Suppressive effect of bacterial polysaccharides on BAFF system is responsible for their poor immunogenicity.

Capsular polysaccharides of encapsulated bacteria are weakly immunogenic T cell-independent type 2 (TI-2) Ags. Recent findings suggest that BAFF system molecules have a critical role in the development of Ab responses against TI-2 Ags. In this study, we investigated the effect of bacterial polysaccharides on B cell responses to BAFF and a proliferation-inducing ligand (APRIL). We determined that B cells exposed to meningococcal type C polysaccharide (MCPS) or group B Streptococcus serotype V (GBS-V) were unresponsive to BAFF- and APRIL-induced Ig secretion. Moreover, MCPS and GBS-V strongly downregulated transmembrane activator and calcium-modulator and cyclophilin ligand interactor, the BAFF and APRIL receptor that is responsible for Ab development against TI-2 Ags. Interestingly, (4-hydroxy-3-nitrophenyl)acetyl-Ficoll (NP-Ficoll), a prototype TI-2 Ag, did not manifest a suppressive effect on B cells. Paradoxically, whereas GBS-V and MCPS inhibited IFN-γ-induced BAFF production from dendritic cells, NP-Ficoll strongly increased BAFF secretion. TLR 9 agonist CpG deoxyoligonucleotide (ODN) was able to reverse the MCPS-mediated transmembrane activator and calcium-modulator and cyclophilin ligand interactor suppression but could not rescue the Ig secretion in BAFF- or APRIL-stimulated B cells. In support of these in vitro observations, it was observed that CpG ODN could help augment the Ab response against NP in mice immunized with a CpG ODN-containing NP-Ficoll vaccine but exhibited only marginal adjuvant activity for MCPS vaccine. Collectively, these results suggest a mechanism for the weak immunogenicity of bacterial polysaccharides and explain the previously observed differences between bacterial polysaccharide and NP-Ficoll immunogenicity.

Anthrax protective antigen delivered by Salmonella enterica serovar Typhi Ty21a protects mice from a lethal anthrax spore challenge.

Bacillus anthracis, the etiological agent of anthrax disease, is a proven weapon of bioterrorism. Currently, the only licensed vaccine against anthrax in the United States is AVA Biothrax, which, although efficacious, suffers from several limitations. This vaccine requires six injectable doses over 18 months to stimulate protective immunity, requires a cold chain for storage, and in many cases has been associated with adverse effects. In this study, we modified the B. anthracis protective antigen (PA) gene for optimal expression and stability, linked it to an inducible promoter for maximal expression in the host, and fused it to the secretion signal of the Escherichia coli alpha-hemolysin protein (HlyA) on a low-copy-number plasmid. This plasmid was introduced into the licensed typhoid vaccine strain, Salmonella enterica serovar Typhi strain Ty21a, and was found to be genetically stable. Immunization of mice with three vaccine doses elicited a strong PA-specific serum immunoglobulin G response with a geometric mean titer of 30,000 (range, 5,800 to 157,000) and lethal-toxin-neutralizing titers greater than 16,000. Vaccinated mice demonstrated 100% protection against a lethal intranasal challenge with aerosolized spores of B. anthracis 7702. The ultimate goal is a temperature-stable, safe, oral human vaccine against anthrax infection that can be self-administered in a few doses over a short period of time.

Effect of increased CRM197 carrier protein dose on meningococcal C bactericidal antibody response.

New multivalent CRM(197)-based conjugate vaccines are available for childhood immunization. Clinical studies were reviewed to assess meningococcal group C (MenC) antibody responses following MenC-CRM(197) coadministration with CRM(197)-based pneumococcal or Haemophilus influenzae type b conjugate vaccines. Infants receiving a total CRM(197) carrier protein dose of ∼50 µg and concomitant diphtheria-tetanus-acellular pertussis (DTaP)-containing vaccine tended to have lower MenC geometric mean antibody titers and continued to have low titers after the toddler dose. Nevertheless, at least 95% of children in the reported studies achieved a MenC serum bactericidal antibody (SBA) titer of ≥ 1:8 after the last infant or toddler dose. SBA was measured using an assay with a baby rabbit or human complement source. Additional studies are needed to assess long-term antibody persistence and MenC CRM(197) conjugate vaccine immunogenicity using alternative dosing schedules.

