Abstract
In a companion paper, a two-step developability assessment is presented to rapidly evaluate low-cost formulations (multi-dose, aluminum-adjuvanted) for new subunit vaccine candidates. As a case study, a non-replicating rotavirus (NRRV) recombinant protein antigen P[4] was found to be destabilized by the vaccine preservative thimerosal, and this effect was mitigated by modification of the free cysteine (C173S). In this work, the mechanism(s) of thimerosal-P[4] protein interactions, along with subsequent effects on the P[4] protein’s structural integrity, are determined. Reversible complexation of ethylmercury, a thimerosal degradation byproduct, with the single cysteine residue of P[4] protein is demonstrated by intact protein mass analysis and biophysical studies. A working mechanism involving a reversible S-Hg coordinate bond is presented based on the literature. This reaction increased the local backbone flexibility of P[4] within the helical region surrounding the cysteine residue and then caused more global destabilization, both as detected by HX-MS. These effects correlate with changes in antibody-P[4] binding parameters and alterations in P[4] conformational stability due to C173S modification. Epitope mapping by HX-MS demonstrated involvement of the same cysteine-containing helical region of P[4] in antibody-antigen binding. Future formulation challenges to develop low-cost, multi-dose formulations for new recombinant protein vaccine candidates are discussed.
Highlights
IntroductionTo reduce vaccine cost and expand immunization coverage, multi-dose formulations are an important component of global immunization programs. Multi-dose vaccine formulations offer several cost advantages over single-dose formats including (1)e Current addresses: Amgen, Thousand Oaks, California 91320. f Current addresses: Merck & Co., West Point, Pennsylvania 19486. g These authors contributed to this work.reduced packaging costs since multiple doses are supplied in a single vial, (2) decreased distribution costs since less cold-chain space is required for storage and transportation, and (3) lower disposal costs for medical waste. These benefits lead to their preferred use in vaccines targeted for use in low and middleincome countries (LMICs), those procured and distributed by UNICEF. Multi-dose vaccine vials play a key role in the success of LMIC’s immunization programs, especially for widely-distributed vaccines used in locations with more limited cold-chain storage space.3creativecommons.org/licenses/by/4.0/).K
We explore in detail the molecular mechanism(s) by which TH interacts with variants of the non-replicating rotavirus (NRRV) P[4] antigen, using material produced in E. coli and K. phaffii, including the effects of TH on the protein antigen’s structural integrity and conformational stability, antibody-binding properties, and local backbone flexibility
A total of three different NRRV P[4] fusion-protein antigens were used in these studies including E. coli expressed parent protein P[4], K. phaffii (P. pastoris) expressed parent protein P[4], and K. phaffii (P. pastoris) expressed P[4] variant (C173S), which are hereafter referred as E. coli P[4], Pp P[4] and Pp P[4]-C173S, respectively
Summary
To reduce vaccine cost and expand immunization coverage, multi-dose formulations are an important component of global immunization programs. Multi-dose vaccine formulations offer several cost advantages over single-dose formats including (1)e Current addresses: Amgen, Thousand Oaks, California 91320. f Current addresses: Merck & Co., West Point, Pennsylvania 19486. g These authors contributed to this work.reduced packaging costs since multiple doses are supplied in a single vial, (2) decreased distribution costs since less cold-chain space is required for storage and transportation, and (3) lower disposal costs for medical waste. These benefits lead to their preferred use in vaccines targeted for use in low and middleincome countries (LMICs), those procured and distributed by UNICEF. Multi-dose vaccine vials play a key role in the success of LMIC’s immunization programs, especially for widely-distributed vaccines used in locations with more limited cold-chain storage space.3creativecommons.org/licenses/by/4.0/).K. Reduced packaging costs since multiple doses are supplied in a single vial, (2) decreased distribution costs since less cold-chain space is required for storage and transportation, and (3) lower disposal costs for medical waste.. Reduced packaging costs since multiple doses are supplied in a single vial, (2) decreased distribution costs since less cold-chain space is required for storage and transportation, and (3) lower disposal costs for medical waste.1 These benefits lead to their preferred use in vaccines targeted for use in low and middleincome countries (LMICs), those procured and distributed by UNICEF.. Thimerosal is currently used as a preservative in many pediatric combination vaccines (e.g., Pentabio, Eupenta, and ComBE Five) prequalified by the World Health Organization for use in LMICs. The most commonly used vaccine preservative is thimerosal, followed by phenoxyethanol and phenol. Since the 1930s, thimerosal has been used as a preservative in many biological products including vaccines. Thimerosal is an anionic, ethylmercury-containing organic compound efficient at preventing microbial contamination with a well-documented record of safe use, despite disproven, controversial associations of thimerosal with neurodevelopmental disorders in young children. Thimerosal is currently used as a preservative in many pediatric combination vaccines (e.g., Pentabio, Eupenta, and ComBE Five) prequalified by the World Health Organization for use in LMICs.
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