A rational strategy for a malarial vaccine development

Twenty years ago we reported the first synthetic peptide-based anti-malarial vaccine named SPf66, which conferred limited protective efficacy in large-scale human field-trials. Our efforts towards a second vaccine generation based on the rational selection of conserved high activity binding peptides (HABPs) whose critical binding residues have to be precisely replaced by others. Introducing peptide bond isosters on these HABPs' critical binding residues constitutes also an important approach. Our results suggest that knowing a parasite's immunologically active peptides, their 3D structure, and their interaction for properly stabilizing MHC-II peptide-TCR complexes constitutes the basis for rationally designing a fully effective, multi-component, multistage subunit-based anti-malarial vaccine.     

Tooth size and morphology in a recent Australian Aboriginal population from Broadbeach,South East Queensland

Odontometric and morphologic observations were made of the dentition of skeletal remains of Australian aborigines from Boradbeach, S.E. Queensland. Tooth size, especially of the molars, was found to be significantly larger than that reported for other recent Aboriginal populations. Tooth morphology also differed, with a higher frequency of five cusped second molars, and a lower frequency of shoveling and Carabelli's cusp than previously reported as typical of Australian aborigines.     

Composition and properties of a group A streptococcal teichoic acid

Teichoic acid-like material extracted by cold trichloroacetic acid from lyophilized whole cells of streptococci from groups A,D,E,O, and T was shown to give a positive precipitin reaction with group antisera. Similar material from cells of groups B,C,F,G,H,K,L,M,N,P,Q,R, and S did not give a positive reaction with group antisera. The group A material also reacted with anti-E serum; however, the opposite did not occur. A similar result was also obtained on the group T material and anti-O serum. The group A teichoic acid was purified by Sephadex column chromatography, and was shown to be free of cell wall peptidoglycan and polysaccharide, and ribitol teichoic acid. It was composed of glycerol, phosphate, alanine, and glucosamine. Alkaline hydrolysis showed the presence of ester-linked alanine and glucosaminylglycerol. Phosphorus was released from ester linkage by alkaline phosphatase. N-acetylglucosamine produced a 72% inhibition of the precipitin test at a level of 10 mumoles, and d-alanine methyl ester was significantly stronger than the l-alanine ester. A single precipitin band was seen with group A serum. The data indicate that teichoic acid of group A streptococci is a polymer composed of glycerol phosphate and containing N-acetylglucosamine and alanine. Antisera to these streptococci contain antibodies specific for the alanine and the glucosamine linkages. The use of serum containing antibodies to alanine-polyglycerophosphate shows that the occurrence of this type of teichoic acid is widespread among the streptococci.     

Molecular mechanisms underlying group A streptococcal pathogenesis

Group A Streptococcus (GAS) is a versatile human pathogen causing diseases ranging from uncomplicated mucosal infections to life-threatening invasive disease. The development of human-relevant animal models of GAS infection and introduction of new technologies have markedly accelerated the pace of discoveries related to GAS host–pathogen interactions. For example, recently investigators have identified pili on the GAS cell surface and learned that they are key components for adherence to eukaryotic cell surfaces. Similarly, the recent development of a transgenic mouse expressing human plasminogen has resulted in new understanding of the molecular processes contributing to invasive infection. Improved understanding of the molecular mechanisms underlying the pathogenesis of GAS pharyngeal, invasive and other infections holds the promise of assisting with the development of novel preventive or therapeutic agents for this prevalent human pathogen.     

Osmotic fragility of the group A streptococcal L form

The osmotic fragility, expressed in terms of survival, of two group A streptococcal L-form strains was examined by suspending the L form in sodium chloride and sucrose solutions of graded concentrations. An immediate and marked reduction in viability followed suspension in sodium chloride solutions of less than 0.7m. A wide distribution of osmotic fragility within the L-form population was observed. The two L-form strains (GL-8 and AED) differed in that the AED L-form strain appeared to be consistently more resistant to osmotic lysis, and survived considerably better in sodium chloride solutions up to 90 minutes. Sucrose solutions of tonicities comparable to those of the sodium chloride solutions used, however, stabilized the labile GL-8 L form completely. Magnesium chloride (0.05m) and serum (10% v/v) substantially increased L-form survival in sodium chloride. The results are interpreted to indicate a difference in the cell envelope of the two L-form strains, the AED limiting membrane possessing a greater intrinsic stability. The significantly greater resistance to sonic oscillation of the AED L form as compared to the GL-8 L form, is in agreement with and supports this conclusion. The possibility that the difference in physical properties of the two L-form strains is related to a difference in chemical composition of their limiting envelopes is discussed.The author wishes to thank Dr. W. Hijmans for his interest and advice, and Miss H. L. Ensering and Miss M. J. W. Kastelein for technical assistance.     

