James B. Dale

Safety and Immunogenicity of 26-Valent Group A Streptococcus Vaccine in Healthy Adult Volunteers

BACKGROUND Group A streptococcus (GAS) causes illness ranging from uncomplicated pharyngitis to life-threatening necrotizing fasciitis, toxic shock, and rheumatic fever. Attempts to develop an M protein-based vaccine have been hindered by the fact that some M proteins elicit both protective antibodies and antibodies that cross-react with human tissues. New molecular techniques have allowed the previous obstacles to be largely overcome. METHODS The vaccine is comprised of 4 recombinant proteins adsorbed to aluminum hydroxide that contain N-terminal peptides from streptococcal protective antigen and M proteins of 26 common pharyngitis, invasive, and/or rheumatogenic serotypes. Thirty healthy adult subjects received intramuscular 26-valent GAS vaccine (400 microg) at 0, 1, and 4 months, with clinical and laboratory follow-up for safety and immunogenicity using assays for tissue cross-reactive antibodies, type-specific M antibodies to 27 vaccine antigens, and functional (opsonization) activity of M protein antibodies. RESULTS The incidence of local reactogenicity was similar to that for other aluminum hydroxide-adsorbed vaccines in adults. No subject developed evidence of rheumatogenicity or nephritogenicity, and no induction of human tissue-reactive antibodies was detected. Overall, 26 of 27 antigenic peptides evoked a >4-fold increase in the geometric mean antibody titer over baseline. The mean log2 fold-increase in serum antibody titer (+/- standard error of the mean) for all 27 antigens was 3.67 +/- 0.21. A significant mean log2 reduction in streptococcal bacterial counts in serum samples obtained after immunization was seen in opsonization assays for all M serotypes. CONCLUSIONS On the basis of epidemiological data demonstrating that the majority of cases of pharyngitis, necrotizing fasciitis, and other invasive streptococcal infections are caused by a limited number of serotypes, this 26-valent vaccine could have significant impact on the overall burden of streptococcal disease.

Immunogenicity of a 26-Valent Group A Streptococcal Vaccine

ABSTRACT A multivalent vaccine containing amino-terminal M protein fragments from 26 different serotypes of group A streptococci was constructed by recombinant techniques. The vaccine consisted of four different recombinant proteins that were formulated with alum to contain 400 μg of protein per dose. Rabbits were immunized via the intramuscular route at 0, 4, and 16 weeks. Immune sera were assayed for the presence of type-specific antibodies against the individual recombinant M peptides by enzyme-linked immunosorbent assay and for opsonic antibodies by in vitro opsonization tests and indirect bactericidal tests. The 26-valent vaccine was highly immunogenic and elicited fourfold or greater increases in antibody levels against 25 of the 26 serotypes represented in the vaccine. The immune sera were broadly opsonic and were bactericidal against the majority of the 26 different serotypes. Importantly, none of the immune sera cross-reacted with human tissues. Our results indicate that type-specific, protective M protein epitopes can be incorporated into complex, multivalent vaccines designed to elicit broadly protective opsonic antibodies in the absence of tissue-cross-reactive antibodies.

Molecular mechanisms of adhesion, colonization, and invasion of group A streptococci

The initial step in establishing a bacterial infection is adhesion of the organism to the epithelium of the host. Group A streptococci use multiple adhesins to attach to host cells and the types of adhesins expressed by a particular strain will determine its tissue specificity. Expression of adhesins is regulated in response to changing environmental and growth conditions. Thus, the array of adhesins expressed by a group A streptococcus will depend on the complement of its adhesin genes and on the environment. Expression of some adhesins may trigger internalization of the streptococci by host cells, which may enable the streptococci to evade antibiotics and to facilitate the penetration of deeper tissues. In this review, we present the different molecular mechanisms of adhesion utilized by group A streptococci and how these interactions lead to colonization and invasion.

