• Vaccine Ontology ID: VO_0004115
• Type: Conjugate vaccine
• Antigen: NTHi detoxified lipooligosaccharide (dLOS) conjugated with outer membrane protein P6
• Adjuvant: Ribi vaccine adjuvant
  • VO ID: VO_0001238
• Preparation: LOS was purified from a clinical isolate of NTHi strain 9274 and five major LOS prototype strainsby a modified phenol–water extraction method. A hydrazinolysis was used to produce the detoxified LOS (dLOS). An adipic acid dihydrazide (ADH, Aldrich Chemical Co., Milwaukee, WI) was then bound covalently to the dLOS to form an adipic hydrazide derivative, AH-dLOS. P6 was isolated from strain 9274 (Wu et al., 2005). P6 was covalently conjugated to dLOS of strain 9274 through adipic acid dihydrazide with different ratios of dLOS to P6. It resulted in two conjugate formulations with weight ratios of dLOS to P6 of 3.7 for dLOS–P6 (I) and 1.6 for dLOS–P6 (II). Binding activity of the conjugates was examined by ELISA with mouse monoclonal antibodies specific to LOS and P6 and a rabbit anti-P6 serum. The results showed that the conjugates bound not only to the LOS antibody but also to both P6 antibodies. It suggested that the conjugates retained epitopes of both LOS and P6 antigens (Wu et al., 2005).
• Virulence: detoxified

Mouse Response

• Vaccination Protocol: Ten mice per group were given a total of three subcutaneous injections at 2-week intervals with conjugates, conjugates with Ribi adjuvant, P6, or a mixture of P6 and dLOS. Blood samples were collected 14 days after the first injection and 7 days after the second and third injections (Wu et al., 2005).
• Immune Response: In mice, the mixture of P6 and dLOS or P6 alone did not elicit anti-LOS IgG responses. In contrast, both conjugates were able to elicit a significant rise of anti-LOS IgG, whereas dLOS–P6 (II) was a better immunogen for eliciting mouse IgG as compared to dLOS–P6 (I) ( P < 0.01). There was a 648-fold rise of anti-LOS IgG elicited by dLOS–P6 (II) and a 14-fold rise by dLOS–P6 (I) after three injections when compared to their pre-immune sera that contained 1 ELISA unit (p < 0.01). Formulation of the conjugates with Ribi adjuvant enhanced the antibody responses in both conjugates, especially for dLOS–P6 (I). In addition, dLOS–P6 (I) alone elicited better responses of anti-LOS IgM (Wu et al., 2005).

Rabbit Response

• Vaccination Protocol: Two or three rabbits per group were immunized subcutaneously and intramuscularly twice on days 0 and 28 with conjugates, conjugates with Ribi adjuvant, P6, or a mixture of P6 and dLOS. Blood samples were collected on days 0, 14 and 38 (Wu et al., 2005).
• Efficacy: Both conjugates were further tested for their immunogenicity in rabbits. The mixture of P6 and dLOS or P6 alone did not elicit significant rises of anti-LOS IgG. In contrast, both conjugates elicited high levels of anti-LOS IgG after the second injection as compared to those in their pre-immune sera (122–243-fold rise). Unlike with mice, formulation of both conjugates with Ribi adjuvant did not further enhance the immunogenicity of the conjugates (Wu et al., 2005).