Effect of Aluminum Adjuvant and Preservatives on Structural Integrity and Physicochemical Stability Profiles of Three Recombinant Subunit Rotavirus Vaccine Antigens

Abstract

A nonreplicating rotavirus vaccine (NRRV) containing 3 recombinant fusion proteins adsorbed to aluminum adjuvant (Alhydrogel [AH]) is currently in clinical trials. The compatibility and stability of monovalent NRRV antigen with key components of a multidose vaccine formulation were examined using physicochemical and immunochemical methods. The extent and strength of antigen-adjuvant binding were diminished by increasing phosphate concentration, and acceptable levels were identified along with alternate buffering agents. Addition of the preservative thimerosal destabilized AH-adsorbed P2-VP8-P[8] as measured by differential scanning calorimetry. Over 3 months at 4 C, AH-adsorbed P2-VP8-P[8] was stable, whereas at 25 C and 37 C, instability was observed which was greatly accelerated by thimerosal addition. Loss of antibody binding (enzyme-linked immunosorbent assay) correlated with loss of structural integrity (differential scanning calorimetry, fluorescence spectroscopy) with concomitant nonnative disulfide bond formation (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and Asn deamidation (liquid chromatography-mass spectrometry peptide mapping). An alternative preservative (2phenoxyethanol) showed similar antigen destabilization. Due to limited availability, only key assays were performed with monovalent P2-VP8-P[4] and P2-VP8-P[6] AH-adsorbed antigens, and varying levels of preservative incompatibility were observed. In summary, monovalent AH-adsorbed NRRV antigens stored at 4 C showed good stability without preservatives; however, future formulation development efforts are required to prepare a stable, preservative-containing, multidose NRRV formulation.

Formulation and preclinical studies with a trivalent rotavirus P2-VP8 subunit vaccine

“…36,37 Several studies have been conducted to characterize the vaccine candidate as well as on going work to correlate in vitro analysis methods and in vivo guinea pig immunogenicity (manuscripts in preparation: McAdams D and White JA). 38,39 In addition to the clinical advancement of this vaccine candidate, the formulation developed is undergoing a long-term controlled stability study to support drug labeling and regulatory filing in the future.…”

Mechanism of Thimerosal-Induced Structural Destabilization of a Recombinant Rotavirus P[4] Protein Antigen Formulated as a Multi-Dose Vaccine

“…NRRV P [4] specific mAb was developed by PATH and obtained from Precision Antibody (Columbia, MD) as described elsewhere. 22,33…”

“…Experimental details of various physicochemical and immunochemical methods used in this study with the P [4] protein antigens, including UV-visible spectroscopy, SDS-PAGE, intact protein MS spectrometry, differential scanning calorimetry (DSC), extrinsic fluorescence spectroscopy, and inhibition ELISA are described in detail elsewhere 22,26,27 and in the companion paper. 23 The experimental setup for Octet bio-layer interferometry and LC-MS peptide mapping methods are provided in the Supplementary Methods.…”

“…18,19 Recent work in our laboratories demonstrated the incompatibility of two APs (thimerosal and 2-phenoxyethanol) with three different recombinant non-replicating rotavirus (NRRV) fusion protein antigens as part of the analytical and formulation development of a trivalent NRRV vaccine candidate. 22 As described in the companion paper 23 and elsewhere, 22,[24][25][26][27] each recombinant NRRV antigen comprises a truncated rotavirus VP4 surface protein (derived from three target RV strains) linked to the tetanus toxoid universal CD4 þ T-cell epitope, and are simply referred to as the P [4], P [6], and P [8] protein antigens. The NRRV vaccine candidate has been shown to be safe and immunogenic in infants and toddlers in early clinical trials conducted by PATH in South Africa, 28,29 and mid to late stage clinical evaluations are ongoing.…”

Novel Approach for Designing Rotavirus Vaccine Candidate Based on Two Plant Viruses

“…Кроме того, большинство смертей, ассоциированных с ротавирусом, фиксируется в развивающихся странах, где эффективность этих вакцин ниже по сравнению с таковой в развитых странах (3,(6)(7)(8)(9). Таким образом, существует потребность в разработке нереплицирующейся вакцины нового поколения против ротавирусной инфекции (10,11). Препараты на основе рекомбинантных антигенов ротавируса (род Rotavirus, сем.…”