Prevalence of Rheumatic Heart Disease in Children and Young Adults in Nicaragua

Rheumatic heart disease (RHD) results in morbidity and mortality that is disproportionate among individuals in developing countries compared to those living in economically developed countries. The global burden of disease is uncertain because most previous studies to determine the prevalence of RHD in children relied on clinical screening criteria that lacked the sensitivity to detect most cases. The present study was performed to determine the prevalence of RHD in children and young adults in León, Nicaragua, an area previously thought to have a high prevalence of RHD. This was an observational study of 3,150 children aged 5 to 15 years and 489 adults aged 20 to 35 years randomly selected from urban and rural areas of León. Cardiopulmonary examinations and Doppler echocardiographic studies were performed on all subjects. Doppler echocardiographic diagnosis of RHD was based on predefined consensus criteria that were developed by a working group of the World Health Organization and the National Institutes of Health. The overall prevalence of RHD in children was 48 in 1,000 (95% confidence interval 35 in 1,000 to 60 in 1,000). The prevalence in urban children was 34 in 1,000, and in rural children it was 80 in 1,000. Using more stringent Doppler echocardiographic criteria designed to diagnose definite RHD in adults, the prevalence was 22 in 1,000 (95% confidence interval 8 in 1,000 to 37 in 1,000). In conclusion, the prevalence of RHD among children and adults in this economically disadvantaged population far exceeds previously predicted rates. The findings underscore the potential health and economic burden of acute rheumatic fever and RHD and support the need for more effective measures of prevention, which may include safe, effective, and affordable vaccines to prevent the streptococcal infections that trigger the disease.

A Systematic and Functional Classification of Streptococcus pyogenes That Serves as a New Tool for Molecular Typing and Vaccine Development

Streptococcus pyogenes ranks among the main causes of mortality from bacterial infections worldwide. Currently there is no vaccine to prevent diseases such as rheumatic heart disease and invasive streptococcal infection. The streptococcal M protein that is used as the substrate for epidemiological typing is both a virulence factor and a vaccine antigen. Over 220 variants of this protein have been described, making comparisons between proteins difficult, and hindering M protein-based vaccine development. A functional classification based on 48 emm-clusters containing closely related M proteins that share binding and structural properties is proposed. The need for a paradigm shift from type-specific immunity against S. pyogenes to emm-cluster based immunity for this bacterium should be further investigated. Implementation of this emm-cluster-based system as a standard typing scheme for S. pyogenes will facilitate the design of future studies of M protein function, streptococcal virulence, epidemiological surveillance, and vaccine development.

Group A Streptococcal Pharyngitis Serotype Surveillance in North America, 2000–2002

Geographic and interseasonal heterogeneity of pharyngeal group A streptococcal (GAS) genotypes (emm types) is poorly characterized. We evaluated emm type and subtype distribution among pediatric pharyngitis isolates obtained from 9 sites in the United States during 2000-2001 (year 1) and from 10 sites in the United States and 1 site in Canada during 2001-2002 (year 2). The 7 predominant types were the same in both years, although their order changed. emm 12, 1, and 28 accounted for 49.2% of year 1 isolates, and emm 1, 12, and 4 accounted for 47.1% of year 2 isolates; 6 types accounted for 72.1% in year 1 and 69.4% in year 2. From year 1 to year 2, the proportions of emm 12 and 28 decreased and emm 1 and 6 increased. Striking intersite and interseasonal variations in the distribution of predominant emm types were observed. We conclude that the most-predominant GAS genotypes were similar for each year despite fluctuations, that intersite and intrasite variations in the distribution of emm types were apparent, and that emm type surveillance is needed as M protein vaccine development proceeds.

Serum opacity factor is a major fibronectin‐binding protein and a virulence determinant of M type 2 Streptococcus pyogenes

Serum opacity factor (SOF) is a fibronectin‐binding protein of group A streptococci that opacifies mammalian sera and is expressed by some strains that cause impetigo, pharyngitis and acute glomerulonephritis. Although SOF is expressed by ≈35% of known serotypes, its role in the pathogenesis of group A streptococcal infections has not been previously investigated. The sof genes from M types 2, 28 and 49 Streptococcus pyogenes were cloned, sequenced, and their deduced amino acid sequences were compared. The gene for FnBA, a fibronectin‐binding protein from Streptococcus dysgalactiae, was also cloned and found to express an opacity factor. The leader sequences, the fibronectin‐binding domains, and the membrane anchor regions of these proteins were highly conserved. Short spans of conserved sequences were interspersed throughout the remaining parts of the proteins. The sof2 gene was insertionally inactivated in an M type 2 S. pyogenes strain, T2MR. The resultant SOF‐negative mutant (YL3) did not express SOF or opacify serum, and exhibited a 71% reduction in binding fibronectin. Complementation of the SOF‐negative defect with sof28 in the recombinant strain YL3(pNZ28) fully restored fibronectin‐binding activity and the ability to opacify serum. To determine whether sof plays a role in virulence, mice were challenged intraperitoneally with these strains. None of the 10 mice infected with YL3(pNZ28) survived and only 1 out of 15 mice challenged with T2MR survived, whereas 12 out of 15 mice infected with YL3 survived. These data clearly indicate that SOF is a virulence factor, and they provide the first direct evidence that a fibronectin‐binding protein contributes to the pathogenesis of group A streptococcal infections in vivo.

