Simultaneous Immunization with Multiple Diverse Immunogens Alters Development of Antigen-Specific Antibody-Mediated Immunity

Abstract

Vaccination remains one of the most successful medical interventions in history, significantly decreasing morbidity and mortality associated with, or even eradicating, numerous infectious diseases. Although traditional immunization strategies have recently proven insufficient in the face of many highly mutable and emerging pathogens, modern strategies aim to rationally engineer a single antigen or cocktail of antigens to generate a focused, protective immune response. However, the effect of cocktail vaccination (simultaneous immunization with multiple immunogens) on the antibody response to each individual antigen within the combination, remains largely unstudied. To investigate whether immunization with a cocktail of diverse antigens would result in decreased antibody titer against each unique antigen in the cocktail compared to immunization with each antigen alone, we immunized mice with surface proteins from uropathogenic Escherichia coli, Mycobacterium tuberculosis, and Neisseria meningitides, and monitored the development of antigen-specific IgG antibody responses. We found that antigen-specific endpoint antibody titers were comparable across immunization groups by study conclusion (day 70). Further, we discovered that although cocktail-immunized mice initially elicited more robust antibody responses, the rate of titer development decreases significantly over time compared to single antigen-immunized mice. Investigating the basic properties that govern the development of antigen-specific antibody responses will help inform the design of future combination immunization regimens.

Keywords: antibodies; bacterial pathogens; humoral immunity; immunization; subunit vaccines.

Figure 1

Study Design for Simultaneous Immunization with Diverse Antigens; (A) Table of antigens used for immunization in this study. Each of these antigens has been tested at least pre-clinically as a vaccine candidate against their respective native host [15,16,20]. (B) Percent sequence identity overlap between each of the immunogens utilized. (C) Immunization schedule and antigen groups for this study denoted by colored symbols (Pink: IreA, Blue: HBHA, Green: fHbp). n = 5 female BALB/c mice/group. (D) Immunization and bleed regimens used for all groups.

Figure 2

Antigen Immunization Combinations Elicit Comparable Antibody Titers by Study Conclusion. Antigen-specific IgG serum antibody titer measured at study conclusion. Endpoint antibody titer against IreA (A), HBHA (C), and fHbp (E) was measured by direct ELISA using serum from each immunization group shown. Quantification of endpoint titers for each immunization group against IreA (B), HBHA (D), and fHbp (F). Colored lines, and associated bar graphs, represent responses in single antigen immunized mice (IreA, pink; HBHA, blue; fHbp, green). Black lines, and associated bar graphs, represent responses in cocktail (triple antigen)-immunized mice. Gray lines represent mice immunized with each combination of two antigens, each group is denoted by a different symbol. (G) Comparison of antigen-specific antibody titers and number (#) of antigens with which a mouse was immunized. Statistical significance was determined by Kruskal-Wallis test. * denotes p < 0.05.

Figure 3

Cocktail Immunization Initially Elicits Higher Antibody Titers. Development of antigen-specific serum IgG antibody titers against IreA (A), HBHA (B), and fHbp (C) by each immunization group. Comparison of endpoint titer between single antigen (colored line)- or triple antigen (black line)-immunized mice determined by ELISA 14 days after primary immunization (left), boost #1 (middle), and boost #2 (right). (D) The EC₅₀ (concentration of serum at which the half-maximal response is observed) against IreA is quantified over time for both IreA-immunized (pink line) and HBHA+fHbp+IreA-immunized (black lines). (E) The EC₅₀ against HBHA is quantified over time for both HBHA-immunized (blue lines) and HBHA+fHbp+IreA-immunized (black lines). (F) The EC₅₀ against fHbp is quantified over time for both fHbp-immunized (green line) and HBHA+fHbp+IreA-immunized (black lines). Statistical significance was determined by Mann-Whitney U test. * denotes p < 0.05.

Figure 4

Cocktail Immunization Alters Development of Antigen-Specific Antibody-Mediated Immunity. Max endpoint serum antibody titer against IreA (A), HBHA (B), and fHbp (C) measured by ELISA in either single antigen- (color) or cocktail-immunized (IreA+fHbp+HBHA; black). Max serum antibody titer observed in all single antigen vs. cocktail-immunized mice (D). Change in serum antibody titers over the course of vaccination. Change in antibody titer between primary immunization and boost #1 (E), between boost #1 and boost #2 (I), and between primary immunization and boost #2 (M) in IreA- vs. IreA+fHbp+HBHA-immunized mice. (F,J,N) As described for IreA, observed changes in HBHA-specific antibody titers. (G,K,O) As described for HBHA, observed changes in fHbp-specific antibody titers. Change in antibody titer between primary immunization and boost #1 (H), between boost #1 and boost #2 (L), and between primary immunization and boost #2 (P) in all single antigen- vs. cocktail-immunized mice. Statistical significance was determined by Mann-Whitney U test. * denotes p < 0.05.