Multilaboratory comparison of Streptococcus pneumoniae opsonophagocytic killing assays and their level of agreement for the determination of functional antibody activity in human reference sera.

Antibody-mediated killing of Streptococcus pneumoniae (pneumococcus) by phagocytes is an important mechanism of protection of the human host against pneumococcal infections. Measurement of opsonophagocytic antibodies by use of a standardized opsonophagocytic assay (OPA) is important for the evaluation of candidate vaccines and required for the licensure of new pneumococcal conjugate vaccine formulations. We assessed agreement among six laboratories that used their own optimized OPAs on a panel of 16 human reference sera for 13 pneumococcal serotypes. Consensus titers, estimated using an analysis-of-variance (ANOVA) mixed-effects model, provided a common reference for assessing agreement among these laboratories. Agreement was evaluated in terms of assay accuracy, reproducibility, repeatability, precision, and bias. We also reviewed four acceptance criterion intervals for assessing the comparability of protocols when assaying the same reference sera. The precision, accuracy, and concordance results among laboratories and the consensus titers revealed acceptable agreement. The results of this study indicate that the bioassays evaluated in this study are robust, and the resultant OPA values are reproducible for the determination of functional antibody titers specific to 13 pneumococcal serotypes when performed by laboratories using highly standardized but not identical assays. The statistical methodologies employed in this study may serve as a template for evaluating future multilaboratory studies.

Neisseria meningitidis type C capsular polysaccharide inhibits lipooligosaccharide-induced cell activation by binding to CD14.

Encapsulated Neisseria meningitidis can invade mucosal barriers and cause systemic diseases. Activation of the innate immune system by conserved meningococcal molecules such as lipooligosaccharides (LOS) is essential for the generation of an effective host immune response. Here we show that the type C capsular polysaccharide of N. meningitidis (MCPS) inhibited LOS-induced interleukin-6 and TNF-alpha secretion from monocytes, and blocked the maturation of dendritic cells induced by LOS, while the capsular polysaccharide from group B streptococcus type III and t(4-hydroxy-3-nitrophenyl) acetyl (NP)-Ficoll had no such effect. MCPS also inhibited the LOS-induced NF-kappaB activation and phosphorylation of signalling molecules such as ERK1/2, p38 and Jun N-terminal kinase. In a direct binding assay, MCPS manifested a concentration-dependent binding to recombinant lipoprotein binding protein and CD14, the two members of the LOS receptor complex. In addition, the binding of LOS to CD14 and lipopolysaccharide binding protein was inhibited by MCPS. We established that MCPS binding to CD14 is responsible for the inhibition of LOS-mediated cell activation because MCPS inhibition of LOS was reversed when access amounts of CD14 were added to culture media of HEK293 cells expressing TLR4 and MD-2, and the magnitude of recovery in LOS stimulation correlated with the increase in CD14 concentration. These results suggest a new virulence property of meningococcal capsular polysaccharides.

Group A streptococcus (GAS) carbohydrate as an immunogen for protection against GAS infection.

Previous studies have shown that human serum containing anti-group A streptococcus carbohydrate (GAS CHO) antibodies were opsonic for different M protein-carrying serotypes. To investigate the role that anti-GAS CHO antibodies play in passive and active protection, mice were immunized subcutaneously or intranasally with GAS CHO conjugated to tetanus toxoid, and mortality and oral colonization were monitored after challenge with live GAS. Compared with control mice, immunized mice were significantly protected against systemic or nasal challenge with GAS. Furthermore, studies of serum samples and throat cultures from Mexican children revealed an inverse relationship between high serum titers of anti-GAS CHO antibodies and the presence of GAS in the throat. Anti-GAS CHO antibodies were also tested for cross-reactivity with human tissues and cytoskeletal proteins. No cross-reactivity was observed in either assay. The present study demonstrates that GAS CHO is both immunogenic and protective against GAS infections.

Doubly branched hexasaccharide epitope on the cell wall polysaccharide of group A streptococci recognized by human and rabbit antisera.

