Devinder Sehgal

Demonstration That Mast Cells, T Cells, and B Cells Bearing the Activating Kit Mutation D816V Occur in Clusters within the Marrow of Patients with Mastocytosis

Mastocytosis is characterized by focal heterotypic clusters of mast cells and lymphocytes in the bone marrow and by a somatically acquired activating Kit mutation, D816V. The relationship of the occurrence of this mutation to the heterotypic clusters of mast cells and lymphocytes in bone marrow is unknown. We hypothesized that these two unique features of mastocytosis were related. To explore this hypothesis, laser capture microdissected mast cells, B cells, and T cells, from both lesional and non-lesional areas of bone marrow biopsy tissues from patients with mastocytosis, were examined for the D816V mutation in their DNA, using HinfI restriction digestion of nested PCR products amplified from extracts of dissected cells. The D816V mutation was detected in mast cells, B cells, and T cells from lesional but not non-lesional areas of bone marrow tissues. B cells obtained from lesional areas of tissue were also assessed for clonality and were found to at least represent an oligoclonal population. Thus, mast cells and lymphocytes within focal aggregates in the bone marrow of those with mastocytosis are more frequently positive for the codon 816 activating mutation. Further, the B cell population is oligoclonal, suggesting that clonal proliferation is unlikely to be the basis of clustering.

Analysis of Mutational Lineage Trees from Sites of Primary and Secondary Ig Gene Diversification in Rabbits and Chickens

Lineage trees of mutated rearranged Ig V region sequences in B lymphocyte clones often serve to qualitatively illustrate claims concerning the dynamics of affinity maturation. In this study, we use a novel method for analyzing lineage tree shapes, using terms from graph theory to quantify the differences between primary and secondary diversification in rabbits and chickens. In these species, Ig gene diversification starts with rearrangement of a single (in chicken) or a few (in rabbit) VH genes. Somatic hypermutation and gene conversion contribute to primary diversification in appendix of young rabbits or in bursa of Fabricius of embryonic and young chickens and to secondary diversification during immune responses in germinal centers (GCs). We find that, at least in rabbits, primary diversification appears to occur at a constant rate in the appendix, and the type of Ag-specific selection seen in splenic GCs is absent. This supports the view that a primary repertoire is being generated within the expanding clonally related B cells in appendix of young rabbits and emphasizes the important role that gut-associated lymphoid tissues may play in early development of mammalian immune repertoires. Additionally, the data indicate a higher rate of hypermutation in rabbit and chicken GCs, such that the balance between hypermutation and selection tends more toward mutation and less toward selection in rabbit and chicken compared with murine GCs.

Distinct Clonal Ig Diversification Patterns in Young Appendix Compared to Antigen-Specific Splenic Clones

The young rabbit appendix is a dynamic site for primary B cell repertoire development. To study diversification patterns during clonal expansion, we collected single appendix B cells from 3- to 9-wk-old rabbits and sequenced rearranged H and L chain genes. Single cells obtained by hydraulic micromanipulation or laser capture microdissection were lysed, PCR amplified, and products directly sequenced. Gene conversion-like changes occurred in rearranged H and L chain sequences by 3–4 wk of age. Somatic mutations were found in the D regions that lack known conversion donors and probably also occurred in the V genes. A few small sets of clonally related appendix B cells were found at 3–5 wk; by 5.5 wk, some larger clones were recovered. The diversification patterns in the clones from appendix were strikingly different from those found previously in splenic germinal centers where an immunizing Ag was driving the expansion and selection process toward high affinity. Clonally related appendix B cells developed different amino acid sequences in each complementarity-determining region (CDR) including CDR3, whereas dominant clones from spleen underwent few changes in CDR3. The variety of combining sites generated by diversification within individual clones suggests that at least some clonal expansion and selection, known to require normal gut flora, may be driven through indirect effects of microbial components rather than solely by their recognition as specific foreign Ags. This diversity of combining sites within B cell clones supports the proposed role of appendix in generating the preimmune repertoire.

