The acylated form of protein D of Haemophilus influenzae is more immunogenic than the nonacylated form and elicits an adjuvant effect when it is used as a carrier conjugated to polyribosyl ribitol phosphate

Abstract

The nonacylated form of protein D (PDm) of Haemophilus influenzae has been shown to induce the production of antibodies that are bactericidal to homologous and heterologous nontypeable H. influenzae (NTHi) strains. In this study, immunization of rats with lipoprotein D (LPD) induced higher levels of anti-protein D immunoglobulin G and A serum antibodies than immunization with PDm, and the bactericidal activities of sera from LPD-immunized rats were greater than those of sera from PDm-immunized rats. Immunization with LPD or PDm did not prevent the development of acute otitis media (AOM) when rats were challenged with 10 4 CFU of an NTHi strain. However, on the eighth day of bacterial challenge, 50% (5 of 10) of LPD-immunized rats had recovered from otitis media and 30% (3 of 10) had negative middle ear cultures, whereas only 30% (3 of 10) of PDm-immunized rats had recovered, though none was culture positive. Immunization with an inactivated homologous bacterial strain elicited 70% protection (i.e., 7 of 10 rats) in the rat otitis media model. LPD and PDm were also conjugated to the H. influenzae type b (Hib) capsular polysaccharide, polyribosyl ribitol phosphate (PRP), to test protein D-conjugated PRP vaccine's potential for protection against Hib infection. When two LPD-conjugated and two PDm-conjugated PRP vaccines, each containing a different protein concentration, and a tetanus toxoid-conjugated vaccine (ACT-HIB) were tested in the experimental model of rat otitis induced with a Hib strain (Minn A), both of the LPD-conjugated and one of the PDmconjugated vaccines induced significant protection from AOM, the level of protection being highest in animals given the vaccine with the highest LPD content. Sera from these rats also manifested the highest anti-PRP and anti-LPD antibody levels and the highest bactericidal activities against a Hib strain and an NTHi strain.

A biphasic epigenetic switch controls immunoevasion, virulence and niche adaptation in non-typeable Haemophilus influenzae

“…3b ): major outer membrane proteins P2 (ref. 26 ), P5 (refs 27 , 28 ) and P6 (refs 29 , 30 ); lipoprotein D 31 and its deacylated derivative PDM 31 32 ; the high molecular weight (HMW) proteins 33 34 ; and the adhesin Hia 35 . Three different antisera were used to probe for relative expression of OMP P5 (anti-P5, anti-LB1 and anti-chimV4), as these sera contained antibodies specific to unique epitopes of the protein 27 28 36 ( Fig.…”

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

“…The role of LPD in the pathogenesis of OM has been demonstrated in a rat model through the use of an LPDdeficient mutant (34) which was 100-fold less virulent than the parental isolate. 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).…”

“…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).…”

“…Several outer membrane proteins of nontypeable Haemophilus influenzae (NTHI) as well as its lipooligosaccharide have been the subject of recent studies designed to assess their utility as potential immunogens against bacterial otitis media (OM) (3,12,13,18,26,28,30,34,37,44,53,66). The vast majority of these studies have been performed with chinchillas as the host and with disease induced by direct inoculation of the middle ear via transbullar challenge.…”

“…Several current candidate immunogens, which have been shown to reduce the incidence or severity or both of OM in the chinchilla or rat host are a mixture of rPCP, P4, and P6 (26); rHtrA (44); the high-molecular-weight (HMW) adhesin proteins (13); detoxified lipooligosaccharide-protein conjugates (28); outer membrane protein P6 (18); and lipoprotein D (LPD) (3,34). Our laboratory has similarly demonstrated that fimbrin, an NTHI adhesin (11,36,57,58) homologous to outer membrane protein P5 (66), can be used as a parenteral immunogen to significantly reduce the incidence and/or severity of OM in similar direct-challenge models.…”