Targeting Group A Streptococcus with a Recombinant Chimeric Vaccine: Integrating ScpA and SpeA Fragments

Our objective was to develop a vaccine against group A streptococci (Streptococcus pyogenes, GAS), the causative agent of a broad spectrum of infections with varying severity. The expression vectors pET27 and pQE30 were used to clone genes encoding recombinant proteins, which were subsequently affinity-purified. Female mice were immunized subcutaneously twice with the purified polypeptides (20 μg/mouse) formulated with Alum adjuvant (2:1) at three-week intervals. Immune sera were analyzed using ELISA to evaluate antigen-specific responses. Three weeks after the final immunization, mice were challenged intraperitoneally with GAS M1 serotype at a dose of 5 × 10⁷ CFU/mouse. Vaccination efficacy was determined by comparing bacterial clearance in vaccinated versus control animals, assessed by bacterial loads in the spleen at 3 and 15 hours post-infection. The vaccine candidate is a hybrid recombinant protein comprising fragments from two essential GAS virulence factors: C5a peptidase (ScpA) and SpeA exotoxin. T and B cell epitopes from conserved regions, common across multiple GAS serotypes, were identified and included into the construct using bioinformatics tools. The integration of these epitopes is designed to confer broad-spectrum protection against GAS strains carrying exotoxin, particularly those linked to invasive infections. Immunogenicity studies in mice revealed a robust humoral immune response targeting both components of the hybrid protein. Further evaluation of protective efficacy demonstrated accelerated bacterial clearance in vaccinated animals, with the SpeA fragment playing a significant protective role. These findings emphasize the potential of this recombinant chimeric vaccine as a promising candidate for the prevention of group A streptococcal infections, addressing the critical need for effective prophylactic strategies against GAS-associated diseases.

1. Aranha M.P., Penfound T.A., Salehi S., Botteaux A., Smeesters P., Dale J.B., Smith J.C. Design of Broadly Cross-Reactive M Protein-Based Group A Streptococcal Vaccines. J. Immunol., 2021, Vol. 207, no. 4, pp. 1138-1149.

2. Azuar A., Jin W., Mukaida S., Hussein W.M., Toth I., Skwarczynski M. Recent Advances in the Development of Peptide Vaccines and Their Delivery Systems Against Group A Streptococcus. Vaccines, 2019, Vol. 7, no. 3, 58. doi: 10.3390/vaccines7030058.

3. Baker M., Gutman D.M., Papageorgiou A.C., Collins C.M., Acharya K.R. Structural features of a zinc binding site in the superantigen streptococcal pyrogenic exotoxin A (SpeA1): implications for MHC class II recognition. Protein Sci., 2001, Vol. 10, no. 6, pp. 1268-1273.

4. Bi S., Xu M., Zhou Y., Xing X., Shen A., Wang B. A Multicomponent Vaccine Provides Immunity against Local and Systemic Infections by Group A Streptococcus across Serotypes. mBio, 2019, Vol. 10, no. 6, e02600-19. doi: 10.1128/mBio.02600-19.

5. BLAST: Basic Local Alignment Search Tool. National Center for Biotechnology Information. Available at: blast.ncbi.nlm.nih.gov/.

6. Chauhan S., Khasa Y.P. Challenges and Opportunities in the Process Development of Chimeric Vaccines. Vaccines, 202, Vol. 11, no. 12, 1828. doi: 10.3390/vaccines11121828.

7. Cheng Q., Carlson B., Pillai S., Eby R., Edwards L., Olmsted S.B., Cleary P. Antibody against surface-bound C5a peptidase is opsonic and initiates macrophage killing of group B streptococci. Infect. Immun., 2001, Vol. 69, no. 4, pp. 2302-2308.

8. Cornaglia G., Ligozzi M., Mazzariol A., Valentini M., Orefici G., Fontana R. Rapid increase of resistance to erythromycin and clindamycin in Streptococcus pyogenes in Italy, 1993–1995. The Italian surveillance group for antimicrobial resistance. Emerg. Infect. Dis., 1996, Vol. 2, pp. 339-342.

9. Desjardins M., Delgaty K.L., Ramotar K., Seetaram C., Toye B. Prevalence and mechanisms of erythromycin resistance in Group A and Group B Streptococcus: Implications for reporting susceptibility results. J. Clin. Microbiol., 2004, Vol. 42, pp. 5620-5623.

