A controlled human infection model of Streptococcus pyogenes pharyngitis (CHIVAS-M75): an observational, dose-finding study
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Summary
Background
Streptococcus pyogenes is a leading cause of infection-related morbidity and mortality. A reinvigorated vaccine development effort calls for new clinically relevant human S pyogenes experimental infection models to support proof of concept evaluation of candidate vaccines. We describe the initial Controlled Human Infection for Vaccination Against S pyogenes (CHIVAS-M75) study, in which we aimed to identify a dose of emm75 S pyogenes that causes acute pharyngitis in at least 60% of volunteers when applied to the pharynx by swab.
Methods
Findings
Between July 10, 2018, and Sept 23, 2019, 25 healthy adults were challenged with emm75 S pyogenes and included in analyses. Pharyngitis was diagnosed in 17 (85%; 95% CI 62–97) of 20 participants at the starting dose level (1–3 × 105 colony-forming units [CFU]/mL). This high proportion prompted dose de-escalation. At the lower dose level (1–3 × 104 CFU/mL), pharyngitis was diagnosed in one of five participants. Immunological, biochemical, and microbiological results supported the clinical picture, with acute symptomatic pharyngitis characterised by pharyngeal colonisation by S pyogenes accompanied by significantly elevated C-reactive protein and inflammatory cytokines (eg, interferon-γ and interleukin-6), and modest serological responses to streptolysin O and deoxyribonuclease B. There were no severe (grade 3) or serious adverse events related to challenge.
Interpretation
We have established a reliable pharyngitis human infection model with reassuring early safety findings to accelerate development of vaccines and other interventions to control disease due to S pyogenes.
Funding
Australian National Health and Medical Research Council.
Introduction
S pyogenes causes an immense communicable and non-communicable global burden of disease. Unpredictable outbreaks and an uncontrolled endemic burden in marginalised communities belie the virtual disappearance of scarlet fever and rheumatic fever from high-income countries.
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Severe infections are seen in all age groups, but disproportionately affect young children (younger than 1 year), older people (especially those aged 65 years or older), and pregnant women.
Aboriginal and Torres Strait Islander people in Australia and Māori people in New Zealand are affected by a persistent and high burden of S pyogenes infections and post-infectious sequelae. Globally, there are more than 30 million prevalent cases of rheumatic heart disease, causing more than 300 000 deaths annually.
With invasive infections such as streptococcal toxic shock syndrome and necrotising fasciitis, at least 500 000 deaths are directly attributable to S pyogenes each year, making it one of the five leading causes of global infection-related mortality.
At the other end of the spectrum, sore throat is among the most common reasons for seeking primary health care, and widespread inappropriate empirical antibiotic treatment is driven largely by the desire to treat S pyogenes pharyngitis and prevent its complications.
Despite this burden of disease, no vaccine is available for prevention.
Evidence before this study
The potential for immunisation to reduce the global burden of Streptococcus pyogenes diseases was recognised centuries ago, when its most conspicuous clinical syndromes were described together as scarlet fever. However, S pyogenes vaccine development has been frustrated by scientific, regulatory, and commercial obstacles. A renewed global vaccine development effort has prioritised development of new experimental human infection models for vaccine evaluation. We searched PubMed for S pyogenes human infection studies published before Sept 15, 2020, with no language restrictions, using combinations of the search terms “streptococcus pyogenes”, “group A streptococcus”, “scarlet fever”, “experimental”, “human infection”, “human challenge”, “protective”, and “vaccine”. Human experiments in the 19th and early 20th century established S pyogenes as the cause of scarlet fever. Three human infection studies, including a total of 172 participants, were done in the 1970s. Each was a double-blind, placebo-controlled trial of monovalent M-protein vaccines for protection against pharyngitis in healthy adult volunteers challenged with homologous serotype S pyogenes strains applied directly to the pharynx using a swab. More than half of the 84 unvaccinated (control) participants had typical symptoms and signs of S pyogenes pharyngitis, such as sore throat, pharyngeal erythema or exudates, lymphadenopathy, and fever. Vaccine efficacy was as high as 89% (95% CI 23–98) in the first study testing a parenteral M1 vaccine.
