Subclinical exposure to Streptococcus pyogenes drives the development of long-lived immunity

Subclinical exposure to Streptococcus pyogenes drives the development of long-lived immunity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Subclinical exposure to Streptococcus pyogenes drives the development of long-lived immunity Manisha Pandey, Despena Vedis, Victoria Ozberk, Merrina Anugraham, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7145918/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Age-related decline in Streptococcus pyogenes infection rates suggests that immunity develops progressively through repeated exposure during early life. However, the intensity or duration of exposure required is unknown, as to why some individuals appear to develop immunity, despite having few or no previously detected infections. Here, drawing on samples from a human challenge model of pharyngeal S. pyogenes infection, we investigate whether symptomatic disease is required for induction of humoral and cellular immunity. Challenge with M75 S. pyogenes induced M75-specific serum IgG and IgA antibodies and memory B cell in both symptomatic and asymptomatic participants, with responses persisting for at least 6 months. Purified IgG from asymptomatic participants exhibited significantly enhanced binding to M75 S. pyogenes and were bactericidal when transferred into a murine model of pharyngeal infection. M75-specific IgG from these participants had an altered Fc glycosylation signature, indicative of enhanced effector function and ability to limit inflammation. However, S. pyogenes challenge had no impact on cellular or humoral immune responses to a conserved cryptic epitope, p*17. These findings show that asymptomatic (or subclinical) exposure to M75 S. pyogenes generates functional immune responses and contributes to the streptococcal immunity that emerges by adulthood. Biological sciences/Immunology/Infectious diseases/Bacterial infection Biological sciences/Microbiology/Bacteriology S. pyogenes natural immunity CHIVAS human pharyngeal challenge antigen-specific B cell responses IgG Fc glycosylation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Streptococcus pyogenes (Group A Streptococcus , Strep A) is a leading human-restricted pathogen with a persistent and profound global disease burden. It causes broad clinical spectrum of illness, spanning superficial through to severe infections and post-infectious syndromes including rheumatic heart disease 1 , 2 . Every year, up to a billion people are affected and more than 500,000 deaths are attributable to S. pyogenes 3 . ‘Strep throat’ or S. pyogenes pharyngitis is a ubiquitous childhood illness, with incidence peaking during the primary school years 4 , 5 . Lower incidence in adults has generally been ascribed to immunity accumulated through repeated exposure in childhood to different S. pyogenes strains 6 . Although the precise mechanisms of immunity against S. pyogenes remain uncertain, several humoral and cellular responses have been strongly implicated in protection against colonization or disease 7 . These responses include antibodies that bind to bacteria, enabling neutrophils and monocytes at the site of infection to recognize and eliminate them through opsonophagocytosis 7 . In individuals resistant to pharyngeal acquisition, such antibodies can also prevent early bacterial colonization by blocking adherence and promoting rapid clearance 8 . Allelic polymorphism of the S. pyogenes M-protein is a major impediment to the development of immunity. To date, over 250 distinct serotypes have been identified based on its amino-terminal hypervariable region (HVR) 9 . Longitudinal studies indicate that naturally acquired antibodies against the M-protein can provide homologous protection but offer limited cross-protection against disease caused by heterologous strains 10 , 11 . While the M cluster concept explains some degree of cross-protective immunity 12 , it remains unclear how most children develop protective immunity by their second decade of life 12 , considering that only around 15% experience symptomatic S. pyogenes pharyngitis in any given year 1 . A potential explanation is that repeated exposures boost immunity to antigens that are broadly conserved across strains, complementing narrower responses to type-specific antigens such as the M-protein HVR. We previously showed that antibodies to the conserved C-repeat region can kill the S. pyogenes irrespective of its M-protein serotype 13 – 15 . However, C-repeat-specific immunity is unlikely to account for naturally acquired immunity because this region is poorly immunogenic (‘cryptic’) during both experimental and natural infections. Antibodies to this region do not develop in children from streptococcal-endemic areas until well into their second decade of life 16 nor do they arise following repeated exposures of mice to S. pyogenes 17 . Given the high prevalence of asymptomatic pharyngeal S. pyogenes colonization or ‘carriage’ in 10–20% of school-age children 18 and that many different serotypes can be in circulation in a community at any time 19 , a reasonable hypothesis is that repeated asymptomatic (or subclinical) exposures shape protective immune responses to S. pyogenes . Here, we explore this hypothesis in a well-characterised cohort of participants from the CHIVAS-M75 S. pyogenes human pharyngeal challenge study 20 – 22 . While acute symptomatic pharyngitis following direct tonsillar inoculation was the most common outcome, a subset remained asymptomatic despite lacking significant pre-existing serotype-specific antibodies to the M75 strain, enabling us to investigate the immunological outcomes of subclinical infections. At 6-months post-challenge, increased levels of memory B cells and antibodies targeting the M75 hypervariable region (HVR) —but not a conserved cryptic M-protein epitope (p*17) —were observed in participants both with and without pharyngitis. Using several approaches to assess antibody function, including bacterial binding, IgG Fc glycosylation traits, and passive antibody transfer into a murine model of pharyngeal infection, we show that subclinical exposures can elicit immune responses that contribute to long-term protection against S. pyogenes . Results Murine antibodies against the M75 hypervariable region bind specifically to M75 S. pyogenes Our initial studies aimed to characterize humoral immune responses elicited by the M-protein peptides, p*17 and M75-HVR (Fig. 1 A). Although p*17 is poorly recognized following infection of mice, it is known that vaccine-induced antibodies can protect against infection 23 – 26 . BALB/c mice were immunized with either p*17-diphtheria toxoid (DT) or M75-HVR-DT, each adjuvanted with aluminum hydroxide (Alum), following a 3-dose schedule as in a Phase 1 clinical trial 27 . Serum from both groups were collected two weeks after the final vaccine dose and analyzed by indirect ELISA and flow cytometry-based binding assays to assess IgG antibody responses against p*17 and M75-HVR peptides, as well as to live S. pyogenes bacteria (Fig. 1 B), respectively. Serum from M75-DT immunized mice specifically recognized the M75 peptide (Fig. 1 C), whereas serum from p*17-DT-immunized mice exclusively recognized p*17 (Fig. 1 D). Using flow cytometry, we demonstrated that serum from M75-DT–immunized mice specifically bound to M75 S. pyogenes , with no detectable reactivity to an M1 strain (Fig. 1 E). In contrast, serum from p*17-DT–immunized mice recognized both M75 and M1 strains (Fig. 1 F), consistent with p*17 being a conserved epitope across S. pyogenes strains. Human pharyngeal challenge with the M75 S. pyogenes elicits memory responses directed against the M-protein hypervariable region. To assess the long-term human immune responses to infection, we examined memory B cell populations and serum antibodies from CHIVAS-M75 participants 20 – 22 . Antigen-specific responses to p*17 and M75-HVR were assessed for 13 participants who developed acute symptomatic pharyngitis following pharyngeal challenge with M75 S. pyogenes . These were compared to responses from 5 participants who remained asymptomatic (non-pharyngitis) and had no detectable M75 S. pyogenes by culture or qPCR from throat swabs collected twice daily from 24 hours after challenge until discharge. Systemic immune responses were longitudinally analyzed at pre-challenge, 1 month, and 6 months post-challenge. To evaluate immunity against S. pyogenes and explore the dynamics of antigen-specific memory B cells and antibody responses post-challenge, we generated fluorescent tetramers and analyzed responses to the p*17 and the type-specific HVR region of the M75 protein. To determine whether a single M75 S. pyogenes infection induced antibodies to the conserved region of the M-protein, we specifically assessed responses to p*17 (Fig. 2 A and B). Flow cytometry-based tetramer analysis was used to characterize p*17-specific memory B cells (CD27 + CD21 + ) within both the IgA and IgG subclasses (Supp. Figure 1 A). The frequency of p*17-specific IgG + memory B cells remained low and unchanged following pharyngeal challenge with M75 S. pyogenes out to 6 months in both symptomatic and asymptomatic participants (Fig. 2 A). Likewise, p*17-specific IgA + cells in the blood were unaffected by the challenge, with no detectable link to disease progression (Fig. 2 B). Antibody response analyses in the same participants similarly revealed that IgG antibody levels targeting p*17 remained low and unchanged across all timepoints in all participants following pharyngeal challenge (Fig. 2 C). Similarly, serum IgA responses to the p*17 antigen were minimal (O.D. < 0.2 at a dilution of 1:20), showing no significant changes post-challenge and no association with disease progression (Fig. 2 D). In those who developed symptomatic disease post-challenge, the frequency of M75-specific IgG + memory B cells increased significantly at 6 months (Fig. 3 A). M75-specific IgA + memory B cells showed an early increase, observed at 1 month, which also endured for at least 6 months post-challenge (Fig. 3 B). In asymptomatic participants there was a trend toward an increase at 6 months for both M75-specific IgG + (2.09 fold-increase, P > 0.05, Fig. 3 A) and IgA + (1.69 fold-increase, P > 0.05, Fig. 3 B) memory B cells. Similarly, antibody analysis showed a rise in M75-specific IgG levels in participants with pharyngitis but was detected only at 6 months post-challenge (Fig. 3 C). This increase was driven specifically by the M75-specific IgG3 subtype (Supp. Figure 1 B) with no significant changes in IgG1, IgG2, or IgG4 levels (Supp. Figure 1 C-E). M75-specific serum IgA antibodies also showed a significant increase detected at 6 months post-challenge (Fig. 3 D). Although a significant antibody increase was observed only in symptomatic participants, the average increase in M75-specific antibody levels was similar in those with and without pharyngitis (Supp. Figure 1 F) (Total IgG: mean values of 1.176 for pharyngitis and 1.242 for non-pharyngitis; IgG3: mean values of 1.431 for pharyngitis and 1.435 for non-pharyngitis; IgA:1.147 for pharyngitis and 1.330 for non-pharyngitis). The increase in IgG + memory B cells binding the M75 antigen correlated with increased M75-specific serum IgG at 6 months relative to pre-challenge (R 2 = 0.438, P = 0.02, Fig. 3 E). Interestingly, the increase in IgA + memory B cells binding the M75 S. pyogenes antigen did not correlate with increased M75-specific serum IgA (R 2 = 0.04, P = 0.39, Fig. 3 F). There was no significant difference in pre-challenge M75-specific memory B cells and antibodies between participants who did and did not develop pharyngitis following challenge (Fig. 3 C and D). Exposure to M75 S. pyogenes induces long-term, strain-specific systemic immunity, as evidenced by sustained serum Ig levels and memory B cell responses involving both IgG and IgA. While occurring in both groups of participants, the immune responses were more pronounced in those who developed clinical disease. IgG Fc glycosylation is associated with clinical manifestations of S. pyogenes infections Post-translational modifications, particularly glycosylation of the constant (Fc) IgG domain, are well-known features of all IgGs and play a critical role in modulating antibody effector functions, including complement activation and Fc receptor engagement 28 , 29 . We initially assessed whether pre-challenge IgG Fc N-glycosylation traits could be a distinguishing factor between participants with and without pharyngitis. We affinity-purified total (non-specific) and M75-specific IgG from asymptomatic and symptomatic participants prior to challenge. M75-specific IgG levels pre-challenge were very low in all participants, but affinity purification enabled enrichment of these antibodies (Fig. 3 C and 4 A). Mass spectrometry-based characterisation revealed that pre-challenge IgG1 antibodies were the major IgG subclass targeting the M75-epitope across all participants. Participants who remained asymptomatic following challenge displayed a distinct baseline Fc glycosylation profile for global (entire IgG pool present in plasma) IgG1 compared to participants who became symptomatic (1.5-fold and 2.16 fold-increase in sialylated and afucosylated N-glycans, P 0.05, respectively, Fig. 4 B, i), whereas no significant differences were detected in overall glycoform distribution (Fig. 4 C, i). Profound changes were observed in the glycosylation profile of M75-specific IgG1 antibodies from asymptomatic participants, marked by a 30-fold decrease in afucosylation (P < 0.05) and a 4.9-fold increase in bisecting GlcNAc moieties (P < 0.01, Fig. 4 D, i), which is largely derived from increased levels of G1FB and G2FB N-glycans (3.1-fold and 13.9-fold increase, P < 0.05 and P < 0.01, respectively, Fig. 4 E, i), compared to M75-specific IgG1 from symptomatic participants. In comparison to