Vaccines against pneumococcal disease for use in developing countries should be safe, effective against a broad range of serotypes and affordable. The existing conjugate vaccines offer protection against the serotypes included in the vaccine which were selected based on their prevalence in North America and Europe, and are predicted to provide incomplete protection against pneumococcal infections in Asia and Africa. In addition, these conjugate vaccines are expensive to produce. The work in this report demonstrates that a vaccine composed of killed whole cell, nonencapsulated pneumococci and formulated with AH, induces a strong antibody and IL-17 response. Both the antigen and adjuvant are relatively inexpensive suggesting that the vaccine will be affordable for use in developing countries.
Previous work with a simple protein antigen, alpha casein, indicated that the strength of adsorption of antigens onto aluminum-containing adjuvants is inversely related to the antibody response to these antigens . A similar relationship was found with a larger and more complex antigen, hepatitis B surface antigen (HBsAg), but the negative effect of a high adsorptive coefficient was not as strong as with alpha casein . The antigen used in the current studies, WCA, consists of killed whole bacterial cells and some soluble bacterial proteins. WCA was mixed with four aluminum-containing adjuvants with different surface properties to determine if differences in adsorptive capacity and adsorptive coefficient could be measured. Although the obtained values should be interpreted with caution because of the complex nature of WCA, they indicate a range of adsorptive properties for the four adjuvants. The highest values were measured for AH while adjuvants with more surface phosphates had a lower affinity for WCA. This suggests that at least some of the molecules in WCA are phosphorylated or associated with phospholipid membranes, and are adsorbed by the ligand exchange mechanism.
The antibody response to the vaccine formulations with the four adjuvants with broadly divergent adsorptive capacities and coefficients for WCA indicated that the aluminum-containing adjuvant potentiated the immune response even when the antigen was not adsorbed. In addition, the strength of adsorption was not a significant factor in immunopotentation. Aluminum adjuvants may enhance the immune response to soluble antigens by adsorbing the antigens onto the adjuvant particles that are more readily phagocytised by antigen-presenting cells . Antigen adsorption by the adjuvant may be less relevant when the antigen comprises killed whole cell bacteria as the bacteria are about 1 micrometer in diameter while the primary particles of the adjuvant are smaller than 50 nm . Since changes in adsorption through phosphate treatment of the adjuvants did not affect the antibody response, subsequent experiments focused on AH and AP.
The protective immune response induced by conjugate vaccines is based on serotype-specific anti-polysaccharide antibodies. In contrast, the immune response against WCA involves antibodies directed against protein antigens and Th17 cells. Antibodies induced by WCA can provide protection against systemic disease, but they do not protect against nasopharyngeal colonization in mice [7, 9]. Nasopharyngeal colonization was inhibited by CD4+ T cells that secrete IL-17, and the concentration of IL-17 in WCA-stimulated whole blood cultures was inversely correlated with the degree of nasopharyngeal colonization following intranasal challenge . Infection of naïve mice with S. pneumonia induced Th17 cells which provided enhanced clearance of the bacteria upon secondary challenge . The protective role of IL-17 resides in the induction of secretion of antimicrobial peptides and chemokines that attract monocytes and neutrophils to the site of infection [23, 24]. IL-17 is also involved in the protection against other extracellular bacterial pathogens such as Bordetella pertussis, intracellular bacterial pathogens including Mycobacterium tuberculosis, and fungal pathogens, indicating an important role against infections at mucosal surfaces and in the lung. However, an excessive IL-17 response may be detrimental and cause extensive tissue damage [23, 24]. It has been suggested that Th17 cells are critical for vaccine-induced memory immune responses, and enhancing and regulating the Th17 response may be important in vaccine design . In our studies, the combination of WCA with AH was critical for the induction of a population IL-17 producing cells following intramuscular injection. Neither WCA alone nor WCA with AP induced a significant IL-17 response, even though AP greatly enhanced the antibody response to WCA. Such a dramatic difference in the quality of the immune response between vaccines formulated with AH and vaccines formulated with AP was unexpected.
The induction of Th17 cells in S. pneumoniae infection is dependent on TLR2 . The ligands for TLR2 include molecular components of Gram-positive bacteria such as lipoproteins [25, 26]. The induction of Th17 cells by WCA/AH and not by WCA/AP suggests that these ligands are not available in the WCA/AP formulation, possibly due to strong electrostatic adsorption.
There are few published reports in which the immune responses to bacterial vaccines formulated with AP vs. AH are directly compared. In one study, acellular pertussis antigens combined with AH induced a stronger antibody response and greater protection upon intranasal challenge with Bordetella pertussis compared with AP, but the basis of the increased protection was not further investigated . Th17 cells are induced during infection with Bordetella pertussis, but antibody-mediated depletion of IL-17 only had a modest effect on the bacterial loads in the lungs of experimentally infected mice . Two types of vaccines, a whole cell and an acellular pertusiss vaccine, are used to protect against whooping cough. Both vaccines are effective, but vaccination of mice with a whole cell pertussis vaccine induced Th17 cells, whereas these cells were not induced by the acellular vaccine [29, 30]. The role of adjuvants was not specifically addressed in these studies.
The basis for the difference in immune response generated by WCA formulated with AH vs. AP is not entirely clear, but it is likely that the greater affinity of AH for WCA proteins contributed to this effect. The adsorptive strength, determined as the adsorptive coefficient, of AH was 2.5 times that of AP. Previous work showed that a high adsorptive strength may interfere with the antibody response and the T cell response, probably because there is insufficient release of antigen from the adjuvant to interact with B cells and for antigen processing and presentation . A similar effect was observed at the lower doses of WCA in which a significantly stronger antibody response was obtained with AP in comparison with AH. At higher doses, the difference between AP and AH disappeared and AH induced a stronger antibody response than AP at the highest antigen dose.
Immunoblot analysis revealed qualitative and quantitative differences in the antigenic proteins recognized by antibodies from the mice injected with different WCA formulations. The antibodies from mice injected with adjuvanted WCA reacted with more proteins than those from mice injected with non-adjuvanted WCA. Antibodies from mice injected with WCA/AH and WCA/AP reacted with an overlapping, but different set of proteins. The surface of AH and AP have opposite charges at pH 6-7 resulting in different affinities for individual proteins within the WCA. This may in turn affect which antigens from this complex protein mixture induce antibodies. Further studies are necessary to determine the biological significance of these differences in antibody specificities.
Long term stability of vaccines is an important consideration. In order to assess the stability of the WCA/AH vaccine formulation, the effect of prolonged storage at 4°C on the immune response was determined. There was no significant difference between the stored and freshly prepared formulations indicating that the WCA/AH is quite stable.