A number of epitope specificities associated with the cell wall polysaccharide antigen of group A streptococci were identified in a polyclonal rabbit antiserum induced in rabbits by whole group A streptococci and in polyclonal convalescent human antisera from children that had recovered from streptococcal A infections. The identification was achieved by using a series of synthetic oligosaccharides, glycoconjugates, and bacterial polysaccharide inhibitors to inhibit the binding of the group A helical polysaccharide to the polyclonal antisera. The exclusively dominant epitope expressed in the convalescent human antisera was the doubly branched extended helical hexasaccharide with the structure alpha-L-Rhap(1-->2)[beta-D-GlcpNAc(1-->3)]alpha-L-Rhap(1-->3)alpha-L-Rhap(1-->2)[beta-D-GlcpNAc(1-->3)]alpha-L-Rhap. The hexasaccharide epitope also bound with the highest immunoreactivity to the rabbit antiserum. In contrast, the human antisera did not show significant binding to the singly branched pentasaccharide with the structure alpha-L-Rhap(1-->2)alpha-L-Rhap(1-->3)alpha-L-Rhap(1-->2)[beta-D-GlcpNAc(1-->3)]alpha-L-Rhap or the branched trisaccharide alpha-L-Rhap(1-->2)[beta-D-GlcpNAc(1-->3)]alpha-l-Rhap, although both these haptens bound significantly to the same rabbit antiserum, albeit with less immunoreactivity than the hexasaccharide. Inhibition studies using streptococcal group A and B rabbit antisera and the inhibitors indicated above also suggested that the group A carbohydrate, unlike the group B streptococcal polysaccharide, does not contain the disaccharide alpha-L-Rhap(1-->2)alpha-L-Rhap motif at its nonreducing chain terminus, stressing the importance of mapping the determinant specificities of these two important streptococcal subcapsular group polysaccharides to fully understand the serological relationships between group A and group B streptococci.

Neisseria gonorrhoeae porin, P.IB, causes release of ATP from yeast actin.

Neisserial porins may play a role in the invasion of the host cell by the bacterium. The protein translocates to the host cell membrane and then to the cytosol during the invasive process, and we have shown it interacts with actin in vitro. Here, we have examined the nucleotide-dependence of the interaction of Neisseria porin, P.IB, with fluorescently labeled yeast G actin. Increasing free ATP between 0 to 0.5 mM retards complex formation between the two proteins. The ATP effect probably results from binding of the nucleotide to actin rather than to porin. Complex formation results in a biphasic release of bound nucleoside triphosphate from actin in the absence of free nucleotide at a rate slower than that of complex formation, but it does not induce hydrolysis of the actin-bound nucleotide. ATP prevents the porin-induced distortion of F-actin structure, and addition of ATP to the complex formed in the absence of free nucleotide induces actin polymerization indicating that P.IB stabilizes nucleotide-free G-actin. Our results suggest that P.IB causes an actin conformation change leading to the production of a polymerization-competent nucleotide-free protein.

A co-operative interaction between Neisseria gonorrhoeae and complement receptor 3 mediates infection of primary cervical epithelial cells.

Little is known about the pathogenesis of gonococcal infection within the lower female genital tract. We recently described the distribution of complement receptor 3 (CR3) on epithelia of the female genital tract. Our studies further indicate that CR3-mediated endocytosis serves as a primary mechanism by which N. gonorrhoeae elicits membrane ruffling and cellular invasion of primary, human, cervical epithelial cells. We have extended these studies to describe the nature of the gonococcus-CR3 interaction. Western Blot analysis demonstrated production of alternative pathway complement components by ecto- and endocervical cells which allows C3b deposition on gonococci and its rapid conversion to iC3b. Anti-iC3b and -factor I antibodies significantly inhibited adherence and invasion of primary cervical cells, suggesting that iC3b covalently bound to the gonococcus serves as a primary ligand for CR3 adherence. However, gonococcal porin and pili also bound to the I-domain of CR3 in a non-opsonic manner. Binding of porin and pili to CR3 were required for adherence to and invasion of cervical epithelia. Collectively, these data suggest that gonococcal adherence to CR3 occurs in a co-operative manner, which requires gonococcal iC3b-opsonization, porin and pilus. In conjunction, these molecules facilitate targeting to and successful infection of the cervical epithelium.