global IgG1, M75-specific IgG1 in participants without pharyngitis displayed significantly lower levels of afucosylated glycans (mean of 0.12 versus 2.13; P < 0.01), along with a significantly higher proportion of bisected glycans (mean of 5.29 versus 1.36; P < 0.01, Fig. 4 B, i and D, i , respectively). IgG Fc N-glycosylation traits at 6 months post-challenge showed similar differences between participants with and without pharyngitis as those observed pre-challenge. Participants who remained asymptomatic following challenge exhibited a 1.31-fold increase in sialylated N-glycans, a 1.98-fold increase in afucosylated N-glycans, and a 1.65-fold increase in bisecting GlcNAc moieties (P > 0.05, P < 0.05 and P < 0.05, respectively; Fig. 4 B, ii ), whereas no significant differences were detected in overall glycoform distribution (Fig. 4 C, ii ). Profound changes were observed in the glycosylation profile of M75-specific IgG1 antibodies from asymptomatic participants, marked by a 17.3-fold decrease in afucosylation (P > 0.05), a 5.4-fold increase in bisecting GlcNAc moieties (P < 0.01), and a 1.3-fold increase in sialylation (P < 0.05, Fig. 4 D, ii ). This is derived not only from increased levels of G1FB, G2FB and G2FS N-glycans (3.9-fold, 11.3-fold and 2.1-fold increase, P < 0.05, P < 0.01 and P < 0.01, respectively), but also from decreased levels of G0F, G1F and G2F (2.5-fold, 1.8-fold and 1.6-fold decrease P < 0.01, P < 0.05 and P < 0.05, respectively, Fig. 4 E, ii ) compared to M75-specific IgG1 from symptomatic participants. The evident differences at post-challenge were further supported by principal component (PC) analyses, which revealed that Fc glycoforms profiles obtained from symptomatic and asymptomatic participants formed two well-defined and distinct clusters (Fig. 4 F, ii ). Compared to pre-challenge levels, no significant differences were detected in overall IgG Fc N-glycosylation traits or glycoform compositions of global and M75-specific IgG1 antibodies from participants with pharyngitis at 6 months post- S. pyogenes challenge (Supp. Figure 2 ). No changes in IgG Fc N-glycosylation were observed in global IgG1 purified from asymptomatic participants at 6 months post-challenge compared to pre-challenge levels either (Figs. 5 A-C). However, at 6 months, M75-specific IgG1 showed a significant increase in agalactosylation (1.5-fold) and sialylation (1.3-fold, P < 0.05, Fig. 5 D) followed by a specific increase in G2FS (Fig. 5 E) in comparison to pre-challenge. These findings strongly indicate that the Fc-glycosylation traits of IgG1 antibodies directed against the M75 antigen are involved in modulating the susceptibility to symptomatic disease following M75 S. pyogenes challenge. Subclinical exposure to S. pyogenes induces long-term, functionally protective IgG immunity To examine the functional properties of serum IgG and to increase the sensitivity of our analyses, we purified total serum IgG and used flow cytometry to assess the S. pyogenes binding at both pre-challenge and 6 months post-challenge in the participants with and without pharyngitis (Fig. 6 A). We observed a 12.1-fold increase in the binding of purified IgG to the homologous M75 S. pyogenes at 6 months post-challenge in participants with symptomatic pharyngitis, relative to their pre-challenge levels (Fig. 6 B). We also observed a significant increase in IgG binding (6.4-fold, P < 0.05) in asymptomatic participants comparing 6-month post challenge relative to their pre-challenge levels (Fig. 6 B). Similar to sera from mice immunized with M75-DT, IgG from CHIVAS trial participants at 6 months post-challenge (in both pharyngitis and non-pharyngitis sufferers) showed no binding to the heterogeneous M1 S. pyogenes (Figs. 1 E and 6 C). These findings indicate that infection with S. pyogenes can elicit a significant rise in serotype-specific antibodies that persist for at least 6 months regardless of the clinical outcome. The temporal changes observed in the IgG Fc N-glycosylation of asymptomatic participants prompted us to examine their functional properties more closely. We utilized a recently developed in vivo passive transfer model to test functional immunity. In this assay, S. pyogenes organisms were incubated with serum IgG antibodies (’pre-opsonized’) and then inoculated intranasally into immunocompromised mice. Clinical signs and bacterial burden in the respiratory tract were subsequently assessed to determine the protective capacity of the antibodies 24 . Due to limited availability of serum, we purified IgG from the 5 asymptomatic participants at pre and 6-months post challenge, pooled the IgG at each time point, and tested it against M75 S. pyogenes in 7–8 mice for each group (Fig. 6 D). Further controls included IgG from rats vaccinated with p*17-K4S2-CRM 26 or IgG from naïve rats. We tested weight loss (as a general measure of disease in S. pyogenes -infected mice 24 ), and bacterial burden from the entire respiratory tract. Mice inoculated with M75 S. pyogenes that had been pre-incubated with IgG from vaccinated rats exhibited significantly less weight loss compared to mice administered S. pyogenes treated with IgG from naive rats (0% versus 8.4%, P < 0.01) (Fig. 6 E). Across all mucosal sites and organs analyzed for bacterial burden, IgG from p*17-vaccinated rats resulted in significant reductions (P < 0.01 – P < 0.005) (Fig. 6 F). We similarly observed that mice inoculated with M75 S. pyogenes pre-incubated with IgG from CHIVAS-M75 participants collected 6 months post-challenge exhibited significantly reduced weight loss compared to those administered with S. pyogenes incubated with pre-challenge IgG (4.64% versus 10.63%, P < 0.05) (Fig. 6 E). This correlated with 273-fold reduction in total S. pyogenes colonization of the respiratory tract (P < 0.005), which included both mucosal sites (nasal shedding and throat swabs) and respiratory organs (nasal-associated lymphoid tissue [NALT] and lungs). Specifically, we observed a 205-fold reduction in S. pyogenes colonization of the respiratory mucosa (P < 0.01) and 314-fold reduction in colonization of respiratory tissues (P < 0.05). There were no significant differences observed between IgG from naïve rats and the human pre-challenge IgG (Fig. 6 F). Thus, serum IgG antibodies generated from subclinical S. pyogenes infection exhibit both strong strain-specific binding and robust functional activity. Discussion This study demonstrates that functional immunity to S. pyogenes can develop following asymptomatic exposure. Pharyngeal human challenge with M75 S. pyogenes elicited class-switched B cells and antibodies (IgG and IgA) in the peripheral blood targeting the type-specific M-protein HVR region. While the immune responses elicited by asymptomatic challenge were lower than those observed in symptomatic participants, IgG from these participants demonstrated clear strain-specific binding and effectively mediated bacterial clearance in a passive transfer murine infection model, whereas pre-challenge IgG did not. Participants with higher frequency of M75-specific IgG memory B cells at 6 months after challenge also had higher levels of binding anti-M75 IgG, suggesting that memory B cells may help maintain or increase IgG antibody levels over time. These results support the hypothesis that immunity to S. pyogenes is gradually acquired throughout childhood by repeated asymptomatic (or subclinical) exposures to circulating strains and not only through symptomatic infections. Recent longitudinal studies have also highlighted that asymptomatic S. pyogenes exposures may be immunologically significant ( i.e. capable of triggering measurable immune responses), challenging the long-held assumption that only symptomatic infections contribute meaningfully to immunity. Studies in the USA by Hysmith et al. 30 and in The Gambia by Keeley et al. 31 each demonstrated antibody binding responses to M and non-M-protein antigens following asymptomatic episodes of S. pyogenes colonization. This observation is confirmed by other studies showing that a significant proportion of people with asymptomatic colonization of the upper respiratory tract develop functional anti-M-protein antibodies 32 , 33 . Notably, a significant fraction of the children assessed in these studies only developed M-type specific serum IgG antibodies between 6 weeks and a year from detection of a positive throat culture 30 , 32 , which aligns with our data showing a delayed immune response following pharyngeal challenge. In the 2-year study by Hysmith et al., new S. pyogenes acquisitions did not significantly boost homologous M-type-specific antibody responses 30 . Therefore, it is possible that pre-screening and exclusion of participants with high serum type-specific M75 IgG in the CHIVAS-M75 study 21 , 22 may have contributed to the observed immunologically significant M75-specific responses following pharyngeal challenge. Serum antibodies against the M-protein enhance the opsonisation and killing of S. pyogenes and are believed to contribute to immunity against homologous strains. We previously observed that a single intranasal infection with S. pyogenes resulted in long-lasting immunity against subsequent homologous challenge in a mouse model 34 . Here we showed that human serum IgG antibodies, purified 6 months post-challenge, recognised the strain-specific HVR, bound specifically to live M75 S. pyogenes , and were bactericidal. The anti-M75 IgG response observed in the present study is predominantly mediated by the IgG1 and IgG3 subclasses, which are particularly effective in pathogen clearance, presenting a number of antigen-specific effector functions, including interaction of antigen-clustered IgG with the complement system, which activates phagocytosis and production of reactive oxygen species by innate immune cells 35 – 37 . Despite its relatively low abundance in relation to IgG1, IgG3 has been increasingly recognized as a key player in the immune defence against various pathogens, including S. pyogenes 38 due to its superior affinity to Fc-gamma receptors 39 . However, excessive IgG3 recognising M-protein antigens may be detrimental and elevated levels have been linked to the S. pyogenes post-infectious syndrome acute rheumatic fever 40 , 41 . Although total and subclass-specific M75-IgG antibody levels prior to challenge did not distinguish between clinical outcomes following experimental challenge, significant differences in IgG Fc N-glycosylation traits both in global and M75-specific IgG1 were observed. Compared to those with symptomatic pharyngitis, asymptomatic participants had higher total IgG1 sialylation, suggesting that the anti-inflammatory properties of Fc sialylation 42 , 43 may also contribute to protection. Additionally, they exhibited essentially non existing levels of afucosylated N-glycans in M75-specific IgG1 Fc, which has been associated with exacerbated inflammatory responses and disease severity following viral infections 44 , 45 . Our data provide evidence that subclinical exposure to S. pyogenes can selectively shape the N-glycan landscape of M75-specific IgG1 antibodies. The glycosylation features of the Fc region significantly influence the ability of IgG antibodies to trigger inflammatory responses and their interaction with FcγIII receptors 46 , 47 . The concurrent increase in both agalactosylation and sialylation suggests a fine-tuned immune adaptation of M75-specific responses: the host may be enhancing IgG1 functionality for bacterial clearance (via agalactosylation 48 ) while simultaneously limiting collateral inflammation and tissue damage (via sialylation and fucosylation 43 , 49 ). This dual modulation could reflect an evolved mechanism to balance pathogen elimination with immune regulation during asymptomatic S. pyogenes exposures. Although the anti-M75 IgG response was predominantly mediated by the IgG1 and IgG3 subclasses post-challenge (as per ELISA data), the IgG Fc N-glycosylation in the non-pharyngitis (asymptomatic) participants appeared to be more prominent on IgG1 and IgG4 as compared to the pharyngitis group (both global and M75-IgG). Nevertheless, significant glycosylation changes were only observed in M75-specific IgG1 and not IgG3/4. The modest immune responses to the conserved C-terminal M-protein peptide p*17 observed in this study are consistent with previous findings in both mice and children from streptococcal-endemic regions 16 . Although C-terminal antigens appear to be cryptic during exposure and infection with S. pyogenes , vaccination with the p*17 antigen conjugated to a carrier protein elicits robust immune responses in animal models. These include high-affinity, class-switched antibodies that recognize a broad range of S. pyogenes strains, effectively reducing bacterial colonization and preventing severe infection 23 – 26 . These observations underscore the likelihood that vaccine-induced protection against S. pyogenes operates through mechanisms distinct from naturally acquired immunity, which is only partially protective. This is evidenced by continued low susceptibility to infection among adults, particularly in parents of young children, individuals in crowded settings (e.g. army barracks), people who inject drugs, those experiencing homelessness, and older adults with accumulating comorbidities. A limitation of our study is the small cohort size, especially the limited number of asymptomatic participants. However, these participants showed significant and strain-specific IgG binding to S. pyogenes equivalent to antibodies from mice immunised with M75 antigen. The high rate of clinical pharyngitis in the challenged adult participants, despite accumulated immunity to antigens other than the type-specific M75 hypervariable region (an exclusion criteria), may suggest the inoculum (dose) and direct swab challenge method may have circumvented or overwhelmed natural defences 21 . Still, the inoculum was several