Susan K. Hoiseth

BACKGROUND: Recombinant forms of Neisseria meningitidis human factor H binding protein (fHBP) are undergoing clinical trials in candidate vaccines against invasive meningococcal serogroup B disease. We report an extensive survey and phylogenetic analysis of the diversity of fhbp genes and predicted protein sequences in invasive clinical isolates obtained in the period 2000-2006. METHODS: Nucleotide sequences of fhbp genes were obtained from 1837 invasive N. meningitidis serogroup B (MnB) strains from the United States, Europe, New Zealand, and South Africa. Multilocus sequence typing (MLST) analysis was performed on a subset of the strains. RESULTS: Every strain contained the fhbp gene. All sequences fell into 1 of 2 subfamilies (A or B), with 60%-75% amino acid identity between subfamilies and at least 83% identity within each subfamily. One fHBP sequence may have arisen via inter-subfamily recombination. Subfamily B sequences were found in 70% of the isolates, and subfamily A sequences were found in 30%. Multiple fHBP variants were detected in each of the common MLST clonal complexes. All major MLST complexes include strains in both subfamily A and subfamily B. CONCLUSIONS: The diversity of strains observed underscores the importance of studying the distribution of the vaccine antigen itself rather than relying on common epidemiological surrogates such as MLST.

SUMMARY Strains of Salmonella sp with complete nonreverting aromatic biosynthesis ( aro ) defects are expected to be nonvirulent, in respect to invasive infection, because they need the aromatic metabolites paraaminobenzoate (for making folate) and dihydroxybenzoate (for making enterochelin) which are not available in host tissues. Derivatives with transposon-generated complete nonreverting aro − defects were prepared from 3 mouse-virulent strains of S typhimurium , namely, FIRN, WRAY, and UCD. The latter 2 parent strains originally were isolated from calves and are known to be calf-virulent. The resultant aromatic-dependent ( aro − ) strains were used to vaccinate 27 calves (2 to 3 weeks old), usually giving 2 doses by the im route (10 9 bacteria) or orally (1.5 × 10 11 ). Vaccination did not cause severe ill effects in any calf. Thus aro − defects cause loss of virulence for calves, as previously shown for mice. Vaccinated and control calves were challenge exposed, usually at 5 weeks of age, by feeding 1.5 × 10 11 cells of 1 of 2 calf-virulent S typhimurium strains, either UCD 108-11 or SL1323. Of the 16 challenge-exposed control calves, all became anorectic and depressed ( cns ), and 15 had diarrhea. Fourteen of the 16 died; all tested tissues were bacteriologically culture-positive for Salmonella at necropsy. Vaccination with the live UCD aro − vaccine strain, SL1479 by either of 2 schedules ( im or orally) appeared effective. Six of the 7 calves vaccinated with 2 im doses of strain SL1479 survived challenge exposure (4 of the 7 not even developing diarrhea), and all of 3 calves which had been vaccinated with 2 oral doses of the live UCD vaccine also survived challenge exposure. In contrast, 3 calves which had been given 1 im dose and then the oral dose of strain SL1479 were not protected—2 of them dying after challenge exposure. The other aro − strains (FIRN and WRAY) proved ineffective or relatively ineffective as live-organism vaccines, at least with the schedules tested. Failure of the FIRN aro − vaccine may reflect the organism's origin from a wild strain which, though mice-virulent, was of a bio type seldom isolated from cattle and perhaps not well adapted for persistence in bovine tissues. There is no reason that can be suggested for the vaccinal failure of 2 oral doses of the WRAY aro − strain. Thus one aro − live bacterial vaccine, the S typhimurium UCD aro − strain SL1479, given im (2 doses) or orally (2 doses), did not cause significant side effects and prevented death or severe illness in challenge exposed calves.

Salmonella typhimurium SL1479, an auxotrophic mutant strain having a complete block in the aromatic biosynthetic pathway and therefore requiring p-aminobenzoic acid and 2,3-dihydroxybenzoate not available in mammalian tissues, was given orally in a dose of 10(10) live bacteria to 4- to 5-week-old calves. Only a mild transient fever response was seen. Strain SL1479 was unable to colonize and persist in the calves for more than 2 weeks. In a vaccination experiment, groups of six calves were (i) orally vaccinated with the live S. typhimurium SL1479 strain, (ii) subcutaneously vaccinated with a heat-inactivated S. typhimurium SVA1232 strain with aluminum hydroxide as adjuvant, or (iii) not vaccinated, to serve as controls. Calves were orally challenged with the live, calf-virulent S. typhimurium SVA44 strain: either 10(6) bacteria (equivalent to 100 25% lethal doses [LD25S]) or 10(9) bacteria (100,000 LD25S doses). The live oral vaccine gave significantly better protection than the heat-vaccinated, subcutaneously injected vaccine since (i) only control calves and calves given the killed vaccine developed profuse diarrhea, (ii) clinically, the mild fever responses seen after challenge in calves given the live vaccine were significantly lower (P less than 0.0005), (iii) autopsies performed 21 days after the challenge infection revealed normal findings in calves given the live vaccine, whereas calves given the killed vaccine showed signs of acute enteritis and chronic salmonellosis, (iv) all 12 calves given either 100 X or 100,000 X the LD25 survived the 21-day observation period; the mean survival time in nonvaccinated calves was 8.0 days; in calves given heat-inactivated vaccine and 100 X the LD25 it was 21.0 days, and in calves given 100,000 X the LD25 it was 11.5 days, (v) the fecal bacterial counts of the challenge S. typhimurium SVA44 strain were significantly lower (P less than 0.0005) in both groups given the live vaccine, and (vi) upon autopsy followed by culture, the qualitative recovery of the challenge strain from the alimentary canals and tissues of calves given the live vaccine was significantly lower (P less than 0.005).