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.

A two-step developability assessment workflow is described to screen variants of recombinant protein antigens under various formulation conditions to rapidly identify stable, aluminum-adjuvanted, multi-dose vaccine candidates. For proof-of-concept, a series of sequence variants of the recombinant non-replicating rotavirus (NRRV) P[8] protein antigen (produced in Komagataella phaffii ) were compared in terms of primary structure, post-translational modifications, antibody binding, conformational stability, relative solubility and preservative compatibility. Based on these results, promising P[8] variants were down-selected and the impact of key formulation conditions on storage stability was examined (e.g., presence or absence of the aluminum-adjuvant Alhydrogel and the preservative thimerosal) as measured by differential scanning calorimetry (DSC) and antibody binding assays. Good correlations between rapidly-generated developability screening data and storage stability profiles (12 weeks at various temperatures) were observed for aluminum-adsorbed P[8] antigens. These findings were extended and confirmed using variants of a second NRRV antigen, P[4]. These case-study results with P[8] and P[4] NRRV variants are discussed in terms of using this vaccine formulation developability workflow to better inform and optimize formulation design with a wide variety of recombinant protein antigens, with the long-term goal of rapidly and cost-efficiently identifying low-cost vaccine formulations for use in low and middle income countries.

Three-photon excitation fluorescence correlation spectroscopy was used to detect oligomerization equilibria of rat liver phosphofructokinase. The fluorescence intensity produced by the three-photon excitation of tryptophan was collected using the DIVER microscope. In this home-built upright microscope, a large area photomultiplier, placed directly below the sample, is used as the detector. The lack of optical elements in the microscope detection path results in a significantly improved detection efficiency in the UV region down to about 300 nm, which encompasses the fluorescence emission from tryptophan. The three-photon excitation autocorrelation decays obtained for phosphofructokinase in the presence of F6P showed the presence of large oligomers. Substitution of F6P with ATP in the buffer medium results in dissociation of the large oligomers, which is reported by the decreased autocorrelation amplitude. The three-photon excitation process was verified from the slope of the log–log plot of intensity against laser power.

Implementation of inactivated polio vaccines (IPV) containing Sabin strains (sIPV) will further enable global polio eradication efforts by improving vaccine safety during use and containment during manufacturing. Moreover, sIPV-containing vaccines will lower costs and expand production capacity to facilitate more widespread use in low- and middle-income countries (LMICs). This review focuses on the role of vaccine formulation in these efforts including traditional Salk IPV vaccines and new sIPV-containing dosage forms. The physicochemical properties and stability profiles of poliovirus antigens are described. Formulation approaches to lower costs include developing multidose and combination vaccine formats as well as improving storage stability. Formulation strategies for dose-sparing and enhanced mucosal immunity include employing adjuvants (e.g. aluminum-salt and newer adjuvants) and/or novel delivery systems (e.g. ID administration with microneedle patches). The potential for applying these low-cost formulation development strategies to other vaccines to further improve vaccine access and coverage in LMICs is also discussed.

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