The ToxAvapA toxin-antitoxin locus contributes to the survival of nontypeable Haemophilus influenzae during infection.

Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen that is a common cause of acute and recurrent mucosal infections. One uncharacterized NTHi toxin-antitoxin (TA) module, NTHI1912-1913, is a host inhibition of growth (higBA) homologue. We hypothesized that this locus, which we designated toxAvapA, contributed to NTHi survival during infection. We deleted toxAvapA and determined that growth of the mutant in defined media was not different from the parent strain. We tested the mutant for persistence during long-term in vitro co-culture with primary human respiratory tissues, which revealed that the ΔtoxAvapA mutant was attenuated for survival. We then performed challenge studies using the chinchilla model of otitis media and determined that mutant survival was also reduced in vivo. Following purification, the toxin exhibited ribonuclease activity on RNA in vitro, while the antitoxin did not. A microarray comparison of the transcriptome revealed that the tryptophan biosynthetic regulon was significantly repressed in the mutant compared to the parent strain. HPLC studies of conditioned medium confirmed that there was no significant difference in the concentration of tryptophan remaining in the supernatant, indicating that the uptake of tryptophan by the mutant was not affected. We conclude that the role of the NTHi toxAvapA TA module in persistence following stress is multifactorial and includes effects on essential metabolic pathways.

Multiplexed immunoassay to assess Shigella-specific antibody responses.

Infection with Shigella spp. results in bacillary dysentery and a systemic and mucosal antibody response. The immune response is directed at multiple antigens, including LPS and the invasin plasmid antigen (Ipa) proteins, and is capable of conferring short-term, serotype-specific protection. Both live-attenuated and several subunit vaccine approaches have focused on inducing a pronounced mucosal immune response directed to the same antigens recognized after natural infection. Traditionally, Shigella-specific antibody responses are measured using ELISA. Although ELISAs are robust immunological assays, limited sample volume and assay costs often precludes its use for immunological evaluation against multiple antigens. To overcome these shortcomings, a novel assay has been developed to simultaneously measure specific antibody levels to six Shigella antigens, including three serotype-specific LPS preparations and three conserved protein antigens in a Luminex™-based system. Coupling methods were optimized to covalently link recombinant Ipa proteins (IpaB, IpaC, and IpaD) and purified LPS from Shigella sonnei, Shigella flexneri 2a, and Shigella dysenteriae 1 to unique bead sets. The antigen-coupled beads maintained stable reactivity with monoclonal antibodies (mAbs) for 6 weeks (protein) to 3 months (LPS). The specificity of the Luminex assay was similar to an ELISA, with the multiplexed assay providing a larger dynamic range. Comparable levels of antigen-specific reactivity were attained in monoplex or multiplex formats indicating limited interference. The correlations (R(2)) between the ELISA and the multiplexed assay along with the repeatability and reproducibility of the assay were very high. Minor changes in species-specific conjugated secondary antibodies allowed the optimized multiplexed assays to be used to assess antigen-specific antibodies in serum or blood eluted from filter paper isolated from mice and guinea pigs highlighting applicability of the assay for seroepidemiology and pre-clinical/clinical vaccine studies.

Safety and immunogenicity of an intranasal Shigella flexneri 2a Invaplex 50 vaccine.

BACKGROUND: Shigella flexneri 2a lipopolysaccharide 50 is a nasally delivered subunit vaccine consisting of a macromolecular complex composed of LPS, IpaB, IpaC and IpaD. The current study examined vaccine safety and immunogenicity across a dose range and the clinical performance of a new intranasal delivery device. METHODS: Volunteers (N=36) were randomized to receive vaccine via the Dolphin™ (Valois of America, Congers, New York) intranasal spray device at one of three doses (240, 480, and 690 µg) on days 0, 14, and 28. Another group (N=8) received the 240 µg dose via pipette. Vaccine safety was actively monitored and antigen-specific humoral and mucosal immune responses were determined. RESULTS: There were no serious adverse events and the majority of adverse events (98%) were mild. Antibody secreting cells (ASC), plasma, and mucosal immune responses to Shigella antigens were detected at all three dose levels with the 690 µg dose inducing the highest magnitude and frequency of responses. Vaccination with comparable doses of Invaplex 50 via the Dolphin™ resulted in higher plasma and ASC immune responses as compared to pipette delivery. CONCLUSION: In this trial the S. flexneri 2a Invaplex 50 vaccine was safe, well-tolerated and induced robust levels of antigen-specific intestinal IgA and ASC responses. The spray device performed well and offered an advantage over pipette intranasal delivery.

Virulence, inflammatory potential, and adaptive immunity induced by Shigella flexneri msbB mutants.

The ability of genetically detoxified lipopolysaccharide (LPS) to stimulate adaptive immune responses is an ongoing area of investigation with significant consequences for the development of safe and effective bacterial vaccines and adjuvants. One approach to genetic detoxification is the deletion of genes whose products modify LPS. The msbB1 and msbB2 genes, which encode late acyltransferases, were deleted in the Shigella flexneri 2a human challenge strain 2457T to evaluate the virulence, inflammatory potential, and acquired immunity induced by strains producing underacylated lipid A. Consistent with a reduced endotoxic potential, S. flexneri 2a msbB mutants were attenuated in an acute mouse pulmonary challenge model. Attenuation correlated with decreases in the production of proinflammatory cytokines and in chemokine release without significant changes in lung histopathology. The levels of specific proinflammatory cytokines (interleukin-1beta [IL-1beta], macrophage inflammatory protein 1alpha [MIP-1alpha], and tumor necrosis factor alpha [TNF-alpha]) were also significantly reduced after infection of mouse macrophages with either single or double msbB mutants. Surprisingly, the msbB double mutant displayed defects in the ability to invade, replicate, and spread within epithelial cells. Complementation restored these phenotypes, but the exact nature of the defects was not determined. Acquired immunity and protective efficacy were also assayed in the mouse lung model, using a vaccination-challenge study. Both humoral and cellular responses were generally robust in msbB-immunized mice and afforded significant protection from lethal challenge. These data suggest that the loss of either msbB gene reduces the endotoxicity of Shigella LPS but does not coincide with a reduction in protective immune responses.