Synthesis, characterization, and immunologic properties of detoxified lipooligosaccharide from nontypeable Haemophilus influenzae conjugated to proteins

Abstract

Nontypeable Haemophilus influenzae (NTHi) is an important cause of otitis media in children and of pneumonitis in adults with depressed resistance. Lipooligosaccharide (LOS) is a major surface antigen of NTHi and elicits bactericidal and opsonic antibodies. We prepared detoxified LOS (dLOS) protein conjugates from NTHi for use as experimental vaccines. LOS from NTHi 9274 was treated with anhydrous hydrazine and had its toxicity reduced to clinically acceptable levels. dLOS was bound to tetanus toxoid (TT) or highmolecular-weight proteins (HMPs) from NTHi through a linker of adipic acid dihydrazide to form dLOS-TT or dLOS-HMP. The molar ratio of the dLOS to protein carriers ranged from 26:1 to 50:1. The antigenicity of the conjugates was similar to that of the LOS alone as determined by double immunodiffusion. Subcutaneous or intramuscular injection of the conjugates elicited a 28-to 486-fold rise in the level of immunoglobulin G antibodies in mice to the homologous LOS after two or three injections and a 169-to 243-fold rise in the level of immunoglobulin G antibodies in rabbits after two injections. The immunogenicity of the conjugates in mice and rabbits was enhanced by formulation with monophosphoryl lipid A plus trehalose dimycolate. In rabbits, conjugate-induced LOS antibodies induced complement-mediated bactericidal activity against the homologous strain 9274 and prototype strain 3189. These results indicate that a detoxified LOS-protein conjugate is a candidate vaccine for otitis media and pneumonitis caused by NTHi.

Intranasal immunization with a lipooligosaccharide-based conjugate vaccine from nontypeable Haemophilus influenzae enhances bacterial clearance in mouse nasopharynx

“…It is worth noting that local IgGs were mainly systemically derived and IgG is the major isotype in serum. A complement-mediated bactericidal assay performed with the mouse sera observed no bactericidal activity, similar to that of systemic immunization [27]. Many factors may result in the lack of bactericidal activity.…”

“…Apicella, University of Iowa, USA [25] (Table 1). Puri¢cation of LOS, detoxi¢cation of the LOS, conjugation of dLOS to TT, and characterization of dLOS-TT from strain 9274 were as described previously [27,30]. The composition of dLOS-TT was 638 Wg of dLOS and 901 Wg of TT ml 31 with a molar ratio of dLOS to TT of 35:1.…”

“…Speci¢c anti-LOS antibodies in nasal wash, saliva, BALFs, fecal extract and serum were determined by ELI-SA with strain 9274 LOS as a coating antigen (10 Wg ml 31 ) [27]. Samples from naive mice served as negative controls.…”

“…To date, at least ¢ve major serotypes of NTHi LOS have been identi¢ed when studied by immunological methods and sodium dodecyl sulfate^polyacrylamide gel electrophoresis (SDS^PAGE) [25,26]. Previously, we used strain 9274 as a source of LOS for a conjugate vaccine [27]. LOS from this strain does not have the terminal lacto-N-neotetraose found in some pathogenic strains and human cells [24].…”

Exploration ofMoraxella catarrhalisouter membrane proteins, CD and UspA, as new carriers for lipooligosaccharide-based conjugates

“…A complement-mediated bactericidal assay was performed [33]. The bactericidal titer against strain 25238 was determined to be the last dilution of serum causing at least 50% killing as compared with that of a control in which there were bacteria and complement without serum.…”