Risk factors in the pathogenesis of invasive group A streptococcal infections: role of protective humoral immunity

Abstract

An impressive change in the epidemiology and severity of invasive group A streptococcal infections occurred in the 1980s, and the incidence of streptococcal toxic shock syndrome cases continues to rise. The reason for the resurgence of severe invasive cases remains a mystery-has there been a change in the pathogen or in host protective immunity? To address these questions, we have studied 33 patients with invasive infection caused by genotypically indistinguishable M1T1 strains of Streptococcus pyogenes who had different disease outcomes. Patients were classified as having severe (n = 21) and nonsevere (n = 12) invasive infections based on the presence or absence of shock and organ failure. Levels of anti-M1 bactericidal antibodies and of anti-streptococcal superantigen neutralizing antibodies in plasma were significantly lower in both groups than in age- and geographically matched healthy controls (P < 0.01). Importantly, the levels of these protective antibodies in plasma samples from severe and nonsevere invasive cases were not different. Together the data suggest that low levels of protective antibodies may contribute to host susceptibility to invasive streptococcal infection but do not modulate disease outcome. Other immunogenetic factors that regulate superantigen responses may influence the severity of systemic manifestations associated with invasive streptococcal infection.

FIG. 1

Low levels of anti-M1 antibodies in patients with severe and nonsevere invasive infections caused by genotypically indistinguishable M1T1 strains of group A streptococci. Antibodies against the streptococcal M1 protein in acute-phase plasma specimens from 33 patients (21 with severe cases and 12 with nonsevere cases) and in 50 age-matched healthy controls from the same geographical area were determined by ELISA. Plasma specimens diluted 1:500 and 1:100 were added to duplicate wells coated with 0.2 μg of SM1(1-26)c peptide per ml. Rabbit antiserum specific for the M1 peptide (diluted 1:100, 1:500, and 1:1,000) was used to generate a standard curve, and FBS (diluted 1:100) served as negative control. Peroxidase-conjugated goat anti-human IgG and goat anti-rabbit IgG were used as secondary antibodies. After addition of peroxidase substrate, the reaction was monitored at 415 nm.

FIG. 2

Low levels of M1-specific opsonophagocytic antibodies in patients with severe and nonsevere invasive group A streptococcal infections. Neutrophil-mediated opsonophagocytosis was performed as detailed in Materials and Methods. Opsonophagocytic activity in acute-phase plasma specimens from 33 patients infected with M1T1 strains (21 with severe cases and 12 with nonsevere cases) and in 20 age-matched healthy controls from the same geographical area was determined as detailed in Materials and Methods. Rabbit antiserum specific for M1 protein (diluted 1:50) was used as a positive control, and FBS was used as a negative control. Plasma specimens from either controls or patients were tested at a 1:50 dilution. The percentage of neutrophils containing phagocytozed bacteria was determined by direct microscopy.

FIG. 3

Low levels of anti-streptococcal superantigen neutralizing antibodies in patients with severe and nonsevere invasive group A streptococcal infections. Neutralizing antibodies against the mixture of superantigens produced by the M1T1 isolates in plasma specimens of patients infected with indistinguishable M1T1 strains were evaluated. PBMC (10⁶ cells/ml) from a healthy donor were stimulated either with phytohemagglutinin (1 μg/ml) or with the partially purified culture supernatant from M1T1 isolates (diluted 1:100 in RPMI) in the presence of 5% FBS or 4% FBS plus 1% plasma. (A) Neutralizing activity in plasma specimens from healthy individuals (n = 20) of different ages. (B) Neutralizing activity in plasma specimens from patients with severe (n = 21) and nonsevere (n = 12) infections and healthy controls (n = 20) against pure superantigens rSpeA and SpeB. Proliferation was assessed after 3 days of culture, and the mean counts per minute ([³H]thymidine uptake) ± SEM for triplicate cultures was calculated. Neutralizing activity is expressed as percent inhibition of mitogenic activity. (C) Results for patients with severe (n = 21) and nonsevere (n = 12) invasive disease or for age-matched healthy individuals who reside in the same area. Each patient plasma was tested for neutralizing activity against the isolate from that patient, or, in the case of the age-matched healthy individuals, supernatants from six representative isolates (three from severe cases and three from nonsevere cases) were used for stimulation. Proliferation was assessed after 3 days of culture, and the mean counts per minute ([³H]thymidine uptake) ± SEM for triplicate cultures was calculated. Neutralizing activity is expressed as percent inhibition of mitogenic activity.