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Outer membrane protein D15 is conserved among Haemophilus influenzae species and may represent a universal protective
S M Loosmore1, Y P Yang, D C Coleman
1Research Centre, Pasteur Merieux Connaught Canada, North York, Ontario, Canada. sloosmore@ca.pmc-vacc.com
Abstract:
We have cloned and sequenced the d15 gene from two strains of Haemophilus influenzae type b (Hib) and two strains of nontypeable H. influenzae (NTHI). The nucleotide and deduced protein sequences of d15 are highly conserved, with only a small variable region identified near the carboxyl terminus of the protein. Analysis of upstream sequences revealed that the H. influenzae d15 gene may be part of a large potential operon of closely spaced open reading frames, including one with significant homology to the Escherichia coli cds gene encoding CDP-diglyceride synthetase. Southern blot analysis demonstrated that the d15 gene is also present in H. influenzae types a, c, d, e, and f and in Haemophilus parainfluenzae. A recombinant D15 (rD15) protein was expressed in good quantity in E. coli from the inducible T7 promoter, and monospecific anti-rD15 antibodies were raised. Immunoblot analysis of H. influenzae serotypes a, b, c, d, e, and f, NTHI, and H. parainfluenzae lysates revealed that they all expressed a cross-reactive D15-like protein. Purified rD15 was found to be highly immunogenic in mice, guinea pigs, and rabbits, and passive transfer of anti-rD15 antibodies protected infant rats from challenge with H. influenzae type b or type a in infant rat models of bacteremia. Thus, D15 is a highly conserved antigen that is protective in animal models and it may be a useful component of a universal subunit vaccine against Haemophilus infection and disease.
Insights
The D15 protein is a highly conserved antigen found across various Haemophilus influenzae strains. Antibodies against D15 protected against infection in animal models, suggesting its potential for a universal Haemophilus vaccine.
Area of Science:
- Microbiology
- Immunology
- Vaccine Development
Background:
- Haemophilus influenzae, particularly type b (Hib), causes significant invasive disease.
- Nontypeable H. influenzae (NTHI) strains are also important pathogens.
- Development of a universal vaccine against Haemophilus species remains a goal.
Purpose of the Study:
- To investigate the d15 gene and its encoded D15 protein in Haemophilus influenzae.
- To assess the immunogenicity and protective potential of the D15 antigen.
Main Methods:
- Cloning and sequencing of the d15 gene from Hib and NTHI strains.
- Southern blot analysis to determine gene distribution across Haemophilus species.
- Recombinant D15 protein expression and antibody generation.
- Immunoblot analysis of bacterial lysates.
- Animal models of bacteremia to evaluate passive protection.
Main Results:
- The d15 gene and D15 protein sequence are highly conserved across H. influenzae strains.
- The d15 gene is present in multiple H. influenzae serotypes and H. parainfluenzae.
- Recombinant D15 protein elicits a strong immune response in animal models.
- Passive immunization with anti-D15 antibodies confers protection against H. influenzae challenge in infant rat models.
Conclusions:
- D15 is a conserved, immunogenic antigen present in various Haemophilus species.
- Anti-D15 antibodies demonstrate protective efficacy in animal models of bacteremia.
- The D15 antigen represents a promising candidate for a universal subunit vaccine against Haemophilus infections.