A genome-wide association scan for asthma in a general Australian population

To date, almost every chromosome has been implicated in genetic susceptibility to asthma to some degree. When compared with single nucleotide polymorphism, microsatellite markers exhibit high levels of heterozygosity and therefore provide higher statistical power in association. The objective of this study was to perform a genome-wide association study using 23,465 in-house microsatellite markers to detect asthma susceptibility regions in the Busselton population. In this study, three separate pooled DNA screenings yielded 18 markers with significantly different estimated frequencies in the three separate “case and control” pools: each pool consisting of 60 males and 60 females. These markers were evaluated by individual typing in 360 cases and 360 controls. Two markers showed significant differences between cases and controls (P = 0.001 and P = 0.003). Regions surrounding the two markers were subjected to high-density association mapping with a total of 14 additional markers. We were able to confirm and fine map the association in these two regions by typing 14 additional microsatellite markers (1805A09 (D18S0325i), P = 0.002; 1806D05 (D18S0181i), P = 0.001). Each region contains a predicted gene that showed strong associations with asthma. Further studies are underway to characterize the novel candidate asthma susceptibility genes identified in this genome-wide study.     

Bactericidal activity of M protein conserved region antibodies against group A streptococcal isolates from the Northern Thai population

Background  

Most group A streptococcal (GAS) vaccine strategies have focused on the surface M protein, a major virulence factor of GAS. The amino-terminus of the M protein elicits antibodies, that are both opsonic and protective, but which are type specific. J14, a chimeric peptide that contains 14 amino acids from the M protein conserved C-region at the carboxy-terminus, offers the possibility of a vaccine which will elicit protective opsonic antibodies against multiple different GAS strains. In this study, we searched for J14 and J14-like sequences and the number of their repeats in the C-region of the M protein from GAS strains isolated from the Northern Thai population. Then, we examined the bactericidal activity of J14, J14.1, J14-R1 and J14-R2 antisera against multiple Thai GAS strains.     

Molecular characterization of new group A streptococcal bacteriophages containing the gene for streptococcal erythrogenic toxin A (speA)

Summary Bacteriophage T12 is the prototype phage carrying the streptococcal erythrogenic toxin A (speA) gene. To examine more closely the phages involved in lysogenic conversion, we examined 300 group A streptococcal strains, and identified and isolated two new phages that carry the speA gene. The molecular sizes of these phage genomes were between 32 and 40 kb, similar to that of phage T12 (35 kb). However, as ascertained by restriction analysis, the physical maps of the new phage genomes were different from phage T12 and from each other. Hybridization analysis also showed that all of these phages were only partially related to one another and the speA gene was always located close to the phage attachment site. Additionally, colony hybridization showed that whereas phage T12 or one of its close relatives is the most common phage associated with the group A streptococci, phage 49 has a much stronger association with the speA gene. A defective phage was also found following pulsed field gel electrophoresis of total phage DNA. This phage appears to be a resident of strain T25₃c and is found only following induction of a T25₃c lysogen. Restriction enzyme analysis of the isolated defective phage DNA suggests that it is the source of the submolar amounts of DNA previously found in association with phage T12 digestion patterns. Additionally, the defective phage may serve as the site of integration of the speA gene-carrying phages described above.     

Molecular insight into invasive group A streptococcal disease

Streptococcus pyogenes is also known as group A Streptococcus (GAS) and is an important human pathogen that causes considerable morbidity and mortality worldwide. The GAS serotype M1T1 clone is the most frequently isolated serotype from life-threatening invasive (at a sterile site) infections, such as streptococcal toxic shock-like syndrome and necrotizing fasciitis. Here, we describe the virulence factors and newly discovered molecular events that mediate the in vivo changes from non-invasive GAS serotype M1T1 to the invasive phenotype, and review the invasive-disease trigger for non-M1 GAS. Understanding the molecular basis and mechanism of initiation for streptococcal invasive disease may expedite the discovery of novel therapeutic targets for the treatment and control of severe invasive GAS diseases.     