orders of magnitude lower than in comparable small and larger animal models, around a third of challenged participants remained asymptomatic, and those participants did develop de novo type-specific antibodies. Beyond these concerns related to the model, this study did not consider responses to conserved non-M-protein antigens such as SpyCEP, SLO and ScpA, which have previously been found in this cohort to have been increased in participants with pharyngitis and to have persisted for at least 3 months post-challenge 50 . In conclusion, our study demonstrated that asymptomatic S. pyogenes infection can induce persistent serotype-specific functional antibodies. This finding could be re-examined in longitudinal S. pyogenes surveillance studies including serial serum sampling and tested in future human studies incorporating challenge and homologous rechallenge. We propose that subclinical exposures do contribute to the development of protective immunity against S. pyogenes in humans. Material and methods The CHIVAS-M75 study The CHIVAS-M75 study protocol, including information on the M75 S. pyogenes challenge strain and key findings, has been detailed in earlier publications 20 – 22 . Ethical approval for the study, including sample collection and associated immunological investigations, was granted by the Alfred Hospital Human Research Ethics Committee (reference number 500/17), and all participants provided written informed consent. Among the 25 participants enrolled in the trial, 19 developed pharyngitis, across two dose levels. The diagnosis of pharyngitis was corroborated by microbiological evidence (via qPCR and culture), elevated biochemical markers (C-reactive protein, cytokines, and chemokines), and serological responses (anti-streptolysin O and anti-DNase B antibodies). Importantly, all participants classified as asymptomatic in the present study showed no signs of S. pyogenes colonization. In the present study, analysis was limited by sample availability. Serum and PBMC were collected from participants at the evening prior to the challenge, then at 1-month and 6-month outpatient visits. Blood was collected in serum separator tubes (BD Vacutainer SST Gold 8.5 mL) and allowed to clot. Within 2 h of collection, tubes were centrifuged at 1500 × g for 15 min at 20°C, and then aliquots were stored at − 80°C. PBMCs were collected using CPT tubes (BD Bioscience). Murine model of S. pyogenes infection All animal protocols were reviewed and approved by the Griffith University Animal Ethics Committee (GU-AEC) in accordance with the National Health and Medical Research Council (NHMRC) of Australia guidelines. Experimental protocols involving SCID (Severe Combined Immunodeficiency) mice were reviewed and approved by Office of the Gene Technology Regulator (OGTR). Mice (female, 4–6 weeks) were sourced from Ozgene, Western Australia. All mice were housed in a PC2-certified animal facility in individually ventilated cages (IVC) with a maximum of 5 mice/cage. For positive controls in the pre-opsonization assay, Sprague Dawley rats (aged 9.5 weeks at the start of dosing) were sourced from Charles River Laboratories, Inc. The animals received three intramuscular injections of the p*17-K4S2-CRM/Alum vaccine over a six-week period, followed by a two-week recovery phase. The adjuvant, aluminium hydroxide (Alhydrogel 2%, Alum), was obtained from Brenntag Biosector, Denmark. The relative humidity ranged between 45–65% and temperature at 20–24°C. Mice and rats were exposed to a 12-h light–dark cycle. Antigens Synthetic peptides from the M75 protein hypervariable region (M75-HVR) and C-terminal region (p*17) regions were synthesized and purified (> 95%) commercially by GenScript and stored lyophilized or in solution at − 20°C. Peptide sequence for M75-HVR is: EEERTFTELPYEARYKAWKSENDELRENYRRTLDKFNTEQGKTTRLEEQN. Peptide sequence for p*17 is: LRRDLDASREAKNQVERALE. Enzyme-linked immunosorbent assays (ELISA) Standard ELISA was used to measure M75 and p*17-specific serum antibody levels. Briefly, peptides were coated onto NUNC MaxiSorp plates (Thermo Fisher). Antigens were resuspended to 5 µg/mL in 0.1 M coating carbonate buffer and coated at 100 µL per well onto 96-well medium-binding ELISA plates (Greiner Bio-One) overnight at room temperature. Plates were then blocked with 200 µL of 2% w/v bovine serum albumin (BSA, Sigma) in phosphate-buffered saline (PBS) overnight at 4°C, followed by five washes in wash buffer (PBS with 0.05% v/v Tween20, PBS-T). Serial dilutions were performed using diluent (0.05% v/v Tween20) at 1:10, 1:20, 1:40, 1:80 dilutions. Samples were diluted 2-fold and antibody levels detected with HRP-conjugated goat anti-human IgA (Invitrogen, USA), IgG (H + L), IgG1, IgG2, IgG3 and IgG4 antibodies (Thermo Fisher, USA). SIGMAFAST OPD substrate was added according to manufacturer’s instructions and absorbance was measured at 450 nm on a Tecan Infinite M200 Pro plate reader (Tecan Group Ltd., Switzerland). Negative controls were included in each plate containing commercial human sera (Sigma) pre-absorbed of anti-M75 S. pyogenes antibodies or containing 0.05% v/v Tween20 only. Tetramer staining for peptide-specific memory B cells Frozen mononuclear PBMCs were thawed at 37ºC in thawing media (RPMI + 10% FCS), then washed and resuspended in flow buffer (PBS + 2%FCS + 2 mM EDTA). Biotinylated peptides synthesised at Genscript were tetramerised by serial addition of premium-grade streptavidin‐PE and streptavidin‐APC (Bio legend, San Diego, CA, USA) at a four‐molar equivalent of biotinylated peptide to one molar equivalent of streptavidin during 2 h at room temperature 51 . The prepared tetramer was diluted to a concentration of 2 µm for use. Typically, 10 6 PBMCs were pre‐incubated with Fc block (Bio legend) for 10 min followed by tetramer staining for 40 min. Cells were washed with flow buffer and then stained with fluorescently labelled antibodies (including CD19, CD20, CD21, CD27, IgD, IgM, IgG and IgA) and viability dye. Cells were fixed with 1% PFA and analyzed on a LSRFortessa (BD Biosciences). These cells were gated as positive for both fluorochromes of an antigen tetramer pair to reduce the inclusion of non-specific binding cells, as previously described 51 . S. pyogenes strains and growth conditions S. pyogenes strain 611024 (emm75, GenBank accession CP033621), isolated in Melbourne from a child presenting with exudative pharyngitis, was used in this study 21 . S. pyogenes strain M1 5448 (emm1) was kindly provided by Professor Mark Walker, from the University of Queensland, isolated in Australia from a case of necrotising fasciitis. For mouse infection experiments, M75 S. pyogenes was serially passaged through mouse spleens to enhance virulence and rendered streptomycin-resistant (200 µg/mL) to enable selective recovery from commensal flora in respiratory mucosa and tissues. S. pyogenes was cultured at 37°C for 16–18 hours. Single colonies were inoculated into Todd Hewitt Broth (THB; Oxoid, Australia) supplemented with 1% Bacto Neopeptone (Thermo Fisher, USA), yeast extract and streptomycin 24 . To enumerate colony-forming units (CFU), cultures were plated on Columbia agar containing 5% defibrinated horse blood and 200 µg/ml streptomycin (CBA 5%) 24 . IgG purification IgG antibodies were purified from the serum of CHIVAS participants and immunized rats using NAb Protein G columns, following the manufacturer’s instructions (Thermo Fisher, USA). The concentration of purified IgG was measured using a NanoDrop spectrophotometer (Thermo Fisher, USA). M75 antigen-specific IgG antibodies were purified using SulfoLink™ affinity chromatography columns (Thermo Fisher Scientific, USA) following the manufacturer’s protocol. Briefly, the columns were prepared by coupling M75-HVR to the SulfoLink resin via sulfhydryl-reactive chemistry. Serum samples were then applied to the columns, allowing M75-HVR-specific IgG to bind selectively. After thorough washing to remove unbound proteins, antigen-specific IgG was eluted using a low pH glycine buffer and immediately neutralized. The purified IgG fractions were concentrated and buffer-exchanged into PBS using centrifugal filters (Amicon Ultra, Millipore). Purity of the eluted M75-specific IgG were assessed by silver protein stain (Thermo Fisher Scientific, USA) and ELISA. Purified antibodies were stored at − 20°C until further use. IgG binding to S. pyogenes For the binding assay, M1 and M75 S. pyogenes strains (0.5 O.D.) were incubated overnight in PBS containing 0.2% skim milk PBS with 100 µg of purified serum IgG collected from CHIVAS participants at pre-challenge and 6 months post-challenge. Following incubation, bacterial cells were washed and stained with anti-human IgG Fab antibody. Cells were fixed with 1% PFA and analyzed on a LSRFortessa (BD Biosciences) and FlowJo™ v11 Software was used for analysis. Pre-opsonization of S. pyogenes with IgG and intranasal challenge in mice M75 S. pyogenes was incubated with purified serum IgG collected from asymptomatic CHIVAS participants at pre-challenge and 6 months post-challenge. As controls, IgG was purified from Sprague Dawley rats either immunised with p*17-DT-K4S2-CRM/Alum or left unimmunised (naïve). Following incubation, the bacterial cells were pre-incubated with a 10 µL inoculum containing 5 × 10 7 CFU/mouse delivered intranasally to anaesthetized naive SCID mice. The protocol was a modification of a previous assay 24 . Throat swabs were collected using flocked swabs (Copan Diagnostics, USA), which were then suspended in PBS, serially diluted, and plated in duplicate on CBA 5% plates. To assess nasal shedding, each mouse had its nares gently pressed onto a CBA 5% plates ten times (five times per half plate), allowing expelled particles to be streaked across the surface. On day 2 post-infection, mice were euthanized using CO₂ asphyxiation. The nasal-associated lymphoid tissue (NALT), a murine analogue to human tonsils, and the lungs were collected, homogenized in PBS using a Bullet Blender Homogenizer (Next Advance, USA), and serially diluted before being plated in duplicate onto CBA 5% plates. Mice were observed daily for clinical signs of illness according to a monitoring sheet approved by the GU-AEC. IgG Fc-glycopeptide profiling by LC-ESI MS/MS Purified pre- and post-challenge global and M75 antigen-specific IgG from pharyngitis (n = 5) and non-pharyngitis (asymptomatic) CHIVAS participants (n = 5) were dried and redissolved in 25 mM ammonium bicarbonate buffer prior to trypsin digestion (enzyme-to-substrate ratio of 1:25) at 37°C. The resulting glycopeptides were analysed using an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific), coupled to an UltiMate 3000 UHPLC fitted with a trap column (PepMap Neo C18, 5 mm x 300 µm, 5 µm particle size) and analytical reversed-phase C18 PepMap Neo (75 µm × 500 mm, 2 µm particle size) UHPLC column (Thermo Scientific) and PicoTip nanospray interface (New Objective). The samples were injected onto the trap column in 100% Loading Buffer (0.1% trichloroacetic acid) at the flow rate of 15 µL/min and the (glyco)peptides were separated at a flow rate of 300 nL/min using linear gradient from 2% solvent A (0.1% formic acid) to 95% solvent B (80% acetonitrile containing 0.1% formic acid) as follows: 1% solvent B until 8 min, 1%-8% from 8 to 9 min, 8–15% from 9 to 20 min, 15–95% from 20 to 23 min, 95% from 23 to 25 min, before the column was re-equilibrated in 1% solvent B from 28 until 40 min. For MS acquisition, a full MS1 scan was performed at 60,000 resolution between m/z 350–1650 (custom AGC target at 250%, 50 ms maximum injection time, 1 microscan). Precursor ions were isolated using an isolation window of 1.6 Da and MS2 fragmentation was performed using normalized stepped-HCD collision energy (15, 30, 45%) at 60,000 resolution. All quantitative data processing was performed using Skyline (version 24.1) based on a pre-defined list of IgG1, IgG2, and IgG3/4 glycopeptide masses corresponding to doubly [M + 2H]2 + and triply [M + 3H]3 + charged precursor masses (Supp. Table 1). MS1 peak-area-under-the curve quantification was performed and the summed areas of integrated peaks (including monoisotopic transitions) for each glycopeptide precursor mass were represented as a percentage (relative intensity) of the total IgG glycopeptides. Statistical analysis Statistical analysis was performed with GraphPad Prism 9 software using a nonparametric, unpaired Mann–Whitney U test (one-tailed; 90% confidence interval) to compare test groups, unpaired t test with Welch’s correction to compare test groups. ARRIVE guidelines 52 were used to calculate sample size for in vivo experiments. A sample size of 8 was shown to provide a power of 0.8 (G*Power) and therefore used for animal experiments involving bacterial challenge. For ELISA analysis, absorbance values at a 1:20 serum dilution were used to compare antigen-specific responses across the three time points. Statistical analysis was conducted using one-way analysis of variance (ANOVA) with Geisser-Greenhouse correction to account for repeated measures. The same approach was applied to evaluate changes in antigen-specific responses over time in both the ELISA and antigen tetramer assays. Correlation coefficients and correlation p values were determined using Spearman’s method (two-sided) with a linear regression line included to indicate linear trends. Declarations Declaration of interests A.C.S. is co-chair of the Australian Strep A Vaccine Initiative (ASAVI) and the Strep A Vaccine Global Consortium (SAVAC). M.F.G. and M.P. are inventors on patents related to S. pyogenes vaccines. Author contributions Conceptualization: A.L., M.F.G., M.P., D.K., A.C.S. and J.O. Methodology and experiments: A.L., D.V., V.O., M.A., D.B., A.C, H.R.F. and K.I.A. Data analysis: A.L., D.V., D.B. M.A., Patient cohorts: A.C.S. and J.O. Original draft: A.L., M.F.G. and M.P. Manuscript review: A.L., D.V., V.O., K.I.A., J.O., M.P., D.K. and M.F.G. Funding acquisition: A.C.S., J.O., A.L., D.K., M.P. and M.F.G. Acknowledgement The author thanks the support of the Flow Cytometry and Australian Cancer Research Foundation (ACRF) International Centre for Cancer Glycomics Mass Spectrometry Facility at the Institute for Biomedicine and Glycomics, Griffith University. A.L and A.C. are supported by an NHMRC project grant (APP1160379) awarded to M.P. V.O is supported by Griffith University Postgraduate Fellowship. M.A. is supported by an NHMRC ideas grant (GNT2018947) awarded to D.K. M.F.G. is supported by an NHMRC Investigator Fellowship (L3, GNT1174091). D.B. is supported by Queensland Advance WRAP grant awarded to A.L. The work described in this article was funded by The Heart Foundation of Australia (101656). The CHIVAS-M75 study was funded by the Australian National Health and Medical Research Council (1099183). J.O. is supported Australian National Health and Medical Research Council Investigator Fellowships and by a Melbourne Children’s Campus Clinician–Scientist fellowship. 