Systematic design and testing of nested (RT-)PCR primers for specific amplification of mouse rearranged/expressed immunoglobulin variable region genes from small number of B cells.

The aim of this study was to develop a highly specific and sensitive (RT-)PCR capable of potentially amplifying the rearranged/expressed VH and VL gene belonging to any mouse immunoglobulin V gene family from a single or a small number of B cells. A database of germline immunoglobulin sequences was used to design 112 primers for a nested (RT-)PCR based strategy to cover all VH, VL, JH, JL, CH and CL gene families/genes from C57BL/6 and BALB/c mice. 93.7% of the primers had 4-fold or less, while 71.4% had no degeneracy. The proportions of germline V genes to which the primers bind with no, up to 1 and up to 2 mismatches are 59.7%, 84.1% and 94.9%, respectively. Most but not all V gene family specific primers designed allow amplification of full-length V genes. The nested primers permit PCR amplification of rearranged V genes belonging to all VH and VL gene families from splenocyte genomic DNA. The V gene family-specific nature of the primers was experimentally confirmed for randomly selected 6 VH and 6 Vkappa families, and all Vlambda genes. The broad V gene family coverage of our primer set was experimentally validated by amplifying the rearranged/expressed VH and VL genes from splenocytes and a panel of 38 hybridomas under conditions where primer mixes and genomic DNA or total RNA was used as starting template. We observed no or low-level cross-family priming. Pooled constant region specific primers allowed efficient RT-PCR amplification of H and L chain isotypes. The expressed VH and VL genes belonging to different V gene families RT-PCR amplified from a mixture of hybridomas in a representative manner. We successfully amplified the expressed VH and Vkappa gene from a single hybridoma cell by RT-PCR and from 10-15 microdissected B cells by genomic PCR. This, first of its kind, comprehensive set of highly sensitive and specific nested primers that provide broad V gene family coverage will open up new avenues and opportunities to study various aspects of mouse B cell biology.

Molecular Dissection of Antibody Responses against Pneumococcal Surface Protein A: Evidence for Diverse DH-Less Heavy Chain Gene Usage and Avidity Maturation

Immunization of human volunteers with a single dose of pneumococcal surface protein A (PspA) stimulates broad cross-reactive Abs to heterologous PspA molecules that, when transferred, protect mice from fatal infection with Streptococcus pneumoniae. In this study, we report the molecular characterization of 36 mouse mAbs generated against the extracellular domain of PspA (PspA3–286) from strain R36A. Abs to PspA3–286 were encoded by diverse VH and Vκ families/genes. The H chain CDR3 and L chain CDR3 lengths were 3–13 (7.8 ± 0.5) and 8–9 (8.7 ± 0.2) codons, respectively. Unexpectedly, seven hybridomas expressed H chains that lack DH gene-derived amino acids. Nontemplate-encoded addition(s) were observed in the H chain expressed in six of these seven hybridomas; Palindromic addition(s) were absent. Absence of DH gene-derived amino acids did not prevent anti-PspA3–286 mAbs from attaining average relative avidity. Avidity maturation occurred during primary IgG anti-PspA3–286 polyclonal Ab response in PspA3–286- and R36A-immunized mice. Compared with PspA3–286-immunized mice, the relative avidity of the primary polyclonal IgG Abs was higher in R36A immunized mice on days 72, 86, and 100. Two pairs of clonally related hybridomas were observed. DH genes expressed in the majority (75.9%) of the hybridomas used reading frame 3. Analysis of replacement/silent mutation ratio in the CDR and framework regions provided evidence for Ag-driven selection in 11 mAbs. Based on epitope localization experiments, the mAbs were classified into 12 independent groups. ELISA additivity assay indicated that members within a group recognized topographically related epitopes. This study provides molecular insights into the biology of DH-less Abs.