10. Fulurija A., Cunningham M.W., Korotkova N., Masterson M.Y., Bansal G.P., Baker M.G., Cannon J.W., Carapetis J.R., Steer A.C. Research opportunities for the primordial prevention of rheumatic fever and rheumatic heart disease-streptococcal vaccine development: a national heart, lung and blood institute workshop report. BMJ Glob. Health, 2023, Vol. 8, Suppl. 9, e013534. doi: 10.1136/bmjgh-2023-013534.

11. Gao N.J., Uchiyama S., Pill L., Dahesh S., Olson J., Bautista L., Maroju S., Berges A., Liu J.Z., Zurich R.H., et al. Site-Specific Conjugation of Cell Wall Polyrhamnose to Protein SpyAD Envisioning a Safe Universal Group A Streptococcal Vaccine. Infect. Microbes Dis., 2010, Vol. 3, pp. 87-100.

12. Gupalova T., Leontieva G., Kramskaya T., Grabovskaya K., Bormotova E., Korjevski D., Suvorov A. Development of experimental GBS vaccine for mucosal immunization. PLoS One, 2018, Vol. 13, no. 5, e0196564. doi: 10.1371/journal.pone.0196564.

13. Kaul R., McGeer A., Norrby-Teglund A., Kotb M., Schwartz B., O’Rourke K, Talbot J., Low D.E.. Intravenous immunoglobulin therapy for streptococcal toxic shock syndrome – a comparative observational study. The Canadian Streptococcal Study Group. Clin. Infect. Dis., 1999, Vol. 28, no. 4, pp. 800-807.

14. Kuo C.F., Tsao N., Hsieh I.C., Lin Y.S., Wu J.J., Hung Y.T. Immunization with a streptococcal multiple-epitope recombinant protein protects mice against invasive group A streptococcal infection. PLoS One, 2017, Vol. 12, no. 3, e0174464. doi: 10.1371/journal.pone.0174464.

15. Lapthorne S., McWade R., Scanlon N., Ní Bhaoill S., Page A., O’Donnell C., Dornikova G., Hannan M., Lynch B., Lynch M., Brady D. Rising clindamycin resistance in group A Streptococcus in an Irish healthcare institution. Access Microbiol., 2024, Vol. 6, no. 6, 000772.v4. doi: 10.1099/acmi.0.000772.v4.

16. Linnér A., Darenberg J., Sjölin J., Henriques-Normark B., Norrby-Teglund A. Clinical efficacy of polyspecific intravenous immunoglobulin therapy in patients with streptococcal toxic shock syndrome: A comparative observational study. Clin. Infect. Dis., 2014. Vol. 59, pp. 851-857.

17. Lynskey N.N., Reglinski M., Calay D., Siggins M.K., Mason J.C., Botto M., Sriskandan S. Multi-functional mechanisms of immune evasion by the streptococcal complement inhibitor C5a peptidase. PLoS Pathog., 2017, Vol. 13, no. 8, e1006493. doi: 10.1371/journal.ppat.1006493.

18. Martín-Delgado M.C., De Lucas Ramos P., García-Botella A., Cantón R., García-Lledó A., HernándezSampelayo T., Gómez-Pavón J., González Del Castillo J., Martín Sánchez F.J., Martínez-Sellés M., Molero García J.M., Moreno Guillén S., Rodríguez-Artalejo F.J., Ruiz-Galiana J., Burillo A., Muñoz P., Calvo Rey C., Catalán-González M., Cendejas-Bueno E., Halperin-Benito V, Recio R., Viñuela-Benítez C., Bouza E. Invasive group A Streptococcus infection (Streptococcus pyogenes): Current situation in Spain. Rev. Esp. Quimioter., 2024, martin30jul2024. doi: 10.37201/req/067.2024.

19. Mascini E.M., Jansze M., Schellekens J.F.P., Musser J.M., Faber J.A.J., Verhoef-Verhage L.A.E., Schouls L., van Leeuwen W.J., Verhoef J., van Dijk H. Invasive group A streptococcal disease in the Netherlands: Evidence for a protective role of anti-exotoxin A antibodies. J. Infect. Dis., 2000, Vol. 181, pp. 631-638.

20. McCabe S., Bjånes E., Hendriks A., Wang Z., van Sorge N.M., Pill-Pepe L., Bautista L., Chu E., Codée J.D.C., Fairman J., Kapoor N., Uchiyama S., Nizet V. The Group A Streptococcal Vaccine Candidate VAX-A1 Protects against Group B Streptococcus Infection via Cross-Reactive IgG Targeting Virulence Factor C5a Peptidase. Vaccines, 2023, Vol. 11, no. 12, 1811. doi: 10.3390/vaccines11121811.

