Synthesis and characterization of lipooligosaccharide-based conjugates as vaccine candidates for Moraxella (Branhamella) catarrhalis

Abstract

Moraxella (Branhamella) catarrhalis is an important cause of otitis media and sinusitis in children and of lower respiratory tract infections in adults. Lipooligosaccharide (LOS) is a major surface antigen of the bacterium and elicits bactericidal antibodies. Treatment of the LOS from strain ATCC 25238 with anhydrous hydrazine reduced its toxicity 20,000-fold, as assayed in the Limulus amebocyte lysate (LAL) test. The detoxified LOS (dLOS) was coupled to tetanus toxoid (TT) or high-molecular-weight proteins (HMP) from nontypeable Haemophilus influenzae through a linker of adipic acid dihydrazide to form dLOS-TT or dLOS-HMP. The molar ratios of dLOS to TT and HMP conjugates were 19:1 and 31:1, respectively. The antigenicity of the two conjugates was similar to that of the LOS, as determined by double immunodiffusion. Subcutaneous or intramuscular injection of both conjugates elicited a 50- to 100-fold rise in the geometric mean of immunoglobulin G (IgG) to the homologous LOS in mice after three injections and a 350- to 700-fold rise of anti-LOS IgG in rabbits after two injections. The immunogenicity of the conjugate was enhanced by formulation with monophosphoryl lipid A plus trehalose dimycolate. In rabbits, conjugate-induced antisera had complement-mediated bactericidal activity against the homologous strain and heterologous strains of M. catarrhalis. These results indicate that a detoxified LOS-protein conjugate is a candidate for immunization against M. catarrhalis diseases.

FIG. 1

Silver-stained SDS-PAGE patterns of LOS and dLOS from M. catarrhalis 25238. Lanes 1 and 2 contain 200 ng each of Salmonella minnesota LPS Ra and Rc, respectively; lanes 3 through 6 contain 200, 100, 50, and 25 ng, respectively, of LOS; lane 7 contains 20 μg of dLOS from M. catarrhalis.

FIG. 2

Double immunodiffusion. The central well contains a rabbit hyperimmune serum against strain 25238 whole cells. Other wells: 1, LOS, 2 mg/ml; 2, dLOS-TT, 103 μg/ml; 3, dLOS-HMP, 220 μg/ml; 4 and 5, dLOS, 1 mg/ml and 200 μg/ml, respectively; 6, HMP, 500 μg/ml.

FIG. 3

Bactericidal activity of conjugate-induced antisera against M. catarrhalis 25238. Rabbit antisera after two vaccinations of LOS, conjugates, and conjugates with Ribi adjuvant were tested, and each group contained two or three rabbits. The bactericidal titers were expressed as the fold increase above the value for preimmune sera based on the serum dilution causing >50% killing of the bacteria and expressed as the geometric mean and standard deviation for each group. The bactericidal titer for the hyperimmune sera elicited by whole cells was 1:1,600.