Molecular basis of B-cell responses

Infections caused by medically important extracellular bacteria are critically controlled by B-cells. Thus, it is important to understand the induction and regulation of B-cell responses against extracellular bacteria. We use unencapsulated Streptococcus pneumoniae as a model system to understand the molecular basis of murine B-cell responses. Unencapsulated strains of S. pneumoniae have numerous potentially immunogenic protein and polysaccharide antigens on its cell surface. Immunity to S. pneumoniae is primarily conferred by antibodies specific to cell surface determinants. The theme of this project is to decipher the molecular basis of spleen-dependent induction and regulation of B-cell responses against S. pneumoniae. The main objectives of the project are to (i) study the architecture and kinetics of development of splenic germinal centers, (ii) investigate the population genetics and selection of B-cells in splenic germinal centers, and (iii) correlate the mutation(s)/mutational pattern(s) observed in the rearranged immunoglobulin variable region genes with the process of affinity maturation. These objectives will be studied during primary as well as secondary B-cell responses.

Molecular cloning, expression and purification of recombinant pneumococcal surface protein A (PspA)

This group initiated its research activities by successfully establishing in vitro conditions for propagation and maintenance of S. pneumoniae in the laboratory. We have concentrated our initial efforts towards studying the anti-protein B-cell responses following immunization with heat-killed unencapsulated S. pneumoniae strain R36A. For this purpose we have selected Pneumococcal Surface Protein A (PspA) as a representative cell surface protein. We choose PspA because it is expressed in great majority of pneumococcal strains and it is a promising vaccine candidate. PspA is made up of three major amino acid sequence domains. The choline-binding domain at the C-terminus attaches the protein to the cell surface. Upstream of this domain is the proline-rich domain, which is thought to span the cell wall and capsule layer. N-terminal to the proline rich domain is the a-helical domain, which is exposed on the bacterial surface and is thought to form an anti-parallel coiled-coil structure reminiscent of many other fibrillar surface proteins on gram-positive bacteria. Passive immunization with anti-PspA antibodies or active immunization with purified full-length or truncated PspA has been shown to protect mice against infection with pneumococci of different serotypes. DNA vaccines expressing the N-terminal half of PspA or its fragments elicits protective immunity against S. pneumoniae. Prior studies of PspA have shown that protective antibodies are reactive with epitopes in the a-helical domain and that most cross-protective epitopes mapped to the C-terminal half of the a-helical region. The cell surface exposed a-helical region of PspA from R36A was PCR amplified (amino acid residue 3 to 286 of mature peptide; pspA-01) using Pfu DNA polymerase. The PCR product was cloned in the expression vector pQE-30 and sequenced. DNA sequence based homology search revealed 77 to 100% identity with PspA gene sequence from other S. pneumoniae strains. A subfragment of PspA (amino acid residue 191 to 260 of the mature peptide; pspA-02) to which majority of the protection eliciting B-cell epitopes have been mapped previously was also cloned and sequenced. pspA-01 and pspA-02 were expressed as 6xHis-tagged recombinant proteins in Escherichia coli and were purified to homogeneity by Ni-NTA affinity chromatography under denaturing conditions with a yield of ~6 mg/l.

