Biological activity of serum antibodies to a nonacylated form of lipoprotein D of Haemophilus influenzae

Abstract

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.

Bactericidal antibody response against P6, protein D, and OMP26 of nontypeableHaemophilus influenzaeafter acute otitis media in otitis-prone children

“…Different from the total antibody results, we found that protein D less frequently induced bactericidal antibody compared with vaccine candidate P6 in otitis-prone children and OMP26 again failed to generate any bactericidal effect. Our finding with protein D is consistent with a prior report by Akkoyunlu et al (1996). Those results along with other ongoing research in our laboratory is a step forward to understand the immunological differences in otitis-prone and nonotitis-prone children.…”

A fluorescent multiplexed bead-based immunoassay (FMIA) for quantitation of IgG against Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis protein antigens

“…Firstly, regarding the overall lower values of the fluorescence readings for these antigens, the kinetics of the response to these proteins must be considered when evaluating these data. For instance, for H. influenzae Protein D, the levels of IgG are low until the age of 2 years, increasing to moderate levels at 2 to 10 years of age and finally reaching higher levels in adulthood (Akkoyunlu et al, 1996). As half of the tested population was aged b2 years, this factor may have contributed to the low levels of IgG found.…”

Protection against Development of Otitis Media Induced by NontypeableHaemophilus influenzaeby Both Active and Passive Immunization in a Chinchilla Model of Virus-Bacterium Superinfection

“…A later study (3) showed that rats immunized with LPD produced bactericidal antibody with activity against both homologous and heterologous NTHI isolates and also experienced an accelerated rate of recovery from experimental OM, thus generating interest in LPD as a vaccine candidate. Similarly, a nonacylated mutated form of LPD (PDm), expressed by and isolated from the periplasmic space of E. coli (32), was found to be more easily produced and purified than LPD (3,32) and also induced IgG and IgA in serum, which had significant bactericidal activity against both homologous and heterologous NTHI isolates (2). While neither LPD nor PDm immunization prevents the development of acute OM in rats, both result in more rapid recovery from disease, with LPD being the more efficacious (3).…”

“…After concentration, the product was stored in phosphatebuffered saline (PBS; pH 6.8)-0.5% Empigen BB at Ϫ20°C until formulated for use. Protein D (PDm) is a recombinant nonacylated form of LPD lacking three N-terminal palmitate residues, whose expression and purification has been described (2,3,32).…”