similar to vaccine 1, but without liposome. Rats of groups 5 and 6 received oral vaccines 5 and 6 consisting of the three antigens with and without ODN#1982, respectively, similar to vaccines 2 and 3, but without liposome. Rats of groups 7, 8 and 9 received oral placebos, namely liposomes (L), ODN#1826 (CpG), and vaccine diluent, i.e. 5% NaHCO3 solution, respectively. All vaccines were given in three doses at 14-day intervals. It was found that the combination of liposome and ODN#1826 in vaccine 1 evoked the highest immune response to V. cholerae antigen compared to other vaccine formulations and placebos, as measured by the appearance of antigen-specific antibody-producing cells in the intestinal lamina propria. The immunogenicity according to the magnitude of the immune response was: V1>V2=V3>V4>V5=V6>V7=V8=V9. The results of this study indicate that CpG-DNA and liposome are effective mucosal adjuvants for an oral cholera vaccine prepared from refined V. cholerae antigens and their combination seems to be synergistic. The potential role of liposome as a vaccine delivery vehicle has been confirmed.

Recombinant American cockroach component, Per a 1, reactive to IgE of allergic Thai patients.

Twelve similar recombinant Per a 1 clones were produced from an American cockroach (CR) cDNA library. The nucleotide sequence of a representative cline, i.e. clone A6, contained 579 base pairs (bp) and a 372 bp open reading frame (2-373) encoding 124 amino acids. A stop codon was found at position 374-376 followed by a 3' end untranslated region with an AATAAA polyadenylation signal and a poly (A) tail. The estimated molecular mass of the 24 amino acid residue protein was 13.8 kDa, with a predicted isoelectric point value of 4.74. Cysteine or N-linked glycosylation was not found. The deduced amino acid sequence of the A6 revealed 84.68-95.97% identity to other previously reported Per a 1 clones and 65.87-69.60% homology to the previously reported Bla g 1 clones. However, while previously reported Per a 1 clones showed homology to ANG12, a precursor protein in the midgut of the female Anopheles gambiae secreted after the blood meal, the A6 DNA sequence was found to have homology (37.1%) to DNA of G2, a putative protein in the midgut of Aedes aegypti (AY 050565). The deduced amino acid sequence of A6 contained a mitochondrial energy transfer protein signature, phosphorylation sites for the cAMP-and cGMP-dependent protein kinase C and casein kinase II. Hydrophobic and hydrophilic characteristics of the A6 deduced peptide indicated that it was a transmembrane protein. This is the first report that Per a 1 is a transmembrane protein. The deduced amino acid sequence of the A6, which contained the sequence LIRSLFGLP, differed in one amino acid from two previously reported epitopes, i.e. LIRALFGL and IRSWFGLP, of Per a 1.0104 which bound 80% and 100%, respectively, to IgE of the allergic patients tested. The A6 DNA sequence was deposited in the GenBank (Accession number AY 259514) and has been designated Per a 1.0105. The A6 expressed protein bound to monoclonal antibodies (MAb 3C2) specific to American cockroach and also bound to IgE of all (100%) of the 20 allergic Thai patients.