Abstract
Mucosal or parenteral immunization with a killed unencapsulated pneumococcal whole cell antigen (WCA) with an adjuvant protects mice from colonization by a TH17 CD4+ cell-mediated mechanism. Using preparative SDS gels, we separated the soluble proteins that compose the WCA in order to identify fractions that were immunogenic and protective. We screened these fractions for their ability to stimulate IL-17A secretion from splenocytes obtained from mice immunized with WCA and adjuvant. We identified 12 proteins within the stimulatory fractions by mass spectrometry; these proteins were then cloned, recombinantly expressed and purified using an Escherichia coli expression system. The ability of these proteins to induce IL-17A secretion was then evaluated by stimulation of mouse splenocytes. Of the four most stimulatory proteins, three were protective in a mouse pneumococcal serotype 6B colonization model. This work thus describes a method for identifying immunogenic proteins from the soluble fraction of pneumococcus and shows that several of the proteins identified protect mice from colonization when used as mucosal vaccines. We propose that, by providing protection against pneumococcal colonization, one or more of these proteins may serve as components of a multivalent pneumococcal vaccine.
Highlights
While pneumococcal conjugate vaccines (PCVs) have resulted in significant reductions in rates of invasive pneumococcal disease due to vaccine-serotypes, infection with Streptococcus pneumoniae remains an important public health issue, in developing countries [1]
We have shown that inactivation methods using betapropiolactone or organic solvents, such as chloroform, that retain the soluble proteins of RM200 produce significantly more potent vaccine preparations, such that 100 times lower whole cell antigen (WCA) concentration confers the same immune and protective responses as compared to a WCA
Preparation in which soluble proteins have been removed [8]. These data led us to explore the hypothesis that proteins with protective potential may be present in the soluble fraction of the WCA: consistent with this hypothesis, we showed that intranasal immunization with the soluble fraction of chloroform-inactivated WCA conferred highly significant reductions in colonization compared with adjuvant-alone immunized mice [8]
Summary
While pneumococcal conjugate vaccines (PCVs) have resulted in significant reductions in rates of invasive pneumococcal disease due to vaccine-serotypes, infection with Streptococcus pneumoniae remains an important public health issue, in developing countries [1]. The impact of PCVs on mucosal disease such as otitis media and pneumonia has been less clear. For these reasons and with the goal of developing a vaccine with lower cost of goods and complexity of manufacture than PCVs, alternative approaches to pneumococcal vaccination are a high global health priority. Our group has shown that either mucosal or parenteral immunization with a killed unencapsulated pneumococcal whole cell vaccine (denoted WCV when administered with appropriate adjuvant) protects mice against pneumococcal colonization with a clinical isolate of serotype 6B and fatal aspiration-sepsis with isolates of serotypes 3 and 5 [6,7,8]. In the mouse model used, congenital absence of antibodies, IFN-c or IL-4 has no effect, whereas depletion of CD4+ T cells or congenital absence of the IL-17A receptor is associated with loss of protection against pneumococcal colonization [10,11]
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