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Supplementary Table Supplementary Table 1 not available with this version. Additional Declarations Yes there is potential Competing Interest. A.C.S. is co-chair of the Australian Strep A Vaccine Initiative (ASAVI) and the Strep A Vaccine Global Consortium (SAVAC). M.F.G. and M.P. are inventors on patents related to S. pyogenes vaccines. Supplementary Files SuppFiguresLepletieretal.CHIVASmanuscript.pdf Supplementary Figures 1 and 2 Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Institute","correspondingAuthor":false,"prefix":"","firstName":"Joshua","middleName":"","lastName":"Osowicki","suffix":""},{"id":489834899,"identity":"07c6c8ae-3871-4682-8232-87c0b70d8a54","order_by":11,"name":"Michael Good","email":"","orcid":"https://orcid.org/0000-0001-8212-248X","institution":"Griffith University","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Good","suffix":""},{"id":489834900,"identity":"c81b9fb0-0c3a-4265-9a8a-4b2e2771f882","order_by":12,"name":"Ailin Lepletier","email":"","orcid":"https://orcid.org/0000-0002-1371-7313","institution":"Griffith University","correspondingAuthor":false,"prefix":"","firstName":"Ailin","middleName":"","lastName":"Lepletier","suffix":""}],"badges":[],"createdAt":"2025-07-17 07:15:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7145918/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7145918/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87710021,"identity":"950aa15b-11bb-4cf1-a5f9-59cf642dba45","added_by":"auto","created_at":"2025-07-28 08:30:47","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":280218,"visible":true,"origin":"","legend":"\u003cp\u003eDomain localization, antibody responses, and S. pyogenes binding following immunization with M75-HVR and p*17 epitope-based vaccines. (A) Schematic representation of M-protein domains indicating the N-terminal position of M75 epitope, which is derived from the HVR region of the M-protein (M75-HVR), and the highly conserved p*17 epitope, derived from the C-terminal (C3) position. (B) Sera collected two weeks after the final immunization of BALB/c mice with p*17-DT or M75-DT adjuvanted with Alum (n=5/group) were assessed using direct ELISA and S. pyogenes binding assays. (C and D) ELISA was used to detect (C) anti-M75 and (D) anti-p*17-specific IgG at O.D. 450 nm. Absorbance values are shown for sera collected from mice immunized with M75-DT/Alum, p*17-DT/Alum or naïve controls at multiple dilutions. (E and F) Flow cytometry-based assay analyzing the binding of sera collected from mice immunized with (E) M75-DT/Alum or (F) p*17-DT/Alum to M1 and M75 live S. pyogenes. Serum obtained from naïve mice was used as controls. i. Representative histograms illustrating S. pyogenes binding by sera from one mouse per group. ii. Box plots depicting IgG1 antibody binding to S. pyogenes, quantified as mean fluorescence intensity (MFI). Unpaired t-tests were used for statistical analysis. n.s. non-significant; ****P\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7145918/v1/a9317125734ee063d586a042.jpeg"},{"id":87710023,"identity":"5682e0b0-886e-4dce-9f7a-23736c1e9561","added_by":"auto","created_at":"2025-07-28 08:30:47","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":363744,"visible":true,"origin":"","legend":"\u003cp\u003eMemory responses to p*17 following human infection with S. pyogenes M75. (A, B). Flow cytometry analysis of p*17-specific (A) IgG+ and (B) IgA+ memory B cells in the peripheral blood of participants in a controlled human infection study. i. Schematic overview of study timeline and collection of peripheral blood mononuclear cells (PBMC) for analysis of B cells in the peripheral blood. ii. Flow cytometry gating strategy for analysis of antigen-specific memory B cells in the peripheral blood. iii. The percentage of p*17-specific memory B cells is shown in the bar graphs at pre-challenge (day -1), 1-month post-challenge, and 6 months post-challenge. (C, D) ELISA was used to detect anti-p*17-specific (C) IgG and (D) IgA antibodies in PBMC-matched serum samples. i. Schematic overview of study timeline and collection of serum for analysis of antibodies. ii. Violin plots show O.D. at 450 nm absorbance values for sera collected at pre-challenge (day -1) and 6 months post-challenge, using a 1:20 dilution. Data are stratified by participants with and without pharyngitis. Each symbol represents an individual participant. Statistical analysis was performed using one-way ANOVA followed by Bonferroni's multiple comparisons test. No significant differences were observed between the groups.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7145918/v1/4fad438d4667f3c59e77de02.jpeg"},{"id":87710026,"identity":"e3c66e6b-99b9-4333-8763-c1a1cf113969","added_by":"auto","created_at":"2025-07-28 08:30:47","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":574758,"visible":true,"origin":"","legend":"\u003cp\u003eMemory responses to M75-HVR following human infection with S. pyogenes M75. (A, B) Flow cytometry analysis of M75-specific (A) IgG+ and (B) IgA+ memory B cells in the peripheral blood of participants in a controlled human infection study. i. Schematic overview of study timeline and collection of PBMC for analysis of B cells. ii. Flow cytometry gating strategy for analysis of antigen-specific memory B cells in the peripheral blood. iii The percentage of M75-specific memory B cells is shown in the bar graphs at pre-challenge (day -1), 1-month post-challenge, and 6 months post-challenge. (C, D) ELISA was used to detect anti-M75-specific (C) IgG and (D) IgA antibodies in PBMC-matched serum samples. i. Schematic overview of study timeline and collection of serum for analysis of antibodies. ii. Violin plots show at O.D. 450 nm absorbance values for sera collected at pre-challenge (day -1) and 6 months post-challenge, using a 1:20 dilution. Data are stratified by participants with and without pharyngitis. Each symbol represents an individual participant. Statistical analysis was performed using one-way ANOVA followed by Bonferroni's multiple comparisons test. *P\u0026lt;0.05, **P\u0026lt;0.01. (E, F) Correlation between the fold change in M75-specific (E) IgG+ and (F) IgA+ memory B cells and the fold change in serum M75-specific antibodies at 6 months post- vs pre-challenge. Full circles = pharyngitis, empty circle non-pharyngitis participants. Correlation coefficient and P value were determined using Spearman’s method with a linear regression line included to indicate linear trends.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7145918/v1/d780fb6f14e09483d6a2571b.jpeg"},{"id":87710028,"identity":"251286ee-b755-4301-b17b-1fc1e3391c76","added_by":"auto","created_at":"2025-07-28 08:30:47","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":700674,"visible":true,"origin":"","legend":"\u003cp\u003eComparative profiling of IgG Fc N-glycosylation in global and M75-specific serum IgG1 from participants with and without pharyngitis at pre-challenge and 6 months post-challenge. (A) Schematic overview of M75-specific IgG purification, digestion and Fc glycan profiling by liquid chromatography tandem mass spectrometry (LC-MS/MS). (B) Bar graphs depict the relative abundance of glycans in total IgG1 antibodies at i. pre-challenge and ii. 6 months post-challenge. (C) Heatmaps illustrate the fractional distribution of individual Fc glycoforms in global IgG1 antibodies at i. pre-challenge and ii. 6 months post-challenge. (D) Bar graphs depict the relative abundance of total glycans in M75-specific antibodies at i. pre-challenge and ii. 6 months post-challenge. (E) Heatmaps illustrate the fractional distribution of individual Fc glycoforms in M75-specific IgG1 antibodies at i. pre-challenge and ii. 6 months post-challenge. Schematic representation of the Fc glycoforms attached to IgG1 differentially distributed across participants with and without pharyngitis. Data represent individual unpaired samples from participants with (full symbols) and without (empty symbols) pharyngitis collected at pre-challenge (green) and 6 months-post challenge (blue) (n = 5/group). Unpaired t-test between participants with and without pharyngitis was used for statistical analysis *P\u0026lt;0.05, **P\u0026lt;0.01. (F) Principal component analysis shows the relationship of Fc glycoforms data from 5 biological replicates across participants with and without pharyngitis collected at i. pre-challenge (green) and ii. 6 months-post challenge (blue).\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7145918/v1/6429ad681254c5b6bb6839c2.jpeg"},{"id":87710033,"identity":"71ecb4f3-7266-4228-9475-96bb551d7d1a","added_by":"auto","created_at":"2025-07-28 08:30:47","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":312753,"visible":true,"origin":"","legend":"\u003cp\u003eFc N-glycosylation profiling of global and M75-specific serum IgG1 pre- and 6 months post-challenge in asymptomatic participants. (A) Schematic overview of M75-specific IgG purification, digestion and Fc glycan profiling by LC-MS/MS. (B) Line graphs depict the relative abundance of glycans in global IgG1 antibodies. (C) Heatmaps illustrate the fractional distribution of individual Fc glycoforms in global IgG1 antibodies. (D) Line graphs depict the relative abundance of glycans in M75-specific IgG1 antibodies in each participant. (E) Heatmaps illustrate the fractional distribution of individual Fc glycoforms in M75-specific IgG1 antibodies. Data represent paired samples collected at the time of challenge (green) and 6 months post-challenge (blue) from participants without pharyngitis (n = 5/group). Paired t-test between pre- and post-challenge was used for statistical analysis *P\u0026lt;0.05, **P\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7145918/v1/f19868792904f1f197d5aa79.jpeg"},{"id":87710964,"identity":"0d13eb45-c1cb-42e6-af26-d7889be49ca6","added_by":"auto","created_at":"2025-07-28 08:38:47","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":438874,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional analysis of purified serum IgG following S. pyogenes binding. (A-C) Flow cytometry-based binding assay shows the binding of IgG purified from sera to (B) M75 and (C) M1 S. pyogenes. Samples were collected at pre-infection (green) and 6 months post-infection (blue) from participants with (filled circles, n=5) and without (empty circles, n=5) pharyngitis. Each circle represents one participant. The level of binding of human IgG antibodies to S. pyogenes is shown as mean fluorescence intensity (MFI). One-way ANOVA followed by Bonferroni's comparisons tests were performed in all statistical analyses. *P\u0026lt;0.05, **P\u0026lt;0.01. (D-F) SCID mice (n=7-8/group) were inoculated intranasally with M75 S. pyogenes incubated with pooled IgG serum from asymptomatic participants (n=5) at pre-challenge and 6 months post-challenge. IgG purified from rat sera immunized with p*17-K4S2-CRM/Alum or left as naïve was used as a control. (E) Graph shows the percentage of weight loss on day 2 following inoculation. (F) S. pyogenes colony-forming units (CFU) were enumerated in (i) total respiratory tract (total CFU obtained from throat swabs, nasal shedding, lungs and nasal-associated lymphoid tissue (NALT) throat swabs, (ii) in mucosal sites (throat swabs and nasal shedding) and (iii) in respiratory organs (CFU obtained lungs NALT and lungs), on day 2 post-challenge. An unpaired t-test was performed to compare weight loss and bacterial burden between mice treated with IgG purified from vaccinated versus naïve rats and from humans at 6 months post- challenge versus pre-challenge. n.s., non-significant; *P\u0026lt;0.05; **P\u0026lt;0.01; ***P\u0026lt;0.005.