Added value of this study
We report the results of a clinical study to establish a new controlled human infection model of S pyogenes pharyngitis, developed in accordance with modern Good Manufacturing Practice and Good Clinical Practice principles. We showed that healthy adult volunteers can be challenged safely with emm75 S pyogenes to produce a convincing streptococcal pharyngitis clinical syndrome in a high proportion of participants, supported by findings of microbiological, biochemical, and immunological investigations.
Implications of all the available evidence
WHO’s 2018 Global Resolution on Rheumatic Fever and Rheumatic Heart Disease listed S pyogenes vaccine research as a key priority for prevention and control. The 2018 WHO roadmap for S pyogenes vaccine development identified the need for human infection models to accelerate vaccine evaluation. This is the only current S pyogenes controlled human infection model, ready to be used as a platform to evaluate new vaccine candidates and therapeutics, and for studying host–pathogen interactions.
In the early 20th century, experimental vaccines were deployed in institutions and the community to prevent scarlet fever, but interest waned as outbreaks became less frequent and rheumatic fever incidence declined in Europe and the USA, and with the arrival of penicillin.
However, the burden of S pyogenes disease has persisted despite its continuing susceptibility to penicillin. Since 2016, reinvigorated global vaccine development efforts have sought to overcome scientific, regulatory, and commercial obstacles that have frustrated previous attempts.
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The 2018 WHO S pyogenes vaccine research and development roadmap called for development of clinically relevant human experimental infection models to support early evaluation of candidate vaccines.
Controlled human infection models are increasingly contributing to vaccine development.
We describe the initial Controlled Human Infection for Vaccination Against S pyogenes (CHIVAS-M75) study, with the primary objective of determining a bacterial inoculum (dose) to cause acute pharyngitis in at least 60% of healthy adult participants when applied by swab to the pharynx.
Methods
Study design and participants
Volunteers meeting eligibility criteria had a transthoracic echocardiogram to exclude subclinical rheumatic heart disease. Participants received AU$50 (average 2019 exchange rate US$0·695) per outpatient visit and $12·50 per h ($300 per day) during the inpatient admission (2019 national minimum wage was $740·80 per 38 h week). The CHIVAS-M75 study protocol and challenge strain selection and manufacture have previously been described in detail.
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This study was approved by the Alfred Hospital Human Research Ethics Committee (500/17). Written informed consent was obtained from all participants. A safety committee with an independent chair reviewed safety data and details of each pharyngitis diagnosis, meeting to approve study continuation after the first participant attended the initial 1 week outpatient follow-up visit and before dose escalation or de-escalation.
Procedures
Manufacture of single-dose vials of emm75 S pyogenes followed principles of Good Manufacturing Practice, including quality control testing by an independent laboratory of 10% of vials produced at each of five dose levels (1–3 × 104, 1–3 × 105, 1–3 × 106, 1–3 × 107, and 1–3 × 108 colony-forming units [CFU]/mL).
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The dose level refers to the concentration (CFU/mL) in a vial. The swabs absorbed approximately 0·1 mL so that the bacterial inoculum (the actual dose) applied to the pharynx was at least 1-log10 lower than the total bacteria in each vial.
on a visual analogue scale, with 0 meaning no pain, 5 meaning moderate pain, and 10 meaning worst pain possible) four times per day; recording of the duration and severity of solicited and unsolicited symptoms; daily blood cultures; and medical assessment for the diagnosis of pharyngitis every 12 h. Paracetamol for analgesia was administered on request. All participants received intramuscular benzathine penicillin G 900 mg once, and oral rifampicin 300 mg twice a day for eight doses, administered promptly upon the diagnosis of pharyngitis or on day 5 for those without pharyngitis. Contact and droplet precautions were used for CHIVAS-M75 participants, who were physically separated from participants in other studies and from each other. Outpatient visits were at 1 week, 1 month, 3 months, and 6 months after discharge with repeat throat swabs, blood, and urine collection, and echocardiography.
