Monoclonal antibodies against Haemophilus lipopolysaccharides

The genus Haemophilus comprises a group of gram-negative bacteria with fastidious growth requirements. Among the Haemophilus species, H. influenzae and H. ducreyi are the most important in human medicine.

H. influenzae is a common agent in respiratory tract infections and causes severe diseases like bacterial meningitidis, pneumonia, otitis and pericarditis among others. Some strains possess a capsular polysaccharide. Six capsular polysaccharides, designated types a-f, have been defined. Most of the serious invasive diseases are caused by H. influenzae capsule type b strains. However, it is evident that in several instances of acute respiratory and middle ear infections, including otitis media, non-encapsulated H. influenzae strains play a pathogenic role. Thus, much attention is being focused on other surface antigens, such as the lipopolysaccharide (LPS). H. ducreyi causes the sexually transmitted infection chancroid which increases the risk of acquisition of the human immunodeficiency virus infection (HIV).

Members of the genus Haemophilus, like other gram-negative bacteria possess a LPS which, in contrast to the LPS from enteric bacteria, lacks the long polysaccharide (O-antigens). The LPS comprises two structural domains: (i) the lipid A moiety, which anchors the molecule to the bacterial outer membrane, (ii) a core-oligosaccharide ketosidically linked to the lipid A. The core may be further divided into two regions: the inner and the outer core moieties. Since LPSs are among the outer-most components of the bacterial membrane, they are available for interaction with host membranes and circulating glycoproteins that initiate immune effector mechanisms. Because of its biological importance, the development of monoclonal antibodies (MAbs) that can selectively neutralize or modulate the effects of LPS is of high priority. Antibodies against conserved antigenic determinants could also confer protection against Haemophilus spp. diseases.

A panel of MAbs (MAHI 3-6, 8, 10 and 11, DH24 and DP8) directed against Haemophilus LPS was generated. The epitopes recognized by the MAbs were delineated and the feasibility of the MAbs, for the analysis of the epitope expression in Haemophilus spp. clinical isolates, was tested. Clinical isolates of H. ducreyi or H. influenzae mutant strains were used as immunogens. The MAbs were specific for epitopes located either on the outer or inner core domains of the LPS, and in lipid A. To determine the binding specificities, the MAbs were tested against a set of chemically-defined LPS antigens native or synthetic oligosaccharides, synthetic oligosaccharides coupled to proteins and glycolipids by employing the following techniques: enzyme immuno assay (EIA).immunoblotting and colony-dot-immunoblotting. At least six main specificities have been defined. None of the MAbs reacted with any enteric LPS, but some reacted with structurally defined LPS from Aeromonas, Vibrio, and Bordetella spp. Two MAbs showed a broad reactivity, binding to conserved epitopes in the inner core of the Haemophilus LPS. The MAb MAHI 3 bound to the LPS from all Haemophilus spp., and recognized the Hepa1-2Hepa1-3Hepa1-5Kdo tetrasaccharide including one of the phosphate groups in lipid A. The MAb DP8 bound to the LPS from all H.ducreyi strains and the proposed epitope is Hepa1-2(Glcb1-4)Hepa1-3Hepa1-5Kdo. Four MAbs (MAHI 5, 6, 8, 11) recognized epitopes on the outer core of H. influenzae LPS associated with terminal Gala1-4Gal residues. MAb DH24 bound to lacto-N-neotetraose series of glycoproteins and this epitope is also located in the outer core moiety of the LPS. MAHI 10 bound to terminal phosphorylated saccharide residues either on the outer or inner core and MAHI 4 to apentasaccharide containing terminal Gal-beta residues. The MAb reactivity was influenced by the acylation pattern of lipid A, which gives the micellar structure needed for a proper binding.

The analysis of the epitope expression in H. influenzae clinical isolates suggested that: (i) the number of epitopes expressed is restricted and (ii) all the strains equally express the epitopes recognized by the MAbs, independent of the source of isolation of the sample.

The availability of anti-LPS MAbs binding to chemically defined and species conservedepitopes has usefulness: (i) as probes for mapping virulent epitopes in clinical isolates. (ii) as toolsfor diagnosis, (iii) in studies on pathogenesis, (iv) for elucidation of LPS structures. (v) for thedevelopment of new therapeutic alternatives, i.e. antibody therapeutics technology to treat infections caused by Haemophilus spp.