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7145918/v1/a0e42a675a7c05276f95353d.jpeg"},{"id":87713716,"identity":"d3585b12-255f-4c25-9433-8ce7a0900916","added_by":"auto","created_at":"2025-07-28 08:54:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3594555,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7145918/v1/cd0bbed2-5baf-4a0d-8dc5-3129dcb2504c.pdf"},{"id":87710963,"identity":"44298f24-e8ff-4b0b-b91d-e87e93e72668","added_by":"auto","created_at":"2025-07-28 08:38:47","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":563865,"visible":true,"origin":"","legend":"Supplementary Figures 1 and 2","description":"","filename":"SuppFiguresLepletieretal.CHIVASmanuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7145918/v1/a019e16bca2868fc37c99c5d.pdf"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nA.C.S. is co-chair of the Australian Strep A Vaccine Initiative (ASAVI) and the Strep A Vaccine Global Consortium (SAVAC). M.F.G. and M.P. are inventors on patents related to S. pyogenes vaccines.","formattedTitle":"\u003cp\u003eSubclinical exposure to \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e drives the development of long-lived immunity\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eStreptococcus pyogenes\u003c/em\u003e (Group A \u003cem\u003eStreptococcus\u003c/em\u003e, Strep A) is a leading human-restricted pathogen with a persistent and profound global disease burden. It causes broad clinical spectrum of illness, spanning superficial through to severe infections and post-infectious syndromes including rheumatic heart disease \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Every year, up to a billion people are affected and more than 500,000 deaths are attributable to \u003cem\u003eS. pyogenes\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. \u0026lsquo;Strep throat\u0026rsquo; or \u003cem\u003eS. pyogenes\u003c/em\u003e pharyngitis is a ubiquitous childhood illness, with incidence peaking during the primary school years \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Lower incidence in adults has generally been ascribed to immunity accumulated through repeated exposure in childhood to different \u003cem\u003eS. pyogenes\u003c/em\u003e strains \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Although the precise mechanisms of immunity against \u003cem\u003eS. pyogenes\u003c/em\u003e remain uncertain, several humoral and cellular responses have been strongly implicated in protection against colonization or disease \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. These responses include antibodies that bind to bacteria, enabling neutrophils and monocytes at the site of infection to recognize and eliminate them through opsonophagocytosis \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. In individuals resistant to pharyngeal acquisition, such antibodies can also prevent early bacterial colonization by blocking adherence and promoting rapid clearance \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAllelic polymorphism of the \u003cem\u003eS. pyogenes\u003c/em\u003e M-protein is a major impediment to the development of immunity. To date, over 250 distinct serotypes have been identified based on its amino-terminal hypervariable region (HVR) \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Longitudinal studies indicate that naturally acquired antibodies against the M-protein can provide homologous protection but offer limited cross-protection against disease caused by heterologous strains \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. While the M cluster concept explains some degree of cross-protective immunity \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, it remains unclear how most children develop protective immunity by their second decade of life \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, considering that only around 15% experience symptomatic \u003cem\u003eS. pyogenes\u003c/em\u003e pharyngitis in any given year \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. A potential explanation is that repeated exposures boost immunity to antigens that are broadly conserved across strains, complementing narrower responses to type-specific antigens such as the M-protein HVR. We previously showed that antibodies to the conserved C-repeat region can kill the \u003cem\u003eS. pyogenes\u003c/em\u003e irrespective of its M-protein serotype \u003csup\u003e\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. However, C-repeat-specific immunity is unlikely to account for naturally acquired immunity because this region is poorly immunogenic (\u0026lsquo;cryptic\u0026rsquo;) during both experimental and natural infections. Antibodies to this region do not develop in children from streptococcal-endemic areas until well into their second decade of life \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e nor do they arise following repeated exposures of mice to \u003cem\u003eS. pyogenes\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eGiven the high prevalence of asymptomatic pharyngeal \u003cem\u003eS. pyogenes\u003c/em\u003e colonization or \u0026lsquo;carriage\u0026rsquo; in 10\u0026ndash;20% of school-age children \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e and that many different serotypes can be in circulation in a community at any time \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, a reasonable hypothesis is that repeated asymptomatic (or subclinical) exposures shape protective immune responses to \u003cem\u003eS. pyogenes\u003c/em\u003e. Here, we explore this hypothesis in a well-characterised cohort of participants from the CHIVAS-M75 \u003cem\u003eS. pyogenes\u003c/em\u003e human pharyngeal challenge study \u003csup\u003e\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. While acute symptomatic pharyngitis following direct tonsillar inoculation was the most common outcome, a subset remained asymptomatic despite lacking significant pre-existing serotype-specific antibodies to the M75 strain, enabling us to investigate the immunological outcomes of subclinical infections. At 6-months post-challenge, increased levels of memory B cells and antibodies targeting the M75 hypervariable region (HVR) \u0026mdash;but not a conserved cryptic M-protein epitope (p*17) \u0026mdash;were observed in participants both with and without pharyngitis. Using several approaches to assess antibody function, including bacterial binding, IgG Fc glycosylation traits, and passive antibody transfer into a murine model of pharyngeal infection, we show that subclinical exposures can elicit immune responses that contribute to long-term protection against \u003cem\u003eS. pyogenes\u003c/em\u003e.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eMurine antibodies against the M75 hypervariable region bind specifically to M75\u003c/b\u003e \u003cb\u003eS. pyogenes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOur initial studies aimed to characterize humoral immune responses elicited by the M-protein peptides, p*17 and M75-HVR (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Although p*17 is poorly recognized following infection of mice, it is known that vaccine-induced antibodies can protect against infection \u003csup\u003e\u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. BALB/c mice were immunized with either p*17-diphtheria toxoid (DT) or M75-HVR-DT, each adjuvanted with aluminum hydroxide (Alum), following a 3-dose schedule as in a Phase 1 clinical trial \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Serum from both groups were collected two weeks after the final vaccine dose and analyzed by indirect ELISA and flow cytometry-based binding assays to assess IgG antibody responses against p*17 and M75-HVR peptides, as well as to live \u003cem\u003eS. pyogenes\u003c/em\u003e bacteria (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), respectively. Serum from M75-DT immunized mice specifically recognized the M75 peptide (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), whereas serum from p*17-DT-immunized mice exclusively recognized p*17 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Using flow cytometry, we demonstrated that serum from M75-DT\u0026ndash;immunized mice specifically bound to M75 \u003cem\u003eS. pyogenes\u003c/em\u003e, with no detectable reactivity to an M1 strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). In contrast, serum from p*17-DT\u0026ndash;immunized mice recognized both M75 and M1 strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF), consistent with p*17 being a conserved epitope across \u003cem\u003eS. pyogenes\u003c/em\u003e strains.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eHuman pharyngeal challenge with the M75\u003c/b\u003e \u003cb\u003eS. pyogenes\u003c/b\u003e \u003cb\u003eelicits memory responses directed against the M-protein hypervariable region.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo assess the long-term human immune responses to infection, we examined memory B cell populations and serum antibodies from CHIVAS-M75 participants \u003csup\u003e\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Antigen-specific responses to p*17 and M75-HVR were assessed for 13 participants who developed acute symptomatic pharyngitis following pharyngeal challenge with M75 \u003cem\u003eS. pyogenes\u003c/em\u003e. These were compared to responses from 5 participants who remained asymptomatic (non-pharyngitis) and had no detectable M75 \u003cem\u003eS. pyogenes\u003c/em\u003e by culture or qPCR from throat swabs collected twice daily from 24 hours after challenge until discharge. Systemic immune responses were longitudinally analyzed at pre-challenge, 1 month, and 6 months post-challenge.\u003c/p\u003e\u003cp\u003eTo evaluate immunity against \u003cem\u003eS. pyogenes\u003c/em\u003e and explore the dynamics of antigen-specific memory B cells and antibody responses post-challenge, we generated fluorescent tetramers and analyzed responses to the p*17 and the type-specific HVR region of the M75 protein. To determine whether a single M75 \u003cem\u003eS. pyogenes\u003c/em\u003e infection induced antibodies to the conserved region of the M-protein, we specifically assessed responses to p*17 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and B). Flow cytometry-based tetramer analysis was used to characterize p*17-specific memory B cells (CD27\u003csup\u003e+\u003c/sup\u003eCD21\u003csup\u003e+\u003c/sup\u003e) within both the IgA and IgG subclasses (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The frequency of p*17-specific IgG\u003csup\u003e+\u003c/sup\u003e memory B cells remained low and unchanged following pharyngeal challenge with M75 \u003cem\u003eS. pyogenes\u003c/em\u003e out to 6 months in both symptomatic and asymptomatic participants (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Likewise, p*17-specific IgA\u003csup\u003e+\u003c/sup\u003e cells in the blood were unaffected by the challenge, with no detectable link to disease progression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Antibody response analyses in the same participants similarly revealed that IgG antibody levels targeting p*17 remained low and unchanged across all timepoints in all participants following pharyngeal challenge (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Similarly, serum IgA responses to the p*17 antigen were minimal (O.D. \u0026lt; 0.2 at a dilution of 1:20), showing no significant changes post-challenge and no association with disease progression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn those who developed symptomatic disease post-challenge, the frequency of M75-specific IgG\u003csup\u003e+\u003c/sup\u003e memory B cells increased significantly at 6 months (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). M75-specific IgA\u003csup\u003e+\u003c/sup\u003e memory B cells showed an early increase, observed at 1 month, which also endured for at least 6 months post-challenge (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In asymptomatic participants there was a trend toward an increase at 6 months for both M75-specific IgG\u003csup\u003e+\u003c/sup\u003e (2.09 fold-increase, P\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and IgA\u003csup\u003e+\u003c/sup\u003e (1.69 fold-increase, P\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) memory B cells.\u003c/p\u003e\u003cp\u003eSimilarly, antibody analysis showed a rise in M75-specific IgG levels in participants with pharyngitis but was detected only at 6 months post-challenge (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). This increase was driven specifically by the M75-specific IgG3 subtype (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) with no significant changes in IgG1, IgG2, or IgG4 levels (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-E). M75-specific serum IgA antibodies also showed a significant increase detected at 6 months post-challenge (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Although a significant antibody increase was observed only in symptomatic participants, the average increase in M75-specific antibody levels was similar in those with and without pharyngitis (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF) (Total IgG: mean values of 1.176 for pharyngitis and 1.242 for non-pharyngitis; IgG3: mean values of 1.431 for pharyngitis and 1.435 for non-pharyngitis; IgA:1.147 for pharyngitis and 1.330 for non-pharyngitis). The increase in IgG\u003csup\u003e+\u003c/sup\u003e memory B cells binding the M75 antigen correlated with increased M75-specific serum IgG at 6 months relative to pre-challenge (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.438, P\u0026thinsp;=\u0026thinsp;0.02, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Interestingly, the increase in IgA\u003csup\u003e+\u003c/sup\u003e memory B cells binding the M75 \u003cem\u003eS. pyogenes\u003c/em\u003e antigen did not correlate with increased M75-specific serum IgA (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.04, P\u0026thinsp;=\u0026thinsp;0.39, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). There was no significant difference in pre-challenge M75-specific memory B cells and antibodies between participants who did and did not develop pharyngitis following challenge (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and D).\u003c/p\u003e\u003cp\u003eExposure to M75 \u003cem\u003eS. pyogenes\u003c/em\u003e induces long-term, strain-specific systemic immunity, as evidenced by sustained serum Ig levels and memory B cell responses involving both IgG and IgA. While occurring in both groups of participants, the immune responses were more pronounced in those who developed clinical disease.