Recombinant, octavalent group A streptococcal M protein vaccine

One of the major obstacles to the development of group A streptococcal M protein vaccines is the multiplicity of M serotypes expressed by these organisms. In this study, we have constructed a recombinant, hybrid M protein that contains type-specific aminoterminal fragments of eight different M proteins. We show that the purified hybrid recombinant protein is immunogenic in rabbits and evokes antibodies that react with native M proteins from the respective streptococcal serotypes. In addition, the immune sera evoked by the octavalent protein opsonized six of the eight serotypes of streptococci, indicating that the majority of the M protein fragments contained protective epitopes that retained their native conformations in the hybrid protein. None of the antisera raised against the octavalent protein crossreacted with human heart tissue. These studies indicate that multivalent, hybrid M proteins may be used to elicit broadly protective immune responses against multiple serotypes of group A streptococci.

Temporal Changes in Streptococcal M Protein Types and the Near-Disappearance of Acute Rheumatic Fever in the United States

BACKGROUND The explanation for the very substantial decrease in the incidence of acute rheumatic fever in the United States, particularly over the past 50 years, is unclear. It has been proposed that certain M types of group A streptococci (GAS) include strains that are particularly rheumatogenic and that others are nonrheumatogenic. METHODS We compared the M type distribution of GAS recovered from children from Chicago, Illinois, with acute pharyngitis during 1961-1968 to that of GAS recovered from Chicago children and children from across the United States in 2000-2004, with attention to changes in M types that previously were associated with rheumatogenic strains. RESULTS The rheumatogenic types 3, 5, 6, 14, 18, 19, and 29 comprised 49.7% of 468 pharyngeal isolates during 1961-1968 but only 10.6% of 450 Chicago isolates during 2000-2004 (P < .001) and 17.9% of 3969 isolates nationwide during 2000-2004 (P < .001). Significant decreases in types 3, 5, and 6 and virtual disappearance of types 14, 18, 19, and 29 occurred between the 2 study periods. No change in the proportion of type 1 isolates, a highly heterogeneous group that includes some rheumatogenic strains, was observed. The nonrheumatogenic GAS types 2, 4, 22, and 28 increased from 4.9% to approximately 28% of pharyngeal isolates in Chicago and nationwide between the 2 study periods (P < .001). CONCLUSIONS These data support the concept of rheumatogenic strains of GAS and indicate that the marked decrease in the incidence of acute rheumatic fever in the United States over the past 4 decades is correlated with the replacement of rheumatogenic types by nonrheumatogenic types in cases of acute streptococcal pharyngitis in children. The reasons underlying the observed change in distribution of M types remain to be elucidated.

Relationship between Expression of the Family of M Proteins and Lipoteichoic Acid to Hydrophobicity and Biofilm Formation in Streptococcus pyogenes

Background Hydrophobicity is an important attribute of bacteria that contributes to adhesion and biofilm formation. Hydrophobicity of Streptococcus pyogenes is primarily due to lipoteichoic acid (LTA) on the streptococcal surface but the mechanism(s) whereby LTA is retained on the surface is poorly understood. In this study, we sought to determine whether members of the M protein family consisting of Emm (M protein), Mrp (M-related protein), Enn (an M-like protein), and the streptococcal protective antigen (Spa) are involved in anchoring LTA in a manner that contributes to hydrophobicity of the streptococci and its ability to form biofilms. Methodology/Principal Findings Isogenic mutants defective in expression of emm, mrp, enn, and/or spa genes of eight different serotypes and their parental strains were tested for differences in LTA bound to surface proteins, LTA released into the culture media, and membrane-bound LTA. The effect of these mutations on the ability of streptococci to form a hydrophobic surface and to generate biofilms was also investigated. A recombinant strain overexpressing Emm1 was also engineered and similarly tested. The serotypes tested ranged from those that express only a single M protein gene to those that express two or three members of the M protein family. Overexpression of Emm1 led to enhanced hydrophobicity and biofilm formation. Inactivation of emm in those serotypes expressing only a single emm gene reduced biofilm formation, and protein-bound LTA on the surface, but did not alter the levels of membrane-bound LTA. The results were more varied in those serotypes that express two to three members of the M protein family. Conclusions/Significance Our findings suggest that the formation of complexes with members of the M protein family is a common mechanism for anchoring LTA on the surface in a manner that contributes to hydrophobicity and to biofilm formation in S. pyogenes, but these activities in some serotypes are dependent on a trypsin-sensitive protein(s) that remains to be identified. The need for interactions between LTA and M proteins may impose functional constraints that limit variations in the sequence of the M proteins, major virulence factors of S. pyogenes.