) were recorded and followed until resolution or stabilisation from the date of participant consent until completion of the 6-month follow-up period. Non-serious adverse events were recorded until the 1-month outpatient visit. All adverse events were graded by severity (mild, moderate, or severe) and their association with S pyogenes challenge (unrelated, or possibly, probably, definitely related).
Outcomes
Statistical analysis
Rules for dose escalation and de-escalation were based on CIs for 20 participants at a given dose level. For example, if 12 of 20 participants had pharyngitis, the 95% CI for the true attack rate would be 36–81% so that a Fisher’s exact test with 0·05 two-sided significance level would have 90% power to detect a difference between two groups of 23 participants for a vaccine with 80% efficacy.
Post-hoc sensitivity analyses considered modified pharyngitis diagnostic criteria: removing tonsil size, removing cervical lymphadenopathy, requiring diagnosis by 48 h after challenge, and requiring a peak CRP of 20 mg/L or more, or 40 mg/L or more.
Analyses are descriptive and presented as proportions, means with associated 95% CIs, and medians with IQRs. Participant data were recorded in a paper file of source documents, stored securely at the clinical trials facility, then transcribed using electronic data capture tools hosted at Murdoch Children’s Research Institute (REDCap, Vanderbilt University, Nashville, TN, USA). Data were analysed using GraphPad Prism (version 8.0).
Role of the funding source
The funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report.
Results
Table 1Participant demographic characteristics and challenge dose data
Data are n (%), mean (SD; range), or mean (SD) unless otherwise specified. CFU=colony-forming unit.
Figure 1CHIVAS-M75 study overview
75 adverse events were recorded in 24 of 25 participants, and 63 (84%) adverse events affecting 23 participants were (possibly, probably, or definitely) related to S pyogenes challenge. Of these 63 challenge-related adverse events, six (10%) were of moderate intensity and the remainder were mild. Typical symptoms of streptococcal pharyngitis accounted for 54 (86%) challenge-related adverse events (sore throat, tender lymphadenopathy, sweats, fever, chills, myalgia, arthralgia, headache, abdominal pain, nausea, vomiting, and malaise). There were no severe (grade 3, preventing usual daily activity or requiring complex treatment) or serious adverse events related to challenge and no local or systemic complications of pharyngitis. Two unrelated serious adverse events occurred in the outpatient follow-up period: Escherichia coli pyelonephritis and suspected sepsis on return from overseas travel, after a dog bite. Participants with pharyngitis responded clinically to antibiotic treatment with no residual symptoms at the 1-week visit, except for very mild sore throat in two participants that had completely resolved by the 1-month visit, and without subsequent relapse. The challenge strain was eradicated in all participants. No secondary cases of disease caused by S pyogenes were reported in study staff or other volunteers.
No clinical episodes were indicative of rheumatic fever or glomerulonephritis, and no signal of subclinical cardiac or renal injury (electrocardiography, echocardiography, urinalysis, urine albumin to creatinine ratio, and serum urea and creatinine) was seen. One participant (075) had emm77 S pyogenes isolated from a throat swab at the 6-month visit (ie, not the emm75 challenge strain). Participant 106 had an abnormal electrocardiogram at the 6-month visit with mild first-degree atrioventricular block and early repolarisation in the anterior leads, varying with heart rate, and intermittent second-degree Mobitz I (Wenckebach) atrioventricular block. Heart block is a feature of rheumatic fever but not in isolation—this participant had no other features of rheumatic fever, rheumatic heart disease, or pharyngitis, and had the typical athletic build and low resting heart rate associated with physiological Wenckebach phenomenon. One participant (017) withdrew from the study before the 1-month outpatient visit and declined further visits. Another participant (013) travelled overseas due to a family emergency and could not attend 3-month and 6-month visits, and participant 075 was unable to attend the 1-month visit.