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eIgG Fc glycosylation is associated with clinical manifestations of\u003c/b\u003e \u003cb\u003eS. pyogenes\u003c/b\u003e \u003cb\u003einfections\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePost-translational modifications, particularly glycosylation of the constant (Fc) IgG domain, are well-known features of all IgGs and play a critical role in modulating antibody effector functions, including complement activation and Fc receptor engagement \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e We initially assessed whether pre-challenge IgG Fc N-glycosylation traits could be a distinguishing factor between participants with and without pharyngitis. We affinity-purified total (non-specific) and M75-specific IgG from asymptomatic and symptomatic participants prior to challenge. M75-specific IgG levels pre-challenge were very low in all participants, but affinity purification enabled enrichment of these antibodies (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Mass spectrometry-based characterisation revealed that pre-challenge IgG1 antibodies were the major IgG subclass targeting the M75-epitope across all participants. Participants who remained asymptomatic following challenge displayed a distinct baseline Fc glycosylation profile for global (entire IgG pool present in plasma) IgG1 compared to participants who became symptomatic (1.5-fold and 2.16 fold-increase in sialylated and afucosylated N-glycans, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and P\u0026thinsp;\u0026gt;\u0026thinsp;0.05, respectively, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, i), whereas no significant differences were detected in overall glycoform distribution (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, i). Profound changes were observed in the glycosylation profile of M75-specific IgG1 antibodies from asymptomatic participants, marked by a 30-fold decrease in afucosylation (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and a 4.9-fold increase in bisecting GlcNAc moieties (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, i), which is largely derived from increased levels of G1FB and G2FB N-glycans (3.1-fold and 13.9-fold increase, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, respectively, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, i), compared to M75-specific IgG1 from symptomatic participants. In comparison to global IgG1, M75-specific IgG1 in participants without pharyngitis displayed significantly lower levels of afucosylated glycans (mean of 0.12 versus 2.13; P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), along with a significantly higher proportion of bisected glycans (mean of 5.29 versus 1.36; P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, i and D, \u003cem\u003ei\u003c/em\u003e, respectively).\u003c/p\u003e\u003cp\u003eIgG Fc N-glycosylation traits at 6 months post-challenge showed similar differences between participants with and without pharyngitis as those observed pre-challenge. Participants who remained asymptomatic following challenge exhibited a 1.31-fold increase in sialylated N-glycans, a 1.98-fold increase in afucosylated N-glycans, and a 1.65-fold increase in bisecting GlcNAc moieties (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, respectively; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, \u003cem\u003eii\u003c/em\u003e), whereas no significant differences were detected in overall glycoform distribution (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, \u003cem\u003eii\u003c/em\u003e). Profound changes were observed in the glycosylation profile of M75-specific IgG1 antibodies from asymptomatic participants, marked by a 17.3-fold decrease in afucosylation (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05), a 5.4-fold increase in bisecting GlcNAc moieties (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and a 1.3-fold increase in sialylation (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, \u003cem\u003eii\u003c/em\u003e). This is derived not only from increased levels of G1FB, G2FB and G2FS N-glycans (3.9-fold, 11.3-fold and 2.1-fold increase, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, respectively), but also from decreased levels of G0F, G1F and G2F (2.5-fold, 1.8-fold and 1.6-fold decrease P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, respectively, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, \u003cem\u003eii\u003c/em\u003e) compared to M75-specific IgG1 from symptomatic participants. The evident differences at post-challenge were further supported by principal component (PC) analyses, which revealed that Fc glycoforms profiles obtained from symptomatic and asymptomatic participants formed two well-defined and distinct clusters (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, \u003cem\u003eii\u003c/em\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eCompared to pre-challenge levels, no significant differences were detected in overall IgG Fc N-glycosylation traits or glycoform compositions of global and M75-specific IgG1 antibodies from participants with pharyngitis at 6 months post-\u003cem\u003eS. pyogenes\u003c/em\u003e challenge (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). No changes in IgG Fc N-glycosylation were observed in global IgG1 purified from asymptomatic participants at 6 months post-challenge compared to pre-challenge levels either (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-C). However, at 6 months, M75-specific IgG1 showed a significant increase in agalactosylation (1.5-fold) and sialylation (1.3-fold, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD) followed by a specific increase in G2FS (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE) in comparison to pre-challenge.\u003c/p\u003e\u003cp\u003eThese findings strongly indicate that the Fc-glycosylation traits of IgG1 antibodies directed against the M75 antigen are involved in modulating the susceptibility to symptomatic disease following M75 \u003cem\u003eS. pyogenes\u003c/em\u003e challenge.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eSubclinical exposure to\u003c/b\u003e \u003cb\u003eS. pyogenes\u003c/b\u003e \u003cb\u003einduces long-term, functionally protective IgG immunity\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo examine the functional properties of serum IgG and to increase the sensitivity of our analyses, we purified total serum IgG and used flow cytometry to assess the \u003cem\u003eS. pyogenes\u003c/em\u003e binding at both pre-challenge and 6 months post-challenge in the participants with and without pharyngitis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). We observed a 12.1-fold increase in the binding of purified IgG to the homologous M75 \u003cem\u003eS. pyogenes\u003c/em\u003e at 6 months post-challenge in participants with symptomatic pharyngitis, relative to their pre-challenge levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). We also observed a significant increase in IgG binding (6.4-fold, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in asymptomatic participants comparing 6-month post challenge relative to their pre-challenge levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Similar to sera from mice immunized with M75-DT, IgG from CHIVAS trial participants at 6 months post-challenge (in both pharyngitis and non-pharyngitis sufferers) showed no binding to the heterogeneous M1 \u003cem\u003eS. pyogenes\u003c/em\u003e (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). These findings indicate that infection with \u003cem\u003eS. pyogenes\u003c/em\u003e can elicit a significant rise in serotype-specific antibodies that persist for at least 6 months regardless of the clinical outcome.\u003c/p\u003e\u003cp\u003eThe temporal changes observed in the IgG Fc N-glycosylation of asymptomatic participants prompted us to examine their functional properties more closely. We utilized a recently developed \u003cem\u003ein vivo\u003c/em\u003e passive transfer model to test functional immunity. In this assay, \u003cem\u003eS. pyogenes\u003c/em\u003e organisms were incubated with serum IgG antibodies (\u0026rsquo;pre-opsonized\u0026rsquo;) and then inoculated intranasally into immunocompromised mice. Clinical signs and bacterial burden in the respiratory tract were subsequently assessed to determine the protective capacity of the antibodies \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Due to limited availability of serum, we purified IgG from the 5 asymptomatic participants at pre and 6-months post challenge, pooled the IgG at each time point, and tested it against M75 \u003cem\u003eS. pyogenes\u003c/em\u003e in 7\u0026ndash;8 mice for each group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Further controls included IgG from rats vaccinated with p*17-K4S2-CRM \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e or IgG from na\u0026iuml;ve rats. We tested weight loss (as a general measure of disease in \u003cem\u003eS. pyogenes\u003c/em\u003e -infected mice \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e), and bacterial burden from the entire respiratory tract. Mice inoculated with M75 \u003cem\u003eS. pyogenes\u003c/em\u003e that had been pre-incubated with IgG from vaccinated rats exhibited significantly less weight loss compared to mice administered \u003cem\u003eS. pyogenes\u003c/em\u003e treated with IgG from naive rats (0% versus 8.4%, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Across all mucosal sites and organs analyzed for bacterial burden, IgG from p*17-vaccinated rats resulted in significant reductions (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01 \u0026ndash; P\u0026thinsp;\u0026lt;\u0026thinsp;0.005) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003eWe similarly observed that mice inoculated with M75 \u003cem\u003eS. pyogenes\u003c/em\u003e pre-incubated with IgG from CHIVAS-M75 participants collected 6 months post-challenge exhibited significantly reduced weight loss compared to those administered with \u003cem\u003eS. pyogenes\u003c/em\u003e incubated with pre-challenge IgG (4.64% versus 10.63%, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). This correlated with 273-fold reduction in total \u003cem\u003eS. pyogenes\u003c/em\u003e colonization of the respiratory tract (P\u0026thinsp;\u0026lt;\u0026thinsp;0.005), which included both mucosal sites (nasal shedding and throat swabs) and respiratory organs (nasal-associated lymphoid tissue [NALT] and lungs). Specifically, we observed a 205-fold reduction in \u003cem\u003eS. pyogenes\u003c/em\u003e colonization of the respiratory mucosa (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and 314-fold reduction in colonization of respiratory tissues (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). There were no significant differences observed between IgG from na\u0026iuml;ve rats and the human pre-challenge IgG (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003eThus, serum IgG antibodies generated from subclinical \u003cem\u003eS. pyogenes\u003c/em\u003e infection exhibit both strong strain-specific binding and robust functional activity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study demonstrates that functional immunity to \u003cem\u003eS. pyogenes\u003c/em\u003e can develop following asymptomatic exposure. Pharyngeal human challenge with M75 \u003cem\u003eS. pyogenes\u003c/em\u003e elicited class-switched B cells and antibodies (IgG and IgA) in the peripheral blood targeting the type-specific M-protein HVR region. While the immune responses elicited by asymptomatic challenge were lower than those observed in symptomatic participants, IgG from these participants demonstrated clear strain-specific binding and effectively mediated bacterial clearance in a passive transfer murine infection model, whereas pre-challenge IgG did not. Participants with higher frequency of M75-specific IgG memory B cells at 6 months after challenge also had higher levels of binding anti-M75 IgG, suggesting that memory B cells may help maintain or increase IgG antibody levels over time. These results support the hypothesis that immunity to \u003cem\u003eS. pyogenes\u003c/em\u003e is gradually acquired throughout childhood by repeated asymptomatic (or subclinical) exposures to circulating strains and not only through symptomatic infections.\u003c/p\u003e\u003cp\u003eRecent longitudinal studies have also highlighted that asymptomatic \u003cem\u003eS. pyogenes\u003c/em\u003e exposures may be immunologically significant (\u003cem\u003ei.e.\u003c/em\u003e capable of triggering measurable immune responses), challenging the long-held assumption that only symptomatic infections contribute meaningfully to immunity. Studies in the USA by Hysmith et al. \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e and in The Gambia by Keeley et al. \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e each demonstrated antibody binding responses to M and non-M-protein antigens following asymptomatic episodes of \u003cem\u003eS. pyogenes\u003c/em\u003e colonization. This observation is confirmed by other studies showing that a significant proportion of people with asymptomatic colonization of the upper respiratory tract develop functional anti-M-protein antibodies \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Notably, a significant fraction of the children assessed in these studies only developed M-type specific serum IgG antibodies between 6 weeks and a year from detection of a positive throat culture \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, which aligns with our data showing a delayed immune response following pharyngeal challenge. In the 2-year study by Hysmith et al., new \u003cem\u003eS. pyogenes\u003c/em\u003e acquisitions did not significantly boost homologous M-type-specific antibody responses \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Therefore, it is possible that pre-screening and exclusion of participants with high serum type-specific M75 IgG in the CHIVAS-M75 study \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e may have contributed to the observed immunologically significant M75-specific responses following pharyngeal challenge.