Prediction of PspA B-cell epitopes using computational approaches

As PspA is a highly immunogenic protein, it is reasonable to assume that it will possess multiple B-cell epitopes. Thus, during an anti-PspA immune response it is likely that B-cells with a variety of immunoglobulin receptors will be recruited. In order to simplify the analysis and to limit the complexity of the PspA-specific B-cell repertoire being analyzed in splenic germinal centers, we attempted to identify regions in PspA that contain one or few B-cell epitopes using computational approaches. We choose PspA-02 for this analysis as majority of the protection eliciting B-cell epitopes have been mapped previously to this region of PspA. These predictions were made based on the assumption that the antigenic determinants would be exposed on the surface of the protein and thus would be located in hydrophilic regions. Several different algorithms were used for analyzing the sequence of PspA-02 for properties like hydrophilicity (Kyte/Doolittle, Hopp/Woods, GES, and von Heijne methods), antigenicity (Parker, Welling, protrusion index, and antigenic index methods), flexibility and surface probability. The Chou–Fasman and Robson–Garnier methods predicted the secondary structure of PspA-02 to be predominantly a-helical as shown previously. A surface probability profile was generated to predict which regions in PspA-02 were most likely to lie on its surface, based on knowledge of which amino acid residues were more likely to be found on the surface of proteins of known structures. Analysis of surface probability profile revealed that all amino acid residues in this region with the exception of a few (209–211 and 238–241) were surface localized. Segmental flexibility, which appears to correlate with known antigenic determinants, was used to predict potentially antigenic sites by locating regions of the protein chain that might be relatively flexible. Segmental flexibility analysis indicated that amino acids 191–208 showed maximum flexibility followed by 250–260 and 211–235. The amino acid residues that were not surface localized were not flexible. Further analysis indicated that PspA-02 was predominantly hydrophilic with peaks at amino acid positions 191–209, 215–238 and 250–260. Similarly, protrusion and Parker antigenicity indices predicted the entire sequence to be antigenic whereas Welling antigenicity index showed dominant peaks at amino acid positions 211–230 and 242–251. Based on the above analyses it can be predicted that the B-cell epitopes are likely to be restricted to the region in and around amino acid positions 191–209, 215–235 and 250–260. Synthetic peptides or recombinant proteins corresponding to these regions will be used as reagents to determine serum antibody titers as well as to identify PspA-specific B-cells and splenic germinal centers.

B-cell response to PspA

In order to understand the molecular basis of B-cell response against PspA it is important to determine the serum anti-PspA antibody titers during primary and secondary B-cell response following immunization with heat-killed R36A. rPspA-01 (~38 kDa) and rPspA-02 (~14 kDa) are currently being used as antigens in ELISA to follow the serum anti-PspA antibody response at days 7, 14 and 21 post-immunization. We have initiated efforts to systematically study the architecture and kinetics of development of PspA-specific splenic germinal centers during the primary and secondary immune response following intraperitoneal immunization with heat-killed R36A using the reagents described above. Splenic sections from immunized mice are currently being analyzed immunohistochemically using antibodies to various differentiation stage and cell type specific markers. As a first step towards investigating the population genetics and selection of B-cells in splenic germinal centers we have designed PCR primers for the second largest immunoglobulin heavy chain V_H family, V_H5. V_H5 family comprises ~20% of all known functional V_H genes. The primers for PCR amplifying the rearranged V_H5 family members are directed against FR1 and J_H regions. We have also designed primers for the two largest immunoglobulin light chain V_k families, V_k4/5 and V_k9/10. V_k4/5 and V_k9/10 families comprise ~40% of all known functional Vk genes. The primers for PCR amplifying the rearranged Vk4/5 and Vk9/10 family members are directed against Lk, Lk-Vk intron and Jk regions. These primers will be used in nested PCR to amplify the rearranged V_H5, V_k4/5 and V_k9/10 family members using DNA recovered from B-cells microdissected from immunohistochemically defined splenic germinal centers. Additional primers sets will be designed to amplify rearranged immunoglobulin heavy and light chain genes belonging to other V_H and V_L families. The sequence of the rearranged V_H and V_L genes from antigen specific B-cells will be analyzed for genealogical relationship, clonal diversity and somatic hypermutation events/patterns.

Publications

Original peer-reviewed articles

1.     ¶*Obiakor H, Sehgal D, Dasso JF, Bonner RF, Malekafzali A and Mage RG (2002) A comparison of hydraulic and laser capture microdissection methods for collection of single B cells, PCR, and sequencing of antibody VDJ. Anal Biochem 306:55-62 (¶on deputation/work done elsewhere, *in press last year, since published).

2.     ¶*Sehgal D, Obiakor H and Mage RG (2002) Distinct clonal Ig diversification patterns in young appendix compared to antigen-specific splenic clones. J Immunol 168:5424-5433 (¶on deputation/work done elsewhere, *in press last year, since published).