21. McKenna S., Huse K.K., Giblin S., Pearson M., Majid A., Shibar M.S., Sriskandan S., Matthews S., Pease J.E. The Role of Streptococcal Cell-Envelope Proteases in Bacterial Evasion of the Innate Immune System. J. Innate Immun., 2022, Vol. 14, no. 2, pp. 69-88.

22. Monteiro R.C., van De Winkel J.G. IgA Fc receptors. Annu. Rev. Immunol., 2003, Vol. 21, pp. 177-204.

23. Moreira M., Ferreira P.R., Sarmento A., Cardoso A.L. Bacterial Tracheitis: A New Presentation of a WellKnown Disease. Cureus, 2024, Vol. 16, no. 7, e63697. doi: 10.7759/cureus.63697.

24. Müller-Alouf H., Geoffroy C., Geslin P., Bouvet A., Felten A., Günther E., Ozegowski J.H., Alouf J.E. Streptococcal pyrogenic exotoxin A, streptolysin O, exoenzymes, serotype and biotype profiles of Streptococcus pyogenes isolates from patients with toxic shock syndrome and other severe infections. Zentralbl. Bakteriol., 1997, Vol. 286, pp. 421-433.

25. Nasr-Eldahan S., Attia Shreadah M., Maher A.M., El-Sayed Ali T., Nabil-Adam A. New vaccination approach using formalin-killed Streptococcus pyogenes vaccine on the liver of Oreochromis niloticus fingerlings. Sci. Rep., 2024, Vol. 14, no. 1, 18341. doi: 10.1038/s41598-024-67198-0.

26. NCBI Home. U.S. National Library of Medicine, National Center for Biotechnology Information. Available at: https://www.ncbi.nlm.nih.gov/.

27. Nixon J., Hennessy J., Baird R.W. Tracking trends in the Top End: clindamycin and erythromycin resistance in Group A Streptococcus in the Northern Territory, 2012-2023. Commun. Dis. Intell. (2018), 2024, 48. doi: 10.33321/cdi.2024.48.31.

28. Oliver-Gutierrez D., van der Veen R.L.P., Ros-Sánchez E., Segura-Duch G., Alonso T., Herranz-Cabarcos A., Matas J., Castro Seco .R, Arcediano M.Á., Zapata M.Á., Oliveres J. Periorbital necrotizing fasciitis: clinical perspectives on nine cases. Eur. J. Clin. Microbiol. Infect. Dis., 2024, Vol. 43, no. 10, pp. 2053-2059.

29. Panchaud A., Guy L., Collyn F., Haenni M., Nakata M., Podbielski A., Moreillon P., Roten C.A. M-protein and other intrinsic virulence factors of Streptococcus pyogenes are encoded on an ancient pathogenicity island. BMC Genomics, 2009, Vol. 10, 198. doi: 10.1186/1471-2164-10-198.

30. Pastural É., McNeil S.A., MacKinnon-Cameron D., Ye L., Langley J.M., Stewart R., Martin L.H., Hurley G.J., Salehi S., Penfound T.A., Halperin S., Dale J.B. Safety and immunogenicity of a 30-valent M protein-based group a streptococcal vaccine in healthy adult volunteers: A randomized, controlled phase I study. Vaccine, 2020, Vol. 38, no. 6, pp. 1384-1392.

31. Pietrocola G., Arciola C.R., Rindi S., Montanaro L., Speziale P. Streptococcus agalactiae Non-Pilus, Cell Wall-Anchored Proteins: Involvement in Colonization and Pathogenesis and Potential as Vaccine Candidates. Front. Immunol., 2018, Vol. 9, 602. doi: 10.3389/fimmu.2018.00602.

32. Rampersadh K., Engel K.C., Engel M.E., Moodley C. A survey of antibiotic resistance patterns among Group A Streptococcus isolated from invasive and non-invasive infections in Cape Town, South Africa. Heliyon, 2024, Vol. 10, no. 13, e33694. doi: 10.1016/j.heliyon.2024.e33694.

33. Reglinski M., Lynskey N.N., Choi Y.J., Edwards R.J., Sriskandan S. Development of a multicomponent vaccine for Streptococcus pyogenes based on the antigenic targets of IVIG. J. Infect., 2016, Vol. 72, pp. 450-459.

