Immunomodulatory and physical effects of oil composition in vaccine adjuvant emulsions

Abstract

Squalene-based oil-in-water emulsions have been used for years in some seasonal and pandemic influenza vaccines. However, concerns have been expressed regarding squalene source and potential biological activities. Little information is available regarding the immunomodulatory activity of squalene in comparison with other metabolizable oils in the context of oil-in-water emulsions formulated with vaccines. The present work describes the manufacture and physical characterization of emulsions composed of different classes of oils, including squalene, long chain triglycerides, a medium chain triglyceride, and a perfluorocarbon, all emulsified with egg phosphatidylcholine. Some differences were apparent among the non-squalene oils in terms of emulsion stability, including higher size polydispersity in the perfluorocarbon emulsion, more rapid visual instability at 60°C for the long-chain triglyceride and perfluorocarbon emulsions, and an increased creaming rate in the medium-chain triglyceride emulsion at 60°C as detected by laser scattering optical profiling. The biological activity of each of these emulsions was compared when formulated with either a recombinant malaria antigen or a split-virus inactivated influenza vaccine. Overall, vaccines containing the squalene emulsion elicited higher antibody titers and more abundant long-lived plasma cells than vaccines containing emulsions based on other oils. Since squalene-based emulsions show higher adjuvant potency compared to the other oils tested, non-squalene oils may be more suitable as carriers of amphiphilic or hydrophobic immunostimulatory molecules (such as TLR agonists) rather than as stand-alone adjuvants.

Figure 1

Emulsion particle size stability at various storage temperatures. Shown are results following storage at (a) 5 °C, (b) RT, (c) 37 °C and (d) 60 °C. Particle size was measured using Malvern Instruments Zetasizer Nano or Automated Particle Sizer. Size measurement is the z-avg, which represents the intensity-biased average size value from dynamic light scattering.

Figure 2

Laser scattering optical profiling analysis of emulsion stability. (a) Laser transmission scans of undiluted grapeseed emulsion in polycarbonate cuvette measured every 10 minutes over 4 hours at 60°C, with blue color indicating initial scans and pink color indicating later scans. (b) Integral transmission profiles of the 25 – 30 mm region (see above) of cuvettes containing emulsion, measured over 4 hours at 60°C.

Figure 2

Laser scattering optical profiling analysis of emulsion stability. (a) Laser transmission scans of undiluted grapeseed emulsion in polycarbonate cuvette measured every 10 minutes over 4 hours at 60°C, with blue color indicating initial scans and pink color indicating later scans. (b) Integral transmission profiles of the 25 – 30 mm region (see above) of cuvettes containing emulsion, measured over 4 hours at 60°C.

Figure 3

Effect of emulsions on antibody response induced by immunization with recombinant malaria antigen. BALB/c mice were immunized twice with PbCSP antigen formulated with emulsions containing squalene, grapeseed, or sesame oil. Antigen-specific IgG were determined by ELISA. The antibody responses from the grapeseed and sesame oil groups were below the limit of detection. In the antigen alone group, only one mouse showed a response above the lower limit of detection. n = 5 per group, and data are shown as mean and s.e.

Figure 4

Effect of adjuvant formulation in humoral immune responses induced by immunization with inactivated split-virus influenza vaccine (Fluzone). BALB/c mice were immunized with Fluzone formulated with emulsions containing squalene, grapeseed, or sesame oil. Antigen-specific IgG (left), IgG1 (middle) and IgG2a (right) were determined by ELISA. Results are shown as the geometric mean endpoint titer (Log₁₀) ± s.e., n = 5 per group.

Figure 5

Effect of adjuvant formulation in humoral immune responses induced by immunization with inactivated split-virus influenza vaccine (Fluzone). BALB/c mice were immunized with Fluzone formulated with emulsions containing squalene, perfluorocarbon, soybean, or Miglyol 810 oil. Antigen-specific IgG (left), IgG1 (middle) and IgG2a (right) were determined by ELISA. Results are shown as the geometric mean endpoint titer (Log₁₀) ± SEM, n = 5 per group except for the squalene group in (b) where n = 4.

Figure 6

IgG-secreting bone marrow plasma cells against Fluzone were determined by ELISPOT and data are represented by mean +/− SEM. (a) * = p-value < 0.05 versus immunization with vaccine alone; # = p-value < 0.05 versus immunization with the vaccine containing the grapeseed emulsion; % = p-value < 0.05 versus immunization with the vaccine containing the sesame emulsion. (b) * = p-value < 0.05 versus immunization with vaccine alone; # = p-value < 0.05 versus immunization with the vaccine containing the perfluorocarbon emulsion; % = p-value < 0.05 versus immunization with the vaccine containing the soybean emulsion; ^ = p-value < 0.05 versus immunization with the vaccine containing the Miglyol emulsion.

Figure 6

IgG-secreting bone marrow plasma cells against Fluzone were determined by ELISPOT and data are represented by mean +/− SEM. (a) * = p-value < 0.05 versus immunization with vaccine alone; # = p-value < 0.05 versus immunization with the vaccine containing the grapeseed emulsion; % = p-value < 0.05 versus immunization with the vaccine containing the sesame emulsion. (b) * = p-value < 0.05 versus immunization with vaccine alone; # = p-value < 0.05 versus immunization with the vaccine containing the perfluorocarbon emulsion; % = p-value < 0.05 versus immunization with the vaccine containing the soybean emulsion; ^ = p-value < 0.05 versus immunization with the vaccine containing the Miglyol emulsion.