Table 2Response of healthy adult volunteers to pharyngeal challenge with emm75 Streptococcus pyogenes
ADB=anti-deoxyribonuclease B. ASO=anti-streptolysin. CFU=colony-forming unit. CRP=C-reactive protein. qPCR=quantitative PCR. Tmax=maximum temperature. ULN=upper limit of normal.
Figure 2Systemic inflammatory response during experimental human pharyngitis
Serial measurements with mean (lines) and 95% CIs (shading) for serum C-reactive protein (the lower reporting limit for the C-reactive protein assay was <5 mg/L), and deviation from baseline for interferon-γ and interleukin-6 for 18 participants with pharyngitis and six participants without pharyngitis. Results for participant 057 are shown separately.
Figure 3Pharyngeal colonisation by emm75 Streptococcus pyogenes during experimental human pharyngitis
Semiquantitative S pyogenes throat swab culture (A) and emm75 quantitative PCR (B) results from throat swabs collected from 20 participants at the 105 CFU dose level and five participants at the 104 CFU dose level from challenge (time 0) to discharge, and at outpatient visits approximately 1 week, 1 month, and 3 months afterwards. Abx=antibiotic treatment. CFU=colony-forming units. *Pharyngitis diagnosed, with next swab timepoint 12 h after antibiotics started. †Cycle threshold values of 35 or greater were considered to be a negative reaction.
Discussion
In this dose-finding, human infection study, swabbing emm75 S pyogenes onto the pharynx induced a convincing acute pharyngitis syndrome in more than 60% of healthy adult volunteers, without challenge-related serious adverse events. We have established a new controlled human infection model of S pyogenes in its only natural host as a platform for evaluating candidate vaccines and novel therapeutics, and for studying host–pathogen interactions.
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We observed a predictable, uncomplicated pharyngitis syndrome, supported by laboratory findings. In the clinic, most of the participants who developed pharyngitis in our study would be assigned a Centor or McIsaac score of 3 or 4.
Exudative pharyngitis and high fever were relatively uncommon, probably due to very early diagnosis, immediate antibiotic treatment, and paracetamol use. As in clinical practice, CRP was elevated in participants with pharyngitis and peak concentrations varied widely.
Pharyngitis was characterised by a T-helper-1 cell inflammatory profile, matching previous in-vitro, animal, and human studies.
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there were three S pyogenes human infection studies in the 1970s using similar protocols.
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Each was a randomised trial of monovalent vaccines for prevention of experimental pharyngitis with homologous strains. Pharyngitis was diagnosed in 44 (52%) of 84 participants in the placebo groups, ranging from 42% to 74% in each study. Vaccine efficacy was as high as 89% in the first study of a parenteral M1 vaccine.
In our study, 85% of participants were diagnosed with pharyngitis at the starting dose level, 1-log10 lower than in the historical studies (using a similar protocol with different strains),
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with 4-log10 to 5-log10 fewer bacteria delivered than in non-human primate models using intranasal instillation.
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With this attack rate in the placebo group, a trial with only 15 participants per group could theoretically show protection conferred by a vaccine with 80% efficacy against pharyngitis, a target listed by WHO-preferred product characteristics for S pyogenes vaccines.
More realistically, future trials will probably follow the successful examples of cholera and typhoid trials in recruiting 25 to 35 participants per group.
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It is not entirely clear why attack rates were higher in our study than in the historical studies. The most likely explanations relate to intrinsic differences between the strains and differences in their handling from collection through to challenge.
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Second, S pyogenes pharyngitis is not easily defined. There is no objective diagnostic test or specific clinical criteria, and pharyngeal colonisation can be asymptomatic. High fever and exudative pharyngitis might occur more frequently in our model if antibiotic treatment is delayed, as in the 1970s studies. However, the binary pharyngitis outcome will remain most relevant as complications, principally rheumatic fever and heart disease, are not related to pharyngitis severity.
As reflected in our study, streptococcal serology using ASO and ADB titres is unreliable and might be attenuated by early antibiotic treatment.