34. Rivera-Hernandez T., Rhyme M.S., Cork A.J., Jones S., Segui-Perez C., Brunner L., Richter J., Petrovsky N., Lawrenz M., Goldblatt D., Collin N., Walker M.J. Vaccine-Induced Th1-Type Response Protects against Invasive Group A Streptococcus Infection in the Absence of Opsonizing Antibodies. mBio, 2020, Vol. 11, no. 2, e00122-20. doi: 10.1128/mBio.00122-20.

35. Rivera-Hernandez T., Pandey M., Henningham A., Cole J., Choudhury B., Cork A.J., Gillen C.M., Ghaffar K.A., West N.P., Silvestri G. Differing Efficacies of Lead Group A Streptococcal Vaccine Candidates and Full-Length M Protein in Cutaneous and Invasive Disease Models. mBio, 2016, Vol. 7, e00618-16. doi: 10.1128/mBio.00618-16.

36. Schalk E., Genseke S., Zautner A.E., Kaasch A.J. Detection of Streptococcus pyogenes in an atypical hematological diagnostic case. Infection, 2024. doi: 10.1007/s15010-024-02335-5.

37. Smeesters P.R., de Crombrugghe G., Tsoi S.K., Leclercq C., Baker C., Osowicki J., Verhoeven C., Botteaux A., Steer A.C. Global Streptococcus pyogenes strain diversity, disease associations, and implications for vaccine development: a systematic review. Lancet Microbe, 2024, Vol. 5, no. 2, pp. e181-e193.

38. Spaulding A.R., Salgado-Pabón W., Kohler P.L., Horswill A.R., Leung D.Y., Schlievert P.M. Staphylococcal and streptococcal superantigen exotoxins. Clin. Microbiol. Rev., 2013, Vol. 26, no. 3, pp. 422-447.

39. Stafslien D.K., Cleary P.P. Characterization of the Streptococcal C5a Peptidase Using a C5a-Green Fluorescent Protein Fusion Protein Substrate. J. Bacteriol., 2000, Vol. 182, no. 11, pp. 3254-3258.

40. Thomas S., Abraham A. Progress in the Development of Structure-Based Vaccines. Methods Mol. Biol., 2022, Vol. 2412, pp. 15-33.

41. Troese M.J., Burlet E., Cunningham M.W., Alvarez K., Bentley R., Thomas N., Carwell S., Morefield G.L. Group A Streptococcus Vaccine Targeting the Erythrogenic Toxins SpeA and SpeB Is Safe and Immunogenic in Rabbits and Does Not Induce Antibodies Associated with Autoimmunity. Vaccines, 2023, Vol. 11, no. 9, 1504. doi: 10.3390/vaccines1109150.

42. Ulrich R.G. Vaccine based on a ubiquitous cysteinyl protease and streptococcal pyrogenic exotoxin A protects against Streptococcus pyogenes sepsis and toxic shock. J. Immune Based Ther. Vaccines, 2008, Vol. 6, 8. doi: 10.1186/1476-8518-6-8.

43. Walkinshaw D.R., Wright M.E.E., Mullin A.E., Excler J.L., Kim J.H., Steer A.C. The Streptococcus pyogenes vaccine landscape. NPJ Vaccines, 2023, Vol. 8, no. 1, 16. doi: 10.1038/s41541-023-00609-x.

44. Wang J., Ma C., Li M., Gao X., Wu H., Dong W., Wei L. Streptococcus pyogenes: Pathogenesis and the Current Status of Vaccines. Vaccines, 2023, Vol. 11, no. 9, 1510. doi: 10.3390/vaccines11091510.

45. World Health Organization. The current evidence for the burden of group a streptococcal diseases. Geneva, Switzerland, 2005, pp. 1-52.

46. Zachariadou L., Papaparaskevas J., Paraskakis I., Efstratiou A., Pangalis A., Legakis N.J., Tassios P.T. Predominance of two M-types among erythromycin-resistant Group A Streptococci from Greek children. Clin. Microbiol. Infect., 2003, Vol. 9, pp. 310-314.

47. Zeppa J.J., Kasper K.J., Mohorovic I., Mazzuca D.M., Haeryfar S.M.M., McCormick J.K. Nasopharyngeal infection by Streptococcus pyogenes requires superantigen-responsive Vβ-specific T cells. Proc. Natl. Acad. Sci. USA, 2017, Vol. 114, no. 38, pp. 10226-10231.