Figure 7

HAI titers after immunization with Fluzone formulated with squalene, grapeseed, or sesame oil emulsion. BALB/c mice were immunized and then serum HAI titers determined four weeks after boosting. (a) HAI titers against the A/Solomon Islands/3/2006 (H1N1) component of Fluzone. (b) HAI titers against the A/Wisconsin/67/2005 (H3N2) component of Fluzone. Data are shown as the (Log₂) titer for each individual animal, with the geometric mean and SEM represented. * = p-value < 0.05 versus immunization with vaccine alone; # = p-value < 0.05 versus immunization with the vaccine containing the grapeseed emulsion; % = p-value < 0.05 versus immunization with the vaccine containing the sesame emulsion.

Figure 7

HAI titers after immunization with Fluzone formulated with squalene, grapeseed, or sesame oil emulsion. BALB/c mice were immunized and then serum HAI titers determined four weeks after boosting. (a) HAI titers against the A/Solomon Islands/3/2006 (H1N1) component of Fluzone. (b) HAI titers against the A/Wisconsin/67/2005 (H3N2) component of Fluzone. Data are shown as the (Log₂) titer for each individual animal, with the geometric mean and SEM represented. * = p-value < 0.05 versus immunization with vaccine alone; # = p-value < 0.05 versus immunization with the vaccine containing the grapeseed emulsion; % = p-value < 0.05 versus immunization with the vaccine containing the sesame emulsion.

Figure 8

HAI titers after immunization with Fluzone formulated with squalene, perfluorocarbon, soybean, or Miglyol 810 oil emulsion. BALB/c mice were immunized and then serum HAI titers determined four weeks after boosting. (a) HAI titers against the A/Solomon Islands/3/2006 (H1N1) component of Fluzone. (b) HAI titers against the A/Wisconsin/67/2005 (H3N2) component of Fluzone. (c) HAI titers against the heterologous A/Brisbane/59/07 influenza strain. (d) HAI titers against the heterologous A/Uruguay/716/07 influenza strain. Data are shown as the (Log₂) titer for each individual animal, with the geometric mean and SEM represented. * = p-value < 0.05 versus immunization with vaccine alone; # = p-value < 0.05 versus immunization with the vaccine containing the perfluorocarbon emulsion; % = p-value < 0.05 versus immunization with the vaccine containing the soybean emulsion; ^ = p-value < 0.05 versus immunization with the vaccine containing the Miglyol emulsion.

Figure 8

HAI titers after immunization with Fluzone formulated with squalene, perfluorocarbon, soybean, or Miglyol 810 oil emulsion. BALB/c mice were immunized and then serum HAI titers determined four weeks after boosting. (a) HAI titers against the A/Solomon Islands/3/2006 (H1N1) component of Fluzone. (b) HAI titers against the A/Wisconsin/67/2005 (H3N2) component of Fluzone. (c) HAI titers against the heterologous A/Brisbane/59/07 influenza strain. (d) HAI titers against the heterologous A/Uruguay/716/07 influenza strain. Data are shown as the (Log₂) titer for each individual animal, with the geometric mean and SEM represented. * = p-value < 0.05 versus immunization with vaccine alone; # = p-value < 0.05 versus immunization with the vaccine containing the perfluorocarbon emulsion; % = p-value < 0.05 versus immunization with the vaccine containing the soybean emulsion; ^ = p-value < 0.05 versus immunization with the vaccine containing the Miglyol emulsion.

Figure 8

HAI titers after immunization with Fluzone formulated with squalene, perfluorocarbon, soybean, or Miglyol 810 oil emulsion. BALB/c mice were immunized and then serum HAI titers determined four weeks after boosting. (a) HAI titers against the A/Solomon Islands/3/2006 (H1N1) component of Fluzone. (b) HAI titers against the A/Wisconsin/67/2005 (H3N2) component of Fluzone. (c) HAI titers against the heterologous A/Brisbane/59/07 influenza strain. (d) HAI titers against the heterologous A/Uruguay/716/07 influenza strain. Data are shown as the (Log₂) titer for each individual animal, with the geometric mean and SEM represented. * = p-value < 0.05 versus immunization with vaccine alone; # = p-value < 0.05 versus immunization with the vaccine containing the perfluorocarbon emulsion; % = p-value < 0.05 versus immunization with the vaccine containing the soybean emulsion; ^ = p-value < 0.05 versus immunization with the vaccine containing the Miglyol emulsion.

Figure 8

HAI titers after immunization with Fluzone formulated with squalene, perfluorocarbon, soybean, or Miglyol 810 oil emulsion. BALB/c mice were immunized and then serum HAI titers determined four weeks after boosting. (a) HAI titers against the A/Solomon Islands/3/2006 (H1N1) component of Fluzone. (b) HAI titers against the A/Wisconsin/67/2005 (H3N2) component of Fluzone. (c) HAI titers against the heterologous A/Brisbane/59/07 influenza strain. (d) HAI titers against the heterologous A/Uruguay/716/07 influenza strain. Data are shown as the (Log₂) titer for each individual animal, with the geometric mean and SEM represented. * = p-value < 0.05 versus immunization with vaccine alone; # = p-value < 0.05 versus immunization with the vaccine containing the perfluorocarbon emulsion; % = p-value < 0.05 versus immunization with the vaccine containing the soybean emulsion; ^ = p-value < 0.05 versus immunization with the vaccine containing the Miglyol emulsion.