The global burden of S pyogenes disease is an unmet public health challenge. We have established a clinically relevant controlled human infection model of S pyogenes pharyngitis. Our model provides new opportunities to explore S pyogenes pathogenesis and the immune response in its only natural host. Importantly, our model is positioned to make a valuable scientific and strategic contribution to vaccine development. Randomised vaccine-challenge trials could deliver the first human evidence of vaccine protection against S pyogenes disease in more than 40 years, building confidence to bridge the gap from preclinical and phase 1 to late-phase field trials.
Contributors
ACS was the chief investigator. JMFW and JDL were the study site principal investigators. JO, ACS, PRS, JRC, MFG, JBD, MB, AJP, JSM, MJW, ACC, TS, AG, MP, and CSW designed the study. KIA, LF, HRF, TR-H, MRN, ALW, AG, SJG, CB, MJW, and NJM contributed to work including challenge strain manufacture, assay development, laboratory processing and analysis, reporting echocardiograms, data interpretation, and statistical analysis. JO and ACS drafted the report. All authors critically reviewed and approved the final version. The authors vouch for the integrity and completeness of the data and analyses, and for the fidelity of the study to the protocol. The first and last authors (JO and ACS) had full access to and verified all the data in the study and were responsible for the decision to submit for publication.
Data sharing
The study protocol is provided in the appendix. Individual participant data will be made available upon requests directed to the corresponding author; after approval of a proposal, data can be shared through a secure online platform.
Declaration of interests
JBD is the inventor of technologies related to the development of S pyogenes vaccines; the University of Tennessee Research Foundation has licensed these technologies to Vaxent, of which JBD is the chief scientific officer and a member. MP and MFG are inventors on patents related to S pyogenes vaccines, and Griffith University (Gold Coast, QLD, Australia) has licensed some of these technologies to Olymvax Pharmaceuticals (China). NJM is an inventor on a patent related to S pyogenes analytical methods and compositions. MJW has a patent pending related to S pyogenes vaccines. All other authors declare no competing interests.
Acknowledgments
This study was supported by a project grant (1099183) from the Australian National Health and Medical Research Council. Abbott (East Brisbane, QLD, Australia) loaned an ID NOW molecular diagnostic instrument free of charge but had no other role in the study. JO was supported by an Australian National Health and Medical Research Council postgraduate scholarship. ACS is supported by fellowships from the Australian Heart Foundation and Viertel Charitable Foundation. LF was supported by a grant from the Belgian Kids’ Fund during her work on this study. JBD is supported by a grant from the US National Institutes of Health National Institute of Allergy and Infectious Diseases. NJM and ALW are supported by a grant from the Maurice Wilkins Centre (New Zealand). AJP is chair of the UK Department of Health and Social Care’s Joint Committee on Vaccination & Immunisation, a UK National Institute for Health Research senior investigator, and a member of the WHO Strategic Advisory Group of Experts. The views expressed in this article do not necessarily represent the views of the Department of Health and Social Care, the Joint Committee on Vaccination & Immunisation, the National Institute for Health Research, or WHO. JO, AJP, JSM, and ACS are members of the Human Infection Challenge Network, which is funded by the UK Global Challenges Research Fund Networks in Vaccines Research and Development, which was co-founded by the Medical Research Council and Biotechnology and Biological Sciences Research Council. First, and foremost, we thank the study participants. We acknowledge the support at the Murdoch Children’s Research Institute (Melbourne, VIC, Australia) and thank Carolyn Stewart, Fiona Williams, and Kate Scarff from the Melbourne Children’s Trial Centre and Phoebe Macleod, Penny Glenn, and Stephanie Drew in the Business Development & Legal Office. For strain manufacturing assistance, we thank Jim Ackland from Global BioSolutions, and Deborah Williamson, Kate Worthing, and staff at the Microbiological Diagnostic Unit Public Health Laboratory and The Peter Doherty Institute for Infection and Immunity at the University of Melbourne.
Supplementary Material
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Published: April 15, 2021
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