\u003c/p\u003e\u003cp\u003eSerum antibodies against the M-protein enhance the opsonisation and killing of \u003cem\u003eS. pyogenes\u003c/em\u003e and are believed to contribute to immunity against homologous strains. We previously observed that a single intranasal infection with \u003cem\u003eS. pyogenes\u003c/em\u003e resulted in long-lasting immunity against subsequent homologous challenge in a mouse model \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Here we showed that human serum IgG antibodies, purified 6 months post-challenge, recognised the strain-specific HVR, bound specifically to live M75 \u003cem\u003eS. pyogenes\u003c/em\u003e, and were bactericidal. The anti-M75 IgG response observed in the present study is predominantly mediated by the IgG1 and IgG3 subclasses, which are particularly effective in pathogen clearance, presenting a number of antigen-specific effector functions, including interaction of antigen-clustered IgG with the complement system, which activates phagocytosis and production of reactive oxygen species by innate immune cells \u003csup\u003e\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Despite its relatively low abundance in relation to IgG1, IgG3 has been increasingly recognized as a key player in the immune defence against various pathogens, including \u003cem\u003eS. pyogenes\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e due to its superior affinity to Fc-gamma receptors \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. However, excessive IgG3 recognising M-protein antigens may be detrimental and elevated levels have been linked to the \u003cem\u003eS. pyogenes\u003c/em\u003e post-infectious syndrome acute rheumatic fever \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAlthough total and subclass-specific M75-IgG antibody levels prior to challenge did not distinguish between clinical outcomes following experimental challenge, significant differences in IgG Fc N-glycosylation traits both in global and M75-specific IgG1 were observed. Compared to those with symptomatic pharyngitis, asymptomatic participants had higher total IgG1 sialylation, suggesting that the anti-inflammatory properties of Fc sialylation \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e may also contribute to protection. Additionally, they exhibited essentially non existing levels of afucosylated N-glycans in M75-specific IgG1 Fc, which has been associated with exacerbated inflammatory responses and disease severity following viral infections \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Our data provide evidence that subclinical exposure to \u003cem\u003eS. pyogenes\u003c/em\u003e can selectively shape the N-glycan landscape of M75-specific IgG1 antibodies. The glycosylation features of the Fc region significantly influence the ability of IgG antibodies to trigger inflammatory responses and their interaction with FcγIII receptors \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. The concurrent increase in both agalactosylation and sialylation suggests a fine-tuned immune adaptation of M75-specific responses: the host may be enhancing IgG1 functionality for bacterial clearance (via agalactosylation \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e) while simultaneously limiting collateral inflammation and tissue damage (via sialylation and fucosylation \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e). This dual modulation could reflect an evolved mechanism to balance pathogen elimination with immune regulation during asymptomatic \u003cem\u003eS. pyogenes\u003c/em\u003e exposures. Although the anti-M75 IgG response was predominantly mediated by the IgG1 and IgG3 subclasses post-challenge (as per ELISA data), the IgG Fc N-glycosylation in the non-pharyngitis (asymptomatic) participants appeared to be more prominent on IgG1 and IgG4 as compared to the pharyngitis group (both global and M75-IgG). Nevertheless, significant glycosylation changes were only observed in M75-specific IgG1 and not IgG3/4.\u003c/p\u003e\u003cp\u003eThe modest immune responses to the conserved C-terminal M-protein peptide p*17 observed in this study are consistent with previous findings in both mice and children from streptococcal-endemic regions \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Although C-terminal antigens appear to be cryptic during exposure and infection with \u003cem\u003eS. pyogenes\u003c/em\u003e, vaccination with the p*17 antigen conjugated to a carrier protein elicits robust immune responses in animal models. These include high-affinity, class-switched antibodies that recognize a broad range of \u003cem\u003eS. pyogenes\u003c/em\u003e strains, effectively reducing bacterial colonization and preventing severe infection \u003csup\u003e\u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. These observations underscore the likelihood that vaccine-induced protection against \u003cem\u003eS. pyogenes\u003c/em\u003e operates through mechanisms distinct from naturally acquired immunity, which is only partially protective. This is evidenced by continued low susceptibility to infection among adults, particularly in parents of young children, individuals in crowded settings (e.g. army barracks), people who inject drugs, those experiencing homelessness, and older adults with accumulating comorbidities.\u003c/p\u003e\u003cp\u003eA limitation of our study is the small cohort size, especially the limited number of asymptomatic participants. However, these participants showed significant and strain-specific IgG binding to \u003cem\u003eS. pyogenes\u003c/em\u003e equivalent to antibodies from mice immunised with M75 antigen. The high rate of clinical pharyngitis in the challenged adult participants, despite accumulated immunity to antigens other than the type-specific M75 hypervariable region (an exclusion criteria), may suggest the inoculum (dose) and direct swab challenge method may have circumvented or overwhelmed natural defences \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Still, the inoculum was several orders of magnitude lower than in comparable small and larger animal models, around a third of challenged participants remained asymptomatic, and those participants did develop de novo type-specific antibodies. Beyond these concerns related to the model, this study did not consider responses to conserved non-M-protein antigens such as SpyCEP, SLO and ScpA, which have previously been found in this cohort to have been increased in participants with pharyngitis and to have persisted for at least 3 months post-challenge \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn conclusion, our study demonstrated that asymptomatic \u003cem\u003eS. pyogenes\u003c/em\u003e infection can induce persistent serotype-specific functional antibodies. This finding could be re-examined in longitudinal \u003cem\u003eS. pyogenes\u003c/em\u003e surveillance studies including serial serum sampling and tested in future human studies incorporating challenge and homologous rechallenge. We propose that subclinical exposures do contribute to the development of protective immunity against \u003cem\u003eS. pyogenes\u003c/em\u003e in humans.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003eThe CHIVAS-M75 study\u003c/p\u003e\u003cp\u003eThe CHIVAS-M75 study protocol, including information on the M75 \u003cem\u003eS. pyogenes\u003c/em\u003e challenge strain and key findings, has been detailed in earlier publications \u003csup\u003e\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Ethical approval for the study, including sample collection and associated immunological investigations, was granted by the Alfred Hospital Human Research Ethics Committee (reference number 500/17), and all participants provided written informed consent. Among the 25 participants enrolled in the trial, 19 developed pharyngitis, across two dose levels. The diagnosis of pharyngitis was corroborated by microbiological evidence (via qPCR and culture), elevated biochemical markers (C-reactive protein, cytokines, and chemokines), and serological responses (anti-streptolysin O and anti-DNase B antibodies). Importantly, all participants classified as asymptomatic in the present study showed no signs of \u003cem\u003eS. pyogenes\u003c/em\u003e colonization. In the present study, analysis was limited by sample availability. Serum and PBMC were collected from participants at the evening prior to the challenge, then at 1-month and 6-month outpatient visits. Blood was collected in serum separator tubes (BD Vacutainer SST Gold 8.5 mL) and allowed to clot. Within 2 h of collection, tubes were centrifuged at 1500 \u0026times; g for 15 min at 20\u0026deg;C, and then aliquots were stored at \u0026minus;\u0026thinsp;80\u0026deg;C. PBMCs were collected using CPT tubes (BD Bioscience).\u003c/p\u003e\u003cp\u003eMurine model of \u003cem\u003eS. pyogenes\u003c/em\u003e infection\u003c/p\u003e\u003cp\u003eAll animal protocols were reviewed and approved by the Griffith University Animal Ethics Committee (GU-AEC) in accordance with the National Health and Medical Research Council (NHMRC) of Australia guidelines. Experimental protocols involving SCID (Severe Combined Immunodeficiency) mice were reviewed and approved by Office of the Gene Technology Regulator (OGTR). Mice (female, 4\u0026ndash;6 weeks) were sourced from Ozgene, Western Australia. All mice were housed in a PC2-certified animal facility in individually ventilated cages (IVC) with a maximum of 5 mice/cage. For positive controls in the pre-opsonization assay, Sprague Dawley rats (aged 9.5 weeks at the start of dosing) were sourced from Charles River Laboratories, Inc. The animals received three intramuscular injections of the p*17-K4S2-CRM/Alum vaccine over a six-week period, followed by a two-week recovery phase. The adjuvant, aluminium hydroxide (Alhydrogel 2%, Alum), was obtained from Brenntag Biosector, Denmark. The relative humidity ranged between 45\u0026ndash;65% and temperature at 20\u0026ndash;24\u0026deg;C. Mice and rats were exposed to a 12-h light\u0026ndash;dark cycle.\u003c/p\u003e\u003cp\u003eAntigens\u003c/p\u003e\u003cp\u003eSynthetic peptides from the M75 protein hypervariable region (M75-HVR) and C-terminal region (p*17) regions were synthesized and purified (\u0026gt;\u0026thinsp;95%) commercially by GenScript and stored lyophilized or in solution at \u0026minus;\u0026thinsp;20\u0026deg;C. Peptide sequence for M75-HVR is: EEERTFTELPYEARYKAWKSENDELRENYRRTLDKFNTEQGKTTRLEEQN. Peptide sequence for p*17 is: LRRDLDASREAKNQVERALE.\u003c/p\u003e\u003cp\u003eEnzyme-linked immunosorbent assays (ELISA)\u003c/p\u003e\u003cp\u003eStandard ELISA was used to measure M75 and p*17-specific serum antibody levels. Briefly, peptides were coated onto NUNC MaxiSorp plates (Thermo Fisher). Antigens were resuspended to 5 \u0026micro;g/mL in 0.1 M coating carbonate buffer and coated at 100 \u0026micro;L per well onto 96-well medium-binding ELISA plates (Greiner Bio-One) overnight at room temperature. Plates were then blocked with 200 \u0026micro;L of 2% w/v bovine serum albumin (BSA, Sigma) in phosphate-buffered saline (PBS) overnight at 4\u0026deg;C, followed by five washes in wash buffer (PBS with 0.05% v/v Tween20, PBS-T). Serial dilutions were performed using diluent (0.05% v/v Tween20) at 1:10, 1:20, 1:40, 1:80 dilutions. Samples were diluted 2-fold and antibody levels detected with HRP-conjugated goat anti-human IgA (Invitrogen, USA), IgG (H\u0026thinsp;+\u0026thinsp;L), IgG1, IgG2, IgG3 and IgG4 antibodies (Thermo Fisher, USA). SIGMAFAST OPD substrate was added according to manufacturer\u0026rsquo;s instructions and absorbance was measured at 450 nm on a Tecan Infinite M200 Pro plate reader (Tecan Group Ltd., Switzerland). Negative controls were included in each plate containing commercial human sera (Sigma) pre-absorbed of anti-M75 \u003cem\u003eS. pyogenes\u003c/em\u003e antibodies or containing 0.05% v/v Tween20 only.\u003c/p\u003e\u003cp\u003eTetramer staining for peptide-specific memory B cells\u003c/p\u003e\u003cp\u003eFrozen mononuclear PBMCs were thawed at 37\u0026ordm;C in thawing media (RPMI\u0026thinsp;+\u0026thinsp;10% FCS), then washed and resuspended in flow buffer (PBS\u0026thinsp;+\u0026thinsp;2%FCS\u0026thinsp;+\u0026thinsp;2 mM EDTA). Biotinylated peptides synthesised at Genscript were tetramerised by serial addition of premium-grade streptavidin‐PE and streptavidin‐APC (Bio legend, San Diego, CA, USA) at a four‐molar equivalent of biotinylated peptide to one molar equivalent of streptavidin during 2 h at room temperature \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. The prepared tetramer was diluted to a concentration of 2 \u0026micro;m for use. Typically, 10\u003csup\u003e6\u003c/sup\u003e PBMCs were pre‐incubated with Fc block (Bio legend) for 10 min followed by tetramer staining for 40 min. Cells were washed with flow buffer and then stained with fluorescently labelled antibodies (including CD19, CD20, CD21, CD27, IgD, IgM, IgG and IgA) and viability dye. Cells were fixed with 1% PFA and analyzed on a LSRFortessa (BD Biosciences). These cells were gated as positive for both fluorochromes of an antigen tetramer pair to reduce the inclusion of non-specific binding cells, as previously described \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cem\u003eS. pyogenes\u003c/em\u003e strains and growth conditions\u003c/p\u003e\u003cp\u003e\u003cem\u003eS. pyogenes\u003c/em\u003e strain 611024 (emm75, GenBank accession CP033621), isolated in Melbourne from a child presenting with exudative pharyngitis, was used in this study \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eS. pyogenes\u003c/em\u003e strain M1 5448 (emm1) was kindly provided by Professor Mark Walker, from the University of Queensland, isolated in Australia from a case of necrotising fasciitis. For mouse infection experiments, M75 \u003cem\u003eS. pyogenes\u003c/em\u003e was serially passaged through mouse spleens to enhance virulence and rendered streptomycin-resistant (200 \u0026micro;g/mL) to enable selective recovery from commensal flora in respiratory mucosa and tissues.