Screening of Oligomeric (Meth)acrylate Vaccine Adjuvants Synthesized via Catalytic Chain Transfer Polymerization

Abstract

This report details the first systematic screening of free-radical-produced methacrylate oligomer reaction mixtures as alternative vaccine adjuvant components to replace the current benchmark compound squalene, which is unsustainably sourced from shark livers. Homo-/co-oligomer mixtures of methyl, butyl, lauryl, and stearyl methacrylate were successfully synthesized using catalytic chain transfer control, where the use of microwave heating was shown to promote propagation over chain transfer. Controlling the mixture material properties allowed the correct viscosity to be achieved, enabling the mixtures to be effectively used in vaccine formulations. Emulsions of selected oligomers stimulated comparable cytokine levels to squalene emulsion when incubated with human whole blood and elicited an antigen-specific cellular immune response when administered with an inactivated influenza vaccine, indicating the potential utility of the compounds as vaccine adjuvant components. Furthermore, the oligomers' molecular sizes were demonstrated to be large enough to enable greater emulsion stability than squalene, especially at high temperatures, but are predicted to be small enough to allow for rapid clearance from the body.

Keywords: adjuvant; catalytic chain transfer; polymerization; screening; squalene; vaccine.

Figure 1

Physicochemical stability of oil-in-water emulsions resulting from methacrylate oligomer mixtures (Table 6). Stable emulsions (SEs) were stored at 5 °C, 25 °C, or 40 °C for up to 18 months and monitored using dynamic light scattering to assess emulsion droplet diameter (top) and size polydispersity index (bottom). Data are represented as mean values with error bars that represent the standard deviation. The MMA (Table 6, Entry 4) and the SMA/LMA co-oligomer (Table 6, Entry 10) mixtures did not formulate due to the higher viscosity of these oligomer mixtures and were therefore excluded.

Figure 2

In vitro stimulation of human whole blood with methacrylate-based oil-in-water emulsions resulted in differential cytokine activity. Heat map representation of log-transformed cytokine concentrations measured in supernatants of heparinized blood stimulated by incubation with stable emulsions (SEs). The various oils were evaluated in comparison with shark squalene emulsion (positive control) and long-chain triglyceride emulsion (negative control). Values represent the mean from 6 donors (3 male and 3 female).

Figure 3

Viability of peripheral blood mononuclear cells (PBMCs) following incubation with methacrylate-based oil-in-water emulsions. PBMCs from a human male donor were employed.

Figure 4

Scaled-up lauryl methacrylate (LMA) emulsion adjuvant activity in mice immunized with split, inactivated H5N1 vaccine antigen. Vaccine antigen (Ag) was mixed with emulsion immediately prior to intramuscular immunization of C57BL/6 (n = 7–8) mice. All mice were immunized twice, three weeks apart. Serum and splenocytes were collected three weeks after the 2nd immunization. Data are represented as box-whisker plots, with bars representing median values, boxes representing 1st–3rd quartiles, and whiskers representing the maximum and minimum values. Statistical comparisons were performed for normally distributed data using one-way ANOVA with Tukey’s correction for multiple comparisons, and for non-normally distributed data, using the Kruskal–Wallis test with Dunn’s correction for multiple comparisons, with p-values of <0.05 reported on the plots. (A) Antigen-specific serum total IgG (IgGT) endpoint titer measured using ELISA. (B) Antigen-specific serum IgG2c endpoint titer measured using ELISA. (C) Antigen-specific serum IgG1 endpoint titer measured using ELISA. (D) Ratio of IgG2c/IgG1 endpoint titers. (E) Antigen-specific IFNγ-secreting splenocytes measured using ELISpot. (F) Antigen-specific IL-5-secreting splenocytes measured using ELISpot. (G) Ratio of IFNγ-secreting cells/IL-5-secreting cells.