Influences of process and formulation parameters on powder flow properties and immunogenicity of spray dried polymer particles entrapping recombinant pneumococcal surface protein A

Particle size, antigen load and its release characteristic are the three the main attributes of polymer particles based vaccine delivery systems. The present studies focus on the formulation of spray dried polylactide microparticles entrapping pneumococcal surface protein A (PspA). Influence of process variables during polymer particle formation were optimized by using half-factorial design. Feed rate and atomization pressure during spray drying were found to be the most important parameters for achieving uniform size particles. Spray drying of preformed particles from different stages of solvent evaporation method resulted in formation of particle having different porosity and protein release profile. Presence of polyvinyl alcohol in the external aqueous phase not only contributed towards regulating the size of particles but also influenced the burst release of protein from particles. Polymer particles entrapping PspA elicited robust IgG responses both in mice and in rats. Antigen load in microparticles correlated with the antibody titer indicating the maintenance of protein integrity during particle formation using spray drying. Both, process engineering and formulation parameters during spray drying influenced the particles in terms of size, load and antigen release characteristics.

Correlation between In Vitro Complement Deposition and Passive Mouse Protection of Anti-Pneumococcal Surface Protein A Monoclonal Antibodies

ABSTRACT The shortcomings of the licensed polysaccharide-based pneumococcal vaccine are driving efforts toward development of a protein-based vaccine that is serotype independent and effective in all age groups. An opsonophagocytic killing assay (OPKA) is used to evaluate the antibody response against polysaccharide-based pneumococcal vaccines. However, the OPKA is not reliable for noncapsular antigens. Thus, there is a need to develop an in vitro surrogate for protection for protein vaccine candidates like pneumococcal surface antigen A (PspA). PspA is a serologically variable cell surface virulence factor. Based on its sequence, PspA has been classified into families 1 (clade 1 and 2), 2 (clades 3, 4 and 5), and 3 (clade 6). Here, we report the characterization of 18 IgG anti-PspA monoclonal antibodies (anti-PspAhkR36A MAbs) generated from mice immunized with heat-killed strain R36A (clade 2). An enzyme-linked immunosorbent assay (ELISA)-based analysis of the reactivity of the MAbs with recombinant PspAs from the 6 clades indicated that they were family 1 specific. This was confirmed by flow cytometry using a hyperimmune serum generated against PspA from R36A. Eight MAbs that bind at least one clade 1- and clade 2-expressing strain were evaluated for complement deposition, bactericidal activity, and passive protection. The anti-PspAhkR36A MAb-dependent deposition of complement on pneumococci showed a positive correlation with passive protection against strain WU2 (r = 0.8783, P = 0.0041). All of our protective MAbs showed bactericidal activity; however, not all MAbs that exhibited bactericidal activity conferred protection in vivo. The protective MAbs described here can be used to identify conserved protection eliciting B cell epitopes for engineering a superior PspA-based vaccine.

Conserved Surface Accessible Nucleoside ABC Transporter Component SP0845 Is Essential for Pneumococcal Virulence and Confers Protection In Vivo

