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

Abstract

More effective rotavirus vaccines are essential for preventing extensive diarrheal morbidity and mortality in children under five years of age in low-resource regions. Nonreplicating rotavirus vaccines (NRRV) administered parenterally provide an alternate vaccination method to the current licensed oral vaccine. Live attenuated vaccines and may generate increased efficacy in low-resource settings because the parenteral administration route bypasses some of the challenges associated with oral administration, including differences in intestinal environments. Work described here supports development of a trivalent NRRV vaccine for parenteral administration to avoid complications of the gastrointestinal route. Recombinant VP8* subunit proteins representing some of the most prevalent strains of rotavirus infecting humans-DS-1 (P[4]), 1076 (P[6]), and Wa (P[8])were combined with an aluminum adjuvant and the P2 epitope of tetanus toxoid to enhance the immune response to this NRRV antigen. Vaccine formulation development included selection of aluminum hydroxide (Alhydrogel®) as an appropriate adjuvant as well as an optimal buffer to maintain antigen stability and optimize antigen binding to the adjuvant. Characterization assays were used to select the lead vaccine formulation and monitor formulation stability. The NRRV liquid formulation was stable for one year at 2°C to 8°C and four weeks at 37°C. Immunogenicity of the NRRV formulation was evaluated using a guinea pig model, where we demonstrated that the adjuvant provided a 20-fold increase in neutralization titer against a homologous antigen and that the P2-fusion also enhanced the serum neutralizing antibody responses. This vaccine candidate is currently being evaluated in human clinical trials.

Rapid Developability Assessments to Formulate Recombinant Protein Antigens as Stable, Low-Cost, Multi-Dose Vaccine Candidates: Case-Study With Non-Replicating Rotavirus (NRRV) Vaccine Antigens

“…As a proof-ofconcept, we focused on non-replicating rotavirus (NRRV) recombinant protein antigens. The preformulation characterization and formulation development of three different E. coli expressed recombinant NRRV protein antigens (P [8], P [6] and P [4]; see nomenclature below) has been recently described. 8e11 In this work, the NRRV antigens were a good case study to assess the utility of newer, more rapid formulation developability assessments (evaluating multiple antigen variants available only in small quantities) compared to more standard, time-consuming formulation development approaches (with preselected antigens available in larger quantities).…”

“…Each NRRV antigen is a recombinant fusion-protein consisting of a truncated version of the rotavirus surface protein VP4 (known as VP8), genetically fused to the tetanus toxoid universal CD4 þ T-cell epitope (P2). The three fusion-protein antigens derive from the VP8 component of three different RV serotypes, P [8], P [6], and P [4]. This leads to the nomenclature P2eVP8eP [8], P2eVP8eP [6], and P2eVP8eP [4], which for simplicity, are often referred to as P [8], P [6], and P [4], respectively.…”

“…9 The development of the series of NRRV antigen variants from K. phaffii, in terms of design rationale, experimental strategies for low-cost vaccine production, and immunological characterization, are presented in detail elsewhere. 16 We used these sequence variants of P [8] and P [4] NRRV as a case study to demonstrate proof-ofconcept for a two-stage formulation developability assessment workflow to rapidly assess formulation variables for recombinant vaccine antigens that could be applied to accelerate development of low-cost vaccine dosage forms targeted for use in low and middle income countries (LMICs). Stage 1 evaluates the physicochemical and immunochemical binding properties of each NRRV protein antigen, and Stage 2 examines the impact of various storage temperatures on conformational stability and antibody binding of downselected P [8] and P [4] NRRV variants, both in the presence and absence of an aluminum-adjuvant (Alhydrogel®, AH) and a vaccine preservative (thimerosal).…”

A Nanoparticle-Based Trivalent Vaccine Targeting the Glycan Binding VP8* Domains of Rotaviruses

“…Several VP8*-based subunit rotavirus vaccine candidates are currently under development, among which a tandem truncated VP8* with a T cell epitope, named P2-VP8 [ 53 ], has been in clinical trials, showing its safety and immunogenicity for parenteral use in humans [ 54 ]. This P2-VP8 vaccine contains only two fused VP8* copies per molecule with a small molecular size of ~20 kDa [ 53 ]. This simple molecular set up of P2-VP8* differs strikingly from our P 24 -VP8* nanoparticle, which has 24 VP8* antigens per nanoparticle and a large molecular mass of 1.25 mDa.…”

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…”