Mouse antibody response to group A streptococcal carbohydrate

In an attempt to more fully understand the generation of antibody diversity to carbohydrate (CHO) Ag, we produced and characterized a panel of hybridoma cell lines specific for group A streptococcal CHO from mice injected with the intact bacteria (minus the hyaluronic acid capsule and cell wall protein Ag). We have analyzed the use of H and L chain V region genes in the early (day 7) and late response (hyperimmune) and have sequenced the dominant VH gene used in several of our hybridomas. Our data allowed us to assess the extent to which the recombination of various V, D, and J gene segments and somatic mutation contribute to antibody diversification in this system. In this report we confirm that a minimum of two VH and four VK gene segments are used to encode this response. We extend this analysis to show that multiple D and J gene segments are used and that a significant amount of junctional variability is tolerated in CDR 3. Our results indicate that the level of somatic mutation in the hyperimmune response is generally low in comparison with the response to haptens and protein Ag. These data also suggest that there is a positive selection for mutation in CDR 1 during the hyperimmune response to group A streptococcal CHO.     

Domains of group A streptococcal M protein that confer resistance to phagocytosis, opsonization and protection: implications for vaccine development

Streptococcus pyogenes (group A streptococcus) colonizes skin and throat tissues resulting in a range of benign and serious human diseases. Opsonization and phagocytosis are important defence mechanisms employed by the host to destroy group A streptococci. Antisera against the cell-surface M protein, of which over 150 different types have been identified, are opsonic and contribute to disease protection. In this issue of Molecular Microbiology, Sandin and colleagues have comprehensively analysed the regions of M5 protein that contribute to phagocytosis resistance and opsonization. Human plasma proteins bound to M5 protein B- and C-repeats were shown to block opsonization, an observation that needs to be carefully considered for the development of M protein-derived vaccines. While safe and efficacious human group A streptococcal vaccines are not commercially available, candidate M protein-derived vaccines have shown promise in murine vaccine models and a recent phase 1 human clinical trial.     

DNase Sda1 provides selection pressure for a switch to invasive group A streptococcal infection

Most invasive bacterial infections are caused by species that more commonly colonize the human host with minimal symptoms. Although phenotypic or genetic correlates underlying a bacterium's shift to enhanced virulence have been studied, the in vivo selection pressures governing such shifts are poorly understood. The globally disseminated M1T1 clone of group A Streptococcus (GAS) is linked with the rare but life-threatening syndromes of necrotizing fasciitis and toxic shock syndrome. Mutations in the GAS control of virulence regulatory sensor kinase (covRS) operon are associated with severe invasive disease, abolishing expression of a broad-spectrum cysteine protease (SpeB) and allowing the recruitment and activation of host plasminogen on the bacterial surface. Here we describe how bacteriophage-encoded GAS DNase (Sda1), which facilitates the pathogen's escape from neutrophil extracellular traps, serves as a selective force for covRS mutation. The results provide a paradigm whereby natural selection exerted by the innate immune system generates hypervirulent bacterial variants with increased risk of systemic dissemination.     

Trends in stature in the South Australian Aboriginal Murraylands

Millennial and secular changes in body height of prehistoric and recent Aboriginal South Australians are investigated. Skeletal remains of 55 male and 40 female individuals who were excavated at Roonka on the River Murray were dated from 9800 to 100 years BP. Stature was reconstructed by using humerus, femur, and tibia ratios to stature derived from Abbie's (1975) data on living Aborigines and the Trotter-Gleser method for blacks. The respective averages were 1,652 mm and 1,665 mm for males and 1,527 mm and 1,549 mm for females. In 1996/1997, statures of 27 adult males and 21 adult females were measured in Aboriginal centers of Gerard and Raukkan (Point McLeay) on the Lower River Murray. These people, as far as it can be ascertained, are the descendants of the people from Roonka. Their statures were adjusted for the stature loss with age, so that the data represent young individuals (≤30 years of age). The average male stature was 1,712 mm, and the average female stature was 1,567 mm. Data collected by Wood Jones and Campbell in 1924 for Aboriginal South Australians show that young adult male stature was 1,668 mm (n = 6), and female stature was 1,552 mm (n = 4). Slopes of regressions of individual statures on radiocarbon dates and on dates of birth are not significantly different from zero. The same is true for regressions of individual long bone lengths on radiocarbon dates. It can be concluded that there was little change in stature of Aboriginal South Australians from prehistoric to recent times. Regressions of individual age-corrected heights on birth dates (1860–1980) of Aboriginal men and women measured in 1924 and in 1996 further indicate no significant increase in height in either sex. Am J Phys Anthropol 106:505–514, 1998. © 1998 Wiley-Liss, Inc.