\u003c/p\u003e\u003cp\u003e\u003cem\u003eS. pyogenes\u003c/em\u003e was cultured at 37\u0026deg;C for 16\u0026ndash;18 hours. Single colonies were inoculated into Todd Hewitt Broth (THB; Oxoid, Australia) supplemented with 1% Bacto Neopeptone (Thermo Fisher, USA), yeast extract and streptomycin \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. To enumerate colony-forming units (CFU), cultures were plated on Columbia agar containing 5% defibrinated horse blood and 200 \u0026micro;g/ml streptomycin (CBA 5%) \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIgG purification\u003c/p\u003e\u003cp\u003eIgG antibodies were purified from the serum of CHIVAS participants and immunized rats using NAb Protein G columns, following the manufacturer\u0026rsquo;s instructions (Thermo Fisher, USA). The concentration of purified IgG was measured using a NanoDrop spectrophotometer (Thermo Fisher, USA).\u003c/p\u003e\u003cp\u003eM75 antigen-specific IgG antibodies were purified using SulfoLink\u0026trade; affinity chromatography columns (Thermo Fisher Scientific, USA) following the manufacturer\u0026rsquo;s protocol. Briefly, the columns were prepared by coupling M75-HVR to the SulfoLink resin via sulfhydryl-reactive chemistry. Serum samples were then applied to the columns, allowing M75-HVR-specific IgG to bind selectively. After thorough washing to remove unbound proteins, antigen-specific IgG was eluted using a low pH glycine buffer and immediately neutralized. The purified IgG fractions were concentrated and buffer-exchanged into PBS using centrifugal filters (Amicon Ultra, Millipore). Purity of the eluted M75-specific IgG were assessed by silver protein stain (Thermo Fisher Scientific, USA) and ELISA. Purified antibodies were stored at \u0026minus;\u0026thinsp;20\u0026deg;C until further use.\u003c/p\u003e\u003cp\u003eIgG binding to \u003cem\u003eS. pyogenes\u003c/em\u003e\u003c/p\u003e\u003cp\u003eFor the binding assay, M1 and M75 \u003cem\u003eS. pyogenes\u003c/em\u003e strains (0.5 O.D.) were incubated overnight in PBS containing 0.2% skim milk PBS with 100 \u0026micro;g of purified serum IgG collected from CHIVAS participants at pre-challenge and 6 months post-challenge. Following incubation, bacterial cells were washed and stained with anti-human IgG Fab antibody. Cells were fixed with 1% PFA and analyzed on a LSRFortessa (BD Biosciences) and FlowJo\u0026trade; v11 Software was used for analysis.\u003c/p\u003e\u003cp\u003ePre-opsonization of \u003cem\u003eS. pyogenes\u003c/em\u003e with IgG and intranasal challenge in mice\u003c/p\u003e\u003cp\u003eM75 \u003cem\u003eS. pyogenes\u003c/em\u003e was incubated with purified serum IgG collected from asymptomatic CHIVAS participants at pre-challenge and 6 months post-challenge. As controls, IgG was purified from Sprague Dawley rats either immunised with p*17-DT-K4S2-CRM/Alum or left unimmunised (na\u0026iuml;ve). Following incubation, the bacterial cells were pre-incubated with a 10 \u0026micro;L inoculum containing 5 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e CFU/mouse delivered intranasally to anaesthetized naive SCID mice. The protocol was a modification of a previous assay \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Throat swabs were collected using flocked swabs (Copan Diagnostics, USA), which were then suspended in PBS, serially diluted, and plated in duplicate on CBA 5% plates. To assess nasal shedding, each mouse had its nares gently pressed onto a CBA 5% plates ten times (five times per half plate), allowing expelled particles to be streaked across the surface. On day 2 post-infection, mice were euthanized using CO₂ asphyxiation. The nasal-associated lymphoid tissue (NALT), a murine analogue to human tonsils, and the lungs were collected, homogenized in PBS using a Bullet Blender Homogenizer (Next Advance, USA), and serially diluted before being plated in duplicate onto CBA 5% plates. Mice were observed daily for clinical signs of illness according to a monitoring sheet approved by the GU-AEC.\u003c/p\u003e\u003cp\u003eIgG Fc-glycopeptide profiling by LC-ESI MS/MS\u003c/p\u003e\u003cp\u003e Purified pre- and post-challenge global and M75 antigen-specific IgG from pharyngitis (n\u0026thinsp;=\u0026thinsp;5) and non-pharyngitis (asymptomatic) CHIVAS participants (n\u0026thinsp;=\u0026thinsp;5) were dried and redissolved in 25 mM ammonium bicarbonate buffer prior to trypsin digestion (enzyme-to-substrate ratio of 1:25) at 37\u0026deg;C. The resulting glycopeptides were analysed using an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific), coupled to an UltiMate 3000 UHPLC fitted with a trap column (PepMap Neo C18, 5 mm x 300 \u0026micro;m, 5 \u0026micro;m particle size) and analytical reversed-phase C18 PepMap Neo (75 \u0026micro;m \u0026times; 500 mm, 2 \u0026micro;m particle size) UHPLC column (Thermo Scientific) and PicoTip nanospray interface (New Objective). The samples were injected onto the trap column in 100% Loading Buffer (0.1% trichloroacetic acid) at the flow rate of 15 \u0026micro;L/min and the (glyco)peptides were separated at a flow rate of 300 nL/min using linear gradient from 2% solvent A (0.1% formic acid) to 95% solvent B (80% acetonitrile containing 0.1% formic acid) as follows: 1% solvent B until 8 min, 1%-8% from 8 to 9 min, 8\u0026ndash;15% from 9 to 20 min, 15\u0026ndash;95% from 20 to 23 min, 95% from 23 to 25 min, before the column was re-equilibrated in 1% solvent B from 28 until 40 min. For MS acquisition, a full MS1 scan was performed at 60,000 resolution between m/z 350\u0026ndash;1650 (custom AGC target at 250%, 50 ms maximum injection time, 1 microscan). Precursor ions were isolated using an isolation window of 1.6 Da and MS2 fragmentation was performed using normalized stepped-HCD collision energy (15, 30, 45%) at 60,000 resolution. All quantitative data processing was performed using Skyline (version 24.1) based on a pre-defined list of IgG1, IgG2, and IgG3/4 glycopeptide masses corresponding to doubly [M\u0026thinsp;+\u0026thinsp;2H]2\u0026thinsp;+\u0026thinsp;and triply [M\u0026thinsp;+\u0026thinsp;3H]3\u0026thinsp;+\u0026thinsp;charged precursor masses (Supp. Table\u0026nbsp;1). MS1 peak-area-under-the curve quantification was performed and the summed areas of integrated peaks (including monoisotopic transitions) for each glycopeptide precursor mass were represented as a percentage (relative intensity) of the total IgG glycopeptides.\u003c/p\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analysis was performed with GraphPad Prism 9 software using a nonparametric, unpaired Mann\u0026ndash;Whitney U test (one-tailed; 90% confidence interval) to compare test groups, unpaired t test with Welch\u0026rsquo;s correction to compare test groups. ARRIVE guidelines \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e were used to calculate sample size for \u003cem\u003ein vivo\u003c/em\u003e experiments. A sample size of 8 was shown to provide a power of 0.8 (G*Power) and therefore used for animal experiments involving bacterial challenge.\u003c/p\u003e\u003cp\u003eFor ELISA analysis, absorbance values at a 1:20 serum dilution were used to compare antigen-specific responses across the three time points. Statistical analysis was conducted using one-way analysis of variance (ANOVA) with Geisser-Greenhouse correction to account for repeated measures. The same approach was applied to evaluate changes in antigen-specific responses over time in both the ELISA and antigen tetramer assays. Correlation coefficients and correlation p values were determined using Spearman\u0026rsquo;s method (two-sided) with a linear regression line included to indicate linear trends.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eDeclaration of interests\u003c/h2\u003e\u003cp\u003eA.C.S. is co-chair of the Australian Strep A Vaccine Initiative (ASAVI) and the Strep A Vaccine Global Consortium (SAVAC). M.F.G. and M.P. are inventors on patents related to \u003cem\u003eS. pyogenes\u003c/em\u003e vaccines.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e\u003cp\u003eConceptualization: A.L., M.F.G., M.P., D.K., A.C.S. and J.O. Methodology and experiments: A.L., D.V., V.O., M.A., D.B., A.C, H.R.F. and K.I.A. Data analysis: A.L., D.V., D.B. M.A., Patient cohorts: A.C.S. and J.O. Original draft: A.L., M.F.G. and M.P. Manuscript review: A.L., D.V., V.O., K.I.A., J.O., M.P., D.K. and M.F.G. Funding acquisition: A.C.S., J.O., A.L., D.K., M.P. and M.F.G.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe author thanks the support of the Flow Cytometry and Australian Cancer Research Foundation (ACRF) International Centre for Cancer Glycomics Mass Spectrometry Facility at the Institute for Biomedicine and Glycomics, Griffith University. A.L and A.C. are supported by an NHMRC project grant (APP1160379) awarded to M.P. V.O is supported by Griffith University Postgraduate Fellowship. M.A. is supported by an NHMRC ideas grant (GNT2018947) awarded to D.K. M.F.G. is supported by an NHMRC Investigator Fellowship (L3, GNT1174091). D.B. is supported by Queensland Advance WRAP grant awarded to A.L. The work described in this article was funded by The Heart Foundation of Australia (101656). The CHIVAS-M75 study was funded by the Australian National Health and Medical Research Council (1099183). J.O. is supported Australian National Health and Medical Research Council Investigator Fellowships and by a Melbourne Children\u0026rsquo;s Campus Clinician\u0026ndash;Scientist fellowship. H.R.F. and K.I.A. are Human Infection Challenge Network for Vaccine Development members.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e\u003cp\u003eThe IgG glycopeptide MS-raw data files have been made available via GlycoPost \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. For the reviewing process, the data is just available with the following information:\u003c/p\u003e\u003cp\u003ePreview Page link:: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://glycopost.glycosmos.org/preview/69820049068637b110aa34\u003c/span\u003e\u003cspan address=\"https://glycopost.glycosmos.org/preview/69820049068637b110aa34\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003cp\u003eID: GPST000603, PIN CODE: 7330.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCarapetis, J. R., Steer, A. C., Mulholland, E. K. \u0026amp; Weber, M. The global burden of group A streptococcal diseases. \u003cem\u003eLancet Infect Dis\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 685-694, doi:10.1016/S1473-3099(05)70267-X (2005).\u003c/li\u003e\n\u003cli\u003eRalph, A. P. \u0026amp; Carapetis, J. R. 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F., Ishihama, Y. \u0026amp; Okuda, S. GlycoPOST realizes FAIR principles for glycomics mass spectrometry data. \u003cem\u003eNucleic Acids Res\u003c/em\u003e \u003cstrong\u003e49\u003c/strong\u003e, D1523-D1528, doi:10.1093/nar/gkaa1012 (2021).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Supplementary Table","content":"\u003cp\u003eSupplementary Table 1 not available with this version.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"S. pyogenes, natural immunity, CHIVAS, human pharyngeal challenge, antigen-specific B cell responses, IgG Fc glycosylation","lastPublishedDoi":"10.21203/rs.3.rs-7145918/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7145918/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAge-related decline in \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e infection rates suggests that immunity develops progressively through repeated exposure during early life. However, the intensity or duration of exposure required is unknown, as to why some individuals appear to develop immunity, despite having few or no previously detected infections. Here, drawing on samples from a human challenge model of pharyngeal \u003cem\u003eS. pyogenes \u003c/em\u003einfection, we investigate whether symptomatic disease is required for induction of humoral and cellular immunity. Challenge with M75 \u003cem\u003eS. pyogenes\u003c/em\u003e induced M75-specific serum IgG and IgA antibodies and memory B cell in both symptomatic and asymptomatic participants, with responses persisting for at least 6 months. Purified IgG from asymptomatic participants exhibited significantly enhanced binding to M75 \u003cem\u003eS. pyogenes \u003c/em\u003eand were bactericidal when transferred into a murine model of pharyngeal infection. M75-specific IgG from these participants had an altered Fc glycosylation signature, indicative of enhanced effector function and ability to limit inflammation. However, \u003cem\u003eS. pyogenes \u003c/em\u003echallenge had no impact on cellular or humoral immune responses to a conserved cryptic epitope, p*17. These findings show that asymptomatic (or subclinical) exposure to M75 \u003cem\u003eS. pyogenes \u003c/em\u003egenerates functional immune responses and contributes to the streptococcal immunity that emerges by adulthood.\u003c/p\u003e","manuscriptTitle":"Subclinical exposure to Streptococcus pyogenes drives the development of long-lived immunity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-28 08:30:42","doi":"10.21203/rs.3.rs-7145918/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d2300541-eeba-496c-9d41-1bae5c3c1413","owner":[],"postedDate":"July 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":52050632,"name":"Biological sciences/Immunology/Infectious diseases/Bacterial infection"},{"id":52050633,"name":"Biological sciences/Microbiology/Bacteriology"}],"tags":[],"updatedAt":"2026-04-21T17:01:40+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-28 08:30:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7145918","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7145918","identity":"rs-7145918","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}