Elderly, very young and immunocompromised individuals are particularly susceptible to infection by Streptococcus pneumoniae (S. pn.). We have previously shown that an immune response to phosphorylcholine (PC), an antigenic determinant expressed on the surface of S. pn. (and many other pathogens) is sufficient to confer protection to challenge by S. pn. We have demonstrated that the mouse VH1 immunoglobulin heavy chain gene is necessary and sufficient to generate a PC-mediated protective immune response against S. pn. Structural determinants of immunoglobulin light chains which may explain the observed differences in the relative affinity/avidity of VH1/VL antibodies for PC have been defined and we are continuing to examine the complimentary contribution of specific VH and VL genes to defining a protective versus an ineffective immune response. We have developed and patented a novel, inexpensive strategy for synthesizing PC-containing compounds which can be used to generate PC-protein conjugates. Immunization of mice with PC-conjugates prepared via this novel synthesis strategy resulted in a long lasting increase in PC-specific antibody titer. Progression towards the development of PC-conjugates as a vaccine for use in humans requires that issues of formulation including adjuvant and carrier protein be addressed. Immunization of mice with EPC-KLH conjugates in combination with Alum (an adjuvant approved for use in humans by the USFDA) as an adjuvant is capable of generating a protective PC-specific immune response. In addition, conjugates of EPC prepared with the CRM197 mutant of DPT (a carrier protein currently in use for vaccines currently approved by USFDA) as a carrier protein and combined with CFA/IFA as adjuvant have also generated a protective PC-specific response in immunized mice. Work continues towards identifying a formulation of EPC-DPT with Alum as an adjuvant that produces a protective PC-specific immune response in vaccinated mice. We are establishing a CRADA with Novartis to examine additional conjugates using pilus proteins from S. pn. as the carrier proteins as well as examining their proprietary adjuvant MF59 in vaccine formulations. Studies to identify potential adverse consequences associated with vaccination have been performed. Gross and histopathological examination of immunized mice failed to show any evidence of vaccine associated immune pathology. A collaborative effort is currently underway to examine the effectiveness of anti-PC antibodies and PC-conjugates to confer protection against challenge by Niesseria sp. as well. In a preliminary study, PC-specific B cells have been identified in peripheral blood lymphocytes of humans and studies to characterize these PC-specific B cells are ongoing. ? Based on our observations in PC transgenic mouse models, we proposed that compromising a central tenet to the clonal selection theory (single B cell, single specificity, and single antigen receptor) was beneficial and necessary for the host. We suggested that in order to prevent the elimination of B cells expressing certain immunoglobulin receptors that were autoreactive but that had been evolutionarily selected to confer protection against various pathogens; these cells expressed two distinct receptors with different specificities. We termed this mechanism of rescuing autoreactive cells receptor dilution. In our current studies we have demonstrated the generality of this mechanism by showing that dual isotype expressing B cells are present in normal, nontransgenic C57BL/6 mice. These studies also indicated that dual isotype expressing B cells most likely arise during normal B cell development in the bone marrow and that a significant proportion of dual isotype expressing B cells may be generated by receptor editing. Consistent with our original hypothesis, the inferred specificities of the VH and VK genes expressed by this small population of B cells are to both self antigens and antigens expressed on numerous pathogens. Ongoing studies continue to extend the receptor dilution model in order to provide a greater understanding about the role of dual receptor expression in establishing and maintaining a protective immune repertoire. ? We have demonstrated that TdT (terminal deoxynucleotidyl transferase) plays an important role in shaping the PC-specific immune repertoire. Our studies established that generation of the dominant M603id (idiotype) anti-PC response to immunization with Proteus morganii is dependent on TdT expression. The impact of TdT expression on shaping the immune repertoire has been extended in subsequent studies in which we have revealed that TdT expression affects the size of the autoreactive, PtC (phosphatidylcholine)-specific B1 B cell population and most likely the VH and VL gene repertoire expressed in these PtC-specific B cells. We continue to examine B cell populations in the TdT-KO mice to determine the identity of the VH and VL genes expressed in this expanded autoreactive PtC-specific population and to determine the mechanism by which TdT shapes the size of and VH/VL gene repertoire of this and other autoreactive B cell populations.
