Use of Proteins Identified through a Functional Genomic Screen To Develop a Protein Subunit Vaccine That Provides Significant Protection against Virulent Streptococcus suis in Pigs

Abstract

Streptococcus suis is a bacterium that is commonly carried in the respiratory tract and that is also one of the most important invasive pathogens of swine, commonly causing meningitis, arthritis, and septicemia. Due to the existence of many serotypes and a wide range of immune evasion capabilities, efficacious vaccines are not readily available. The selection of S. suis protein candidates for inclusion in a vaccine was accomplished by identifying fitness genes through a functional genomics screen and selecting conserved predicted surface-associated proteins. Five candidate proteins were selected for evaluation in a vaccine trial and administered both intranasally and intramuscularly with one of two different adjuvant formulations. Clinical protection was evaluated by subsequent intranasal challenge with virulent S. suis While subunit vaccination with the S. suis proteins induced IgG antibodies to each individual protein and a cellular immune response to the pool of proteins and provided substantial protection from challenge with virulent S. suis, the immune response elicited and the degree of protection were dependent on the parenteral adjuvant given. Subunit vaccination induced IgG reactive against different S. suis serotypes, indicating a potential for cross protection.

Keywords: Streptococcus suis; adjuvant; functional genomics; subunit; vaccine.

FIG 1

SDS-PAGE and Western blots of the five candidate vaccine proteins. The five candidate proteins were expressed in E. coli and purified as described in Materials and Methods. The purified proteins were run on an SDS-polyacrylamide gel (A) and also transferred to membranes and probed with either serum from a pig experimentally infected with S. suis serotype 2 (B) or sera from pigs raised in a pathogen-free environment as a negative control (C). The numbers on the left are molecular masses (in kilodaltons).

FIG 2

Titers of IgG antibodies to the individual subunit proteins among vaccinated pigs in groups 1 and 2 on day 14 (2 weeks after priming) and days 21 and 28 (1 and 2 weeks after the boost, respectively). Pigs in groups 1 and 2 (6 pigs each) were vaccinated with the 5 candidate proteins on days 0 and 14 of the experiment. Both groups were given the 5 proteins intranasally with polyethyleneimine as the adjuvant; in addition, group 1 pigs were given the 5 proteins intramuscularly with AddaVax and Carbopol as the adjuvant, while group 2 pigs were given the 5 proteins intramuscularly with Emulsigen-D as the adjuvant. Titers were determined via indirect ELISA with plates coated with the individual proteins using 2-fold serial dilutions of serum. The resulting OD data were modeled as a nonlinear function of the log₁₀ dilution using the log (agonist)-versus-response variable slope four-parameter logistic model. Endpoints were interpolated by using 4 times the average OD of the day 0 sample for each respective pig as the cutoff.

FIG 3

ELISpot assay data showing the number of IFN-γ-secreting cells detected in PBMCs isolated from pigs in the indicated groups on days 14 (2 weeks after priming), 21 (1 week after the boost), and 28 (2 weeks after the boost). Pigs in groups 1 and 2 (6 pigs each) were vaccinated with the 5 candidate proteins on days 0 and 14 of the experiment. Both groups were given the 5 proteins intranasally with polyethyleneimine as the adjuvant; in addition, group 1 pigs were given the 5 proteins intramuscularly with AddaVax and Carbopol as the adjuvant, while group 2 pigs were given the 5 proteins intramuscularly with Emulsigen-D as the adjuvant. Pigs in groups 3 to 5 were controls given the adjuvants alone (groups 3 and 4, 3 pigs each) or PBS (group 5, 4 pigs). PBMCs collected on days 14, 21, and 28 were seeded at 2.5 × 10⁵ cells per well in duplicate and stimulated with a pool of the 5 candidate proteins. Control wells were stimulated with medium alone or pokeweed mitogen (data not shown). The means and standard errors of the means for each of the treatment groups are denoted. *, statistically significant differences between groups (P < 0.05).

FIG 4

Cytokines produced by PBMCs isolated from pigs in the indicated groups on day 28 (2 weeks after the boost). Pigs in groups 1 and 2 (6 pigs each) were vaccinated with the 5 candidate proteins on days 0 and 14 of the experiment. Both groups were given the 5 proteins intranasally with polyethyleneimine as the adjuvant; in addition, group 1 pigs were given the 5 proteins intramuscularly with AddaVax and Carbopol as the adjuvant, while group 2 pigs were given the 5 proteins intramuscularly with Emulsigen-D as the adjuvant. Pigs in groups 3 to 5 were controls given the adjuvants (groups 3 and 4, 3 pigs each) alone or PBS (group 5, 4 pigs). PBMCs collected on day 28 were stimulated in vitro with a pool of the 5 candidate proteins, and the supernatants were collected to evaluate the levels of cytokines secreted by the cells by multiplex cytokine ELISA. Data are presented as box and dot plots representing the mean cytokine concentration for the group (box) and individual cytokine concentration for each pig (dots) (in picograms per milliliter). Significantly different cytokine concentrations among groups are identified with different letters (P < 0.05).

FIG 5

Survival rates of pigs vaccinated with 5 subunit proteins with different adjuvant formulations (groups 1 and 2) compared to those of pigs given adjuvant alone (groups 3 and 4) or PBS (group 5). Pigs in groups 1 and 2 (6 pigs each) were vaccinated with the 5 candidate proteins on days 0 and 14 of the experiment. Both groups were given the 5 proteins intranasally with polyethyleneimine as the adjuvant; in addition, group 1 pigs were given the 5 proteins intramuscularly with AddaVax and Carbopol as the adjuvant, while group 2 pigs were given the 5 proteins intramuscularly with Emulsigen-D as the adjuvant. Pigs in groups 3 to 5 were controls given the adjuvants alone (groups 3 and 4, 3 pigs each) or PBS (group 5, 4 pigs).

FIG 6

Cross-reactive IgG antibody to whole S. suis bacteria of serotypes that commonly cause systemic disease from group 1 and 2 pigs on day 28 (2 weeks after the boost). Pigs in groups 1 and 2 (6 pigs each) were vaccinated with the 5 candidate proteins on days 0 and 14 of the experiment. Both groups were given the 5 proteins intranasally with polyethyleneimine as the adjuvant; in addition, group 1 pigs were given the 5 proteins intramuscularly with AddaVax and Carbopol as the adjuvant, while group 2 pigs were given the 5 proteins intramuscularly with Emulsigen-D as the adjuvant. IgG reactivity was determined via indirect ELISA with plates coated with heat-inactivated whole bacteria. Serum samples collected on day 28 from each pig were diluted 1:500 and used in the assay. Data are reported as the mean ± SEM optical density at 405 nm. Bacterial strains are listed on the x axis, with the serotype being given in parentheses.