GMMA as an Alternative Carrier for a Glycoconjugate Vaccine against Group A Streptococcus

Abstract

Group A Streptococcus (GAS) causes about 500,000 annual deaths globally, and no vaccines are currently available. The Group A Carbohydrate (GAC), conserved across all GAS serotypes, conjugated to an appropriate carrier protein, represents a promising vaccine candidate. Here, we explored the possibility to use Generalized Modules for Membrane Antigens (GMMA) as an alternative carrier system for GAC, exploiting their intrinsic adjuvant properties. Immunogenicity of GAC-GMMA conjugate was evaluated in different animal species in comparison to GAC-CRM₁₉₇; and the two conjugates were also compared from a techno-economic point of view. GMMA proved to be a good alternative carrier for GAC, resulting in a higher immune response compared to CRM₁₉₇ in different mice strains, as verified by ELISA and FACS analyses. Differently from CRM₁₉₇, GMMA induced significant levels of anti-GAC IgG titers in mice also in the absence of Alhydrogel. In rabbits, a difference in the immune response could not be appreciated; however, antibodies from GAC-GMMA-immunized animals showed higher affinity toward purified GAC antigen compared to those elicited by GAC-CRM₁₉₇. In addition, the GAC-GMMA production process proved to be more cost-effective, making this conjugate particularly attractive for low- and middle-income countries, where this pathogen has a huge burden.

Keywords: GMMA; Group A Carbohydrate; Group A Streptococcus; glycoconjugate.

Figure 1

CRM₁₉₇ and GMMA as carriers for GAC PS. (A) Reaction schemes: oxidized GAC was linked to lysines of CRM₁₉₇ by reductive amination. (D) ADH-derivatized GAC was conjugated to oxidized LPS core sugars of GMMA via reductive amination chemistry. (B,E) Conjugates were characterized by HPLC-SEC analysis in comparison to the corresponding unconjugated carriers. (C) GAC-CRM₁₉₇ and CRM₁₉₇ were compared by SDS-PAGE, (F) with GAC-GMMA and GMMA by DLS.

Figure 2

Comparative immunogenicity study of GAC-CRM₁₉₇ and GAC-GMMA conjugates in mice. One experiment was performed: eight CD1 mice per group were immunized IP at days 0 and 28 with 1.5 μg GAC in the presence of 2 mg/mL of Alhydrogel (Al³⁺). Sera were collected at days −1 (PI, pre-immune), 27 and 42. (A) Summary graphs of anti-GAC IgG geometric mean units (bars) and individual antibody levels (dots) are reported. Mann–Whitney two-tailed test was used to compare the immune response elicited by the two conjugates that resulted as significantly different (p < 0.05). (B) FACS analysis against GAS strain GAS51∆M1 performed on Day 42 pooled sera. Grey-shaded histogram represents the pre-immune sera used as negative control.

Figure 3

Comparative immunogenicity study of GAC-CRM₁₉₇ and GAC-GMMA conjugates in rabbits. One experiment was performed: six New Zealand White female rabbits per group were immunized IM at days 0, 21 and 3 with 5 μg GAC in the presence of 0.7 mg/mL of Alhydrogel (Al³⁺). Sera were collected at days −1 (PI, pre-immune), 20, 34 and final bleed on day 49. (A) Summary graphs of anti-GAC IgG geometric mean units (bars) and individual antibody levels (dots) are reported. Mann–Whitney two-tailed test was used to compare the immune response elicited by the two conjugates that resulted as nonsignificantly different (p > 0.05). (B) FACS analysis GAS strain GAS51∆M1 performed on Day 49 pooled sera. Grey-shaded histogram represents the pre-immune sera used as negative control. (C) 3D view of antibody–antigen capture profiles (Gyrolab Viewer) showing the distribution of the antibodies in the column for the two conjugates.

Figure 4

Comparative immunogenicity study of GAC-CRM₁₉₇ and GAC-GMMA conjugates in mice with and without Alhydrogel. One experiment was performed: eight BalbC mice per group were immunized SC at days 0 and 22 with 2 μg GAC (with or without Alhydrogel, 0.4 mg/mL Al³⁺), and sera were collected on day 36. (A) Summary graph of anti-GAC IgG geometric mean titer (bars) and individual antibody levels (dots) are reported. Mann–Whitney two-tailed test was used to compare the immune response elicited by two different antigens, and p values for the comparison between unadjuvanted conjugates and GAC alone are highlighted (significant difference for p < 0.05). (B) FACS analysis against GAS strain NCTC 8198 performed on day 36 pooled sera. Grey-shaded histogram represents the sera from Alhydrogel group used as negative control.

Figure 5

Techno-economic comparison of GMMA and CRM₁₉₇ as a carrier for conjugate vaccine production. Relative scales of the GMMA production, CRM₁₉₇ production, GAC-GMMA conjugation and GAC-CRM₁₉₇ conjugation processes required to produce 500 million conjugate vaccine doses per year (A). The scale was expressed in fermenter working volume for GMMA and CRM₁₉₇ production and in conjugation with reactor working volume for GAC-GMMA and GAC-CRM₁₉₇ conjugation. In all cases, all unit operations were scaled proportionally to the fermenters and conjugation reactors. Relative productivity of the GMMA production, CRM₁₉₇ production, GAC-GMMA conjugation and GAC-CRM₁₉₇ conjugation processes (B). Productivity was expressed in doses produced per L of reactor scale per year. The relative drug substance production cost per dose, normalized to the amount of GAC used per dose of vaccine (C).