The gene for protein D, a membrane-associated protein with specific affinity for human immunoglobulin D, was cloned from a nontypeable strain of Haemophilus influenzae. The gene was expressed in Escherichia coli

Protein D, a novel surface protein of the bacterial species Haemophilus influenzae with specific affinity for human immunoglobulin (Ig) D was detected in all 127 H. influenzae strains studied. All strains representing different serotypes of encapsulated strains and different biotypes of nonencapsulated strains bound 125I-labeled IgD to a high degree (38 to 74%). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis showed that protein D from all H. influenzae strains had the same apparent molecular weight (i.e., 42,000) and reacted with all three different anti-protein D monoclonal antibodies. By Scatchard analysis, the number of protein D residues on a nontypeable H. influenzae strain was estimated to be approximately 2,800 per organism. The equilibrium constant for the reaction between a human IgD myeloma protein and IgD was found to be 5.8 x 10(8) M-1. Also, all strains of H. haemolyticus and H. aegypticus strains tested bound IgD, 21 to 28% and 41 to 48%, respectively. In extracts of those bacteria, a 42,000-molecular-weight protein reactive with IgD and all three anti-protein D monoclonal antibodies was found. In H. parainfluenzae, H. aphrophilus, H. paraphrophilus, and Actinobacillus actinomycetemcomitans, a 42,000-molecular-weight protein that was reactive with one to three of three anti-protein D monoclonal antibodies but not reactive with human IgD was detected with Western blot analysis. Other Haemophilus species (H. ducreyi, H. parasuis, H. parahaemolyticus, H. segnis, and H. haemoglobinophilus) did not react with human monoclonal IgD or anti-protein D antibodies. On the basis of the wide distribution of protein D among H. influenzae strains, we suggest that protein D could be a vaccine candidate.

Protein D, a surface-exposed 42-kDa membrane lipoprotein, is well conserved among both type b and nontypeable Haemophilus influenzae strains, and it is considered a vaccine against H. influenzae infections. Here, we report the large-scale purification of a nonacylated form of protein D (PDm) from the periplasmic space of Escherichia coli overexpressing PDm. Screening of human sera for levels of antibodies to PDm demonstrated that the immunoglobulin G (IgG) antibody level is above background levels in infants less than 6 months of age. Following a drop to background values in the age group 6 months to 1 year, IgG antibody levels start to increase, together with IgA antibody levels, after 1 year of age. The first appearance of serum IgM antibodies is in 6-month-to 1-year-old infants whose IgG antibody levels have dropped to the postnatal background level. Affinity-purified antibodies from humans and from PDm-immunized rats detected epitopes of protein D which are normally exposed on the bacterial surface. Affinity-isolated human anti-PDm antibodies eluted in acidic buffer were not bactericidal against H. influenzae. Loss of bactericidal activity may occur in this buffer, as was demonstrated in pooled human sera with high bactericidal activity after incubation in the same buffer. Hyperimmunization of rats with PDm induced high levels of serum IgG and IgA antibodies against PDm and significant bactericidal activity against homologous and heterologous H. influenzae strains.

Protein D is a surface-exposed lipoprotein of the gram-negative bacterium Haemophilus influenzae with affinity for human immunoglobulin D myeloma protein. The gene encoding protein D (hpd) in a serotype b strain of H. influenzae was cloned. Escherichia coli carrying the hpd gene bound human myeloma immunoglobulin D. Nucleotide sequence analysis identified an 1,092-bp open reading frame that was more than 99%o identical to the hpd gene from a nontypeable H. influenzae strain. In the deduced amino acid sequences for protein D, only 2 of 364 amino acid residues differed. The restriction fragment length polymorphism of the hpd region in different strains was analyzed by Southern blot analyses of PstIor EcoRI-digested genomic DNA from 100 H. influenzae strains. The analysis was performed by using isolated fragments of the cloned hpd gene, originating from the nontypeable H. influenzae 772, as probes. All strains tested had DNA sequences with a high degree of homology to the hpd probes. The analysis also showed that restriction endonuclease sites within the gene were more conserved than sites adjacent to the hpd gene. An interesting difference between type b strains and unencapsulated strains was observed. The majority of ype b strains seem to have a 1.4-kbp DNA fragment upstream of the hpd gene that is absent in nontypeable strains. On the basis of the high degree of conservation of the hpd gene among H. influenzae strains, we conclude that protein D is a possible vaccine candidate. Haemophilus influenzae is a common colonizer of the mucosa of the upper respiratory tract and causes diseases by local spread or invasion. H. influenzae serotype b (Hib) is a * Corresponding author.

Protein D, a novel surface protein of the bacterial species Haemophilus influenzae with affinity for human IgD, was isolated after solubilization with sonication and Sarcosyl-extraction by a single SDS-PAGE step. From 1 ml of packed bacteria was prepared 0.25 mg of purified protein D. The apparent m.w. of protein D was estimated to 42,000 by SDS-PAGE and gel chromatography. Edman degradation cycles of protein D produced no amino acid phenylthiohydantoin derivatives and the amino-terminal end of the single protein D polypeptide chain is thus probably blocked. Protein D differs from all previously described outer membrane proteins (protein 1 to 6) of H. influenzae. Thus, protein D did not react with antibodies against protein 1 or protein 2 and the latter proteins did not bind IgD. Protein D was found to exhibit unique Ig-binding properties. Thus, in dot blots protein D bound four different human IgD myeloma proteins but not IgG, IgM, IgA, IgE, or some additional proteins. On the IgD molecule, constant parts of the H chains both in the Fab and Fc fragments appear responsible for the interaction with protein D. This novel Ig-binding reagent promises to be of theoretical and practical interest in immunologic and microbiologic research.