Prospective Longitudinal Analysis of Immune Responses in Pediatric Subjects After Pharyngeal Acquisition of Group A Streptococci

Abstract

Background.: Despite the significant burden of disease associated with infection by group A streptococcus (GAS), little is known about the human immune response to GAS antigens after natural infection.

Methods.: We evaluated 195 serum samples obtained prospectively over a consecutive 24-month period from 41 pediatric subjects who experienced a new pharyngeal GAS acquisition. An enzyme-linked immunoassay was used to determine the kinetics and antigen specificity of antibodies against 13 shared GAS antigens and 18 type-specific M peptides. The majority of the antigens tested are currently being considered as vaccine candidates.

Results.: Twelve M types of GAS were recovered from 41 subjects who experienced 51 new GAS acquisitions that elicited antibody responses against at least 1 of the 31 antigens tested (immunologically significant new GAS acquisitions). The immune responses to the 13 shared antigens were highly variable. Increases in antibody levels were detected against a mean of 3.5 shared antigens (range, 1-8). Antibody responses to the homologous M peptide were observed in 32 (63%) of the 51 episodes. Seven subjects acquired more than 1 M type of GAS. There were no new immunologically significant acquisitions of an M type against which the subject had preexisting antibodies to the homologous M peptide. Of the subjects with new GAS acquisition, 65% were asymptomatic, yet immune responses were detected against 1 or more GAS antigens. Immune responses to streptolysin O and/or deoxyribonuclease B were observed after 67% of the new GAS acquisitions. Persistently positive (>12 weeks) throat culture results were returned for 20% of the 41 subjects despite immune responses to homologous M peptides and/or shared antigens.

Conclusions.: The availability of throat culture results, GAS isolates, and serial serum samples collected prospectively over a 2-year period of observation provided a unique opportunity for us to assess the serologic status of pediatric subjects before and after new pharyngeal acquisitions of GAS. With the exception of antibody responses to the homologous M peptides, no clear pattern of immune responses against the remaining GAS antigens was seen. There were no new immunologically significant acquisitions of emm types of GAS against which the subjects had preexisting elevated levels of antibodies against the homologous M peptide. The observation that 65% of new GAS acquisitions caused no symptoms yet were immunologically significant suggests that the majority of infections are not detected, which would result in missed opportunities for primary prevention of rheumatic fever and rheumatic heart disease with appropriate antimicrobial therapy.

Keywords: M protein; group A streptococcus; human immune responses; shared antigens.

Figure 1.

Examples of M peptide antibody responses and variable responses to shared group A streptococcus (GAS) antigens. The subject number is indicated for each set of data. Study weeks are represented on the horizontal axis. The numbers directly under the horizontal axis indicate theemm types of GAS recovered from throat cultures during the study, and N indicates a negative culture result. Dashed lines represent antigen-specific antibody levels that did not change during the observation period indicated. (A) Immune responses to the M1 peptide, deoxyribonuclease B (DNaseB), and C5a peptidase (SCPA) that were also sustained after the acquisition of M1 GAS were found in the serum from subject 784. The antibody response to streptolysin O (SLO) was much lower in magnitude but was sustained. (B) Subject 11 acquired a serum opacity factor (SOF)-positive M4 and mounted a brisk immune response to the M4 peptide, M-related peptide II (MrpII), SLO, DNaseB, SOF, SCPA, and SpyAD. (C) Subject 2 entered the study with a throat culture positive for M3 and elevated levels of antibodies against the M3 peptide. In week 13, the throat culture was positive for M89 GAS, and the subject responded with increases in antibody levels against the M89 peptide, SLO, and SOF. (D) No change in antibody levels against SLO or DNaseB in response to M2 GAS were found in the serum from subject 14, but immune responses to the M2 peptide and MrpI were mounted. Abbreviation: O.D., optical density.

Figure 2.

Examples of antigen-specific immune responses in 4 subjects after a new acquisition of group A streptococcus (GAS). The subject number is indicated for each set of data. Study weeks are represented on the horizontal axis. The numbers directly under the horizontal axis indicate theemm types of GAS recovered from throat cultures during the study. Dashed lines represent antigen-specific antibody levels that did not increase during the observation period indicated. (A) Subject 567 mounted brisk antibody responses to streptolysin O (SLO), deoxyribonuclease B (DNaseB), and SpyAD but no response to the M89 peptide after a culture positive for M89 GAS. (B) Subject 14 provides an example of sequential acquisitions of 3 different M serotypes of GAS over the course of the study. Throat cultures were positive on several occasions for M1 GAS, and the serum contained high levels of M1 peptide antibodies. By week 40, M1 was replaced by M12 GAS, and there was an immune response to the M12 peptide. At week 65, a throat culture was positive for M2 GAS, and there was an antibody response to the M2 peptide. (C) Subject 19 acquired 3 different M types (M2, M89, and M3) over the 2-year observation period. After each episode, there was an immune response to the homologous M peptide. The acquisition of M2 also triggered an antibody response against M-related peptide I (MrpI), and the acquisitions of serum opacity factor (SOF)-positive M2 and M89 were associated with increases in SOF antibodies. This subject also had persistently elevated levels of antibodies to DNaseB, C5a peptidase (SCPA), and SpyAD that did not change markedly from week 0 to 108. (D) An additional example of multiple GAS acquisitions was subject 760, who acquired 2 differentemm types. A culture positive for M1 was associated with increases in antibodies against M1, SCPA, and SpyAD. Approximately 1 year later, a throat culture was positive for M6, which was associated with an increase in M6 peptide antibodies and more pronounced increases in antibody levels against SpyAD and SCPA. Abbreviations: CHO, group A streptococcus carbohydrate; O.D., optical density.

Figure 3.

Immune responses to shared antigens after new pharyngeal acquisitions of group A streptococcus (GAS). Antibody responses to combinations of streptolysin O (SLO), deoxyribonuclease B (DNaseB), C5a peptidase (SCPA), or any 1 additional shared antigen were tabulated after all new GAS acquisitions (A) and asymptomatic acquisitions (B). Abbreviation: SA, streptococcal antigen.

Figure 4.

Antigen-specific immune responses in subjects with a new pharyngeal group A streptococcus (GAS) acquisition. Shown are the percentages of acquisitions that resulted in a specific antibody response, calculated using the number of GAS isolates predicted as the denominator to express each antigen (seeTable 1). Abbreviations: DNaseB, deoxyribonuclease B; FBP, fibronectin-binding protein; GAC, group A carbohydrate; J14, C-repeat M peptide; Mrp, M-related peptide; SCPA, C5a peptidase; SLO, streptolysin O; SOF, serum opacity factor; SpyCEP, serine protease; SSE, serine esterase.