Streptococcus pneumoniae is a leading cause of bacterial pneumonia, sepsis and meningitis. Surface accessible proteins of S. pneumoniae are being explored for the development of a protein-based vaccine in order to overcome the limitations of existing polysaccharide-based pneumococcal vaccines. To identify a potential vaccine candidate, we resolved surface-associated proteins of S. pneumoniae TIGR4 strain using two-dimensional gel electrophoresis followed by immunoblotting with antisera generated against whole heat-killed TIGR4. Ten immunoreactive spots were identified by mass spectrometric analysis that included a putative lipoprotein SP0845. Analysis of the inferred amino acid sequence of sp0845 homologues from 36 pneumococcal strains indicated that SP0845 was highly conserved (>98% identity) and showed less than 11% identity with any human protein. Our bioinformatic and functional analyses demonstrated that SP0845 is the substrate-binding protein of an ATP-binding cassette (ABC) transporter that is involved in nucleoside uptake with cytidine, uridine, guanosine and inosine as the preferred substrates. Deletion of the gene encoding SP0845 renders pneumococci avirulent suggesting that it is essential for virulence. Immunoblot analysis suggested that SP0845 is expressed in in vitro grown pneumococci and during mice infection. Immunofluorescence microscopy and flow cytometry data indicated that SP0845 is surface exposed in encapsulated strains and accessible to antibodies. Subcutaneous immunization with recombinant SP0845 induced high titer antibodies in mice. Hyperimmune sera raised against SP0845 promoted killing of encapsulated pneumococcal strains in a blood bactericidal assay. Immunization with SP0845 protected mice from intraperitoneal challenge with heterologous pneumococcal serotypes. Based on its surface accessibility, role in virulence and ability to elicit protective immunity, we propose that SP0845 may be a potential candidate for a protein-based pneumococcal vaccine.

Delivery of polysaccharides using polymer particles: implications on size-dependent immunogenicity, opsonophagocytosis, and protective immunity.

Bacterial capsular polysaccharides are components of many modern vaccines, but they are weakly immunogenic. Herein, we describe the delivery of pneumococcal capsular polysaccharide serotype-1 (PCP-1) in polylactide polymeric particles to enhance its immunogenicity. Immunization with PCP-1-entrapped particles elicited long-term memory antibody responses from a single intramuscular injection. PCP-1-entrapped nanoparticles (NPs) elicited significantly higher anti-PCP-1 IgG responses than that observed with soluble and microparticles (MPs) formulations. Delivering PCP-1 and pneumococcal proteins in same particles did not improve the IgG response. The sera of animals immunized with PCP-1-entrapped particles promoted efficient opsonophagocytosis of pneumococci by macrophages. Single-dose immunization with PCP-1-entrapped particles conferred a long-term serotype-specific protection against lethal pneumococcal challenge. The higher immunogenicity of PCP-1 nanoparticles showed correlation with enhanced uptake by antigen-presenting cells. The results highlight the potential of polymeric nanoparticles as an efficient means of presenting polysaccharide antigens to the immune system.

Pneumococal Surface Protein A (PspA) Regulates Programmed Death Ligand 1 Expression on Dendritic Cells in a Toll-Like Receptor 2 and Calcium Dependent Manner

Pneumonia leads to high mortality in children under the age of five years worldwide, resulting in close to 20 percent of all deaths in this age group. Therefore, investigations into host-pathogen interactions during Streptococcus pneumoniae infection are key in devising strategies towards the development of better vaccines and drugs. To that end, in this study we investigated the role of S. pneumoniae and its surface antigen Pneumococcal surface protein A (PspA) in modulating the expression of co-stimulatory molecule Programmed Death Ligand 1 (PD-L1) expression on dendritic cells (DCs) and the subsequent effects of increased PD-L1 on key defence responses. Our data indicate that stimulation of DCs with PspA increases the surface expression of PD-L1 in a time and dose dependent manner. Characterization of mechanisms involved in PspA induced expression of PD-L1 indicate the involvement of Toll-Like Receptor 2 (TLR2) and calcium homeostasis. While calcium release from intracellular stores positively regulated PD-L1 expression, calcium influx from external milieu negatively regulated PD-L1 expression. Increase in PD-L1 expression, when costimulated with PspA and through TLR2 was higher than when stimulated with PspA or through TLR2. Further, knockdown of TLR2 and the intermediates in the TLR signaling machinery pointed towards the involvement of a MyD88 dependent pathway in PspA induced PD-L1 expression. Incubation of DCs with S. pneumoniae resulted in the up-regulation of PD-L1 expression, while infection with a strain lacking surface PspA failed to do so. Our data also suggests the role of PspA in ROS generation. These results suggest a novel and specific role for PspA in modulating immune responses against S. pneumoniae by regulating PD-L1 expression.