NTHI is an opportunistic pathogen capable of causing respiratory infections, yet it is commonly found in a harmless association with humans. Research efforts have focused on examining the antigenicity and immunogenicity of surface antigens that might offer potential as components for an effective vaccine against infections caused by NTHI.
In this study, we have reported the cloning, sequencing, and expression of a protein which has been called OMP26. Sequence analysis has revealed that the gene for this protein is nearly identical to a gene identified in the H. influenzae
Rd genome that may be related to the skp/ompH
gene family (1
). The fact that a cleavable signal peptide sequence exists in the preprotein suggests that the mature protein is destined for export across the cytoplasmic membrane via the general secretory pathway (27
). Previous experiments involving OMP extraction and immunolabeling of NTHI with antisera to OMP26 showed surface binding of gold-conjugated particles under electron microscopy (19
), suggesting that it is a component of the outer membrane of NTHI. However, there is good evidence that in E. coli
Skp/OmpH is a soluble periplasmic protein (30
). Clearly, further studies are needed to determine if Skp/OmpH has undergone adaptive changes in H. influenzae
so as to locate to the outer membrane or is to some extent peripherally associated with the outer membrane in all species. Skp is highly basic and in E. coli
binds strongly to lipopolysaccharide (LPS) when released artificially (for example, by lysis) from the periplasm (30
). Perhaps it also binds to LPS in vivo during the export of LPS and/or porin proteins (23
The role of Skp proteins and their homologues in bacterial cellular functions remains speculative. Thome and colleagues (29
) presented evidence supporting a role for such proteins in the export of outer membrane proteins and/or other macromolecules, but the exact nature of the mechanism(s) involved remains to be elucidated. It has been postulated that they might behave like the chaperonins, since they appear to be active on either side of the plasma membrane in E. coli
). A recent study demonstrated that the Skp protein from E. coli
appears to act as a periplasmic molecular chaperone (3
) which may assist OMP transport from the cytoplasmic membrane to the outer membrane. The fact that the skp
gene is located in a complex operon governing the early steps of LPS synthesis suggests a role for this family of proteins in the synthesis of LPS (7
). Indeed, the gene organization of the skp
gene and homologous sequences appears to be highly conserved in gram-negative bacteria examined to date, which suggests an important function (1
). The role of OMP26 in NTHI strains has yet to be determined.
The significance of the consistency of the relatively defined amino acid changes observed in the various strains has yet to be determined. The change to Asn and Lys from Asp and Arg at positions 146 and 157, respectively, probably does not affect the cross-protective immune response. Immunization with the native OMP26 from NTHI-289 cleared the NTHI-II strain following pulmonary challenge in a previous study (19
). The significance of some other amino acid differences in under investigation. In Fig. , only two of the sequences were derived from strains originating outside of Australia (however, it should be noted that the Australian strains were selected from a collection dating over a 10-year period and from two geographical locations). Two other sequences of the same regions of the Haemophilus
genome appear in the GenBank database. A 129-amino-acid segment from H. influenzae
Eagan (accession no. U60832
) and H. influenzae
isolate 33 (accession no. U60831
) show 100 and 96% homologies, respectively. Interestingly, three of the substitutions in isolate 33 were shared with isolates in this study.
Immunization studies with the native OMP26 protein showed significant potential for this protein antigen as a vaccine candidate against pulmonary infections by NTHI in animal studies (19
). The availability of the recombinant protein, precursor and mature, facilitates a greater understanding of the antigenicity and immunogenicity of OMP26 and provides a basis for the development of the potential of OMP26 as a candidate for a vaccine against NTHI infections. Bacterial clearance from the lungs in rats following mucosal immunization with either killed bacteria or protein has been used to identify and investigate potential vaccine antigens (4
). Antibody responses to antigens that arise as a result of either infection or immunization play a role in facilitating bacterial clearance. This is achieved by a number of mechanisms including bactericidal activity and opsonophagocytosis by polymorphonuclear leukocytes (26
); however, in mucosal defenses the role of IgA in inhibiting microbial adherence is a significant first line defense against infection (22
). With respect to antibody titer and specificity, immunization with the recombinant OMP26 that included the leader sequence induced higher titers of both IgG and IgA that recognized both recombinant proteins equivalently. Immunization with the equivalent of the processed OMP26 (no leader peptide) still resulted in a significant antibody response; however, the titers were significantly lower, although the specificity for both forms of the recombinant were similar for IgG but not IgA. This finding suggests that the influence of antibody on the differences seen in the rate of the bacterial clearance may not be a result of differences in specificity but could have been associated with concentration of antibody to OMP26.
The significance of cell-mediated immunity in mucosal defenses is now well recognized (8
). Enhanced pulmonary clearance of NTHI (33
) and Pseudomonas aeruginosa
) has been found in rat models following transfer of T cells from immunized rats to naive rats. A study of T- and B-cell responses following immunization with the OMP designated P2 from NTHI found that enhancement of the T-cell response in the presence of suppressed B-cell responses was capable of increasing bacterial clearance (18
). The results presented in the present study also suggest that a better T-cell response following immunization, as observed with rOMP26, may have contributed to the enhanced clearance of NTHI.
In this study, we have sequenced and cloned a 26-kDa protein from NTHI, determined that the gene is present in all isolates assessed to date, and shown through sequence analysis that the protein is highly conserved, with variation occurring as specific amino acid substitutions. Immunization with rOMP26, which included the leader peptide, effectively enhanced bacterial clearance and induced a significant antibody response and a better T-cell response. Future studies will determine if immunization with this protein will clear other respiratory infections caused by NTHI, such as otitis media, and further investigate and develop the potential of OMP26 as a vaccine candidate.