Antibodies to Keyhole Limpet Hemocyanin Cross-React with an Epitope on the Polysaccharide Capsule of Cryptococcus neoformans and Other Carbohydrates: Implications for Vaccine Development

Abstract

Cryptococcus neoformans causes a life-threatening meningoencephalitis in AIDS patients. Mice immunized with a glycoconjugate vaccine composed of the glucuronoxylomannan (GXM) component of the cryptococcal capsular polysaccharide conjugated to tetanus toxoid produce Abs that can be either protective or nonprotective. Because nonprotective Abs block the efficacy of protective Abs, an effective vaccine must focus the Ab response on a protective epitope. Mice immunized with peptide mimetics of GXM conjugated to keyhole limpet hemocyanin (KLH) with glutaraldehyde developed Abs to GXM. However, control peptides P315 and P24 conjugated to KLH also elicited Abs to GXM. GXM-binding Abs from mice immunized with P315-KLH were inhibited by KLH treated with glutaraldehyde (KLH-g), but not by P315. Furthermore, KLH-g inhibited binding of GXM by serum of mice immunized with GXM-TT, indicating that glutaraldehyde treatment of KLH reveals an epitope(s) that cross-reacts with GXM. Vaccination with KLH-g or unmodified KLH elicited Abs to GXM, but did not confer protection against C. neoformans, suggesting the cross-reactive epitope on KLH was not protective. This was supported by the finding that 4H3, a nonprotective mAb, cross-reacted strongly with KLH-g. Sera from mice immunized with either native KLH or KLH-g cross-reacted with several other carbohydrate Ags, many of which have been conjugated to KLH for vaccine development. This study illustrates how mAbs can be used to determine the efficacy of potential vaccines, in addition to describing the complexity of using KLH and glutaraldehyde in the development of vaccines to carbohydrate Ags.

Variable-Region-Identical Antibodies Differing in Isotype Demonstrate Differences in Fine Specificity and Idiotype

“…The reaction was stopped by the addition of an equal volume of 0.05 M sodium borohydride (Sigma-Aldrich) for 30 min at room temperature. KLH-p was then dialyzed against sterile PBS for 48 h with daily changes as described (27).…”

“…Haptenic macromolecules have been conjugated to KLH using gluteraldehyde (25). KLH-g contains an epitope that has antigenic mimicry to a number of carbohydrate Ags including GXM (27). As observed with GXM, V region-identical Abs with different IgG C regions demonstrated differences in the relative strength of binding to KLH-g, which followed the order IgG1 Ͼ Ͼ IgG2b, IgG3 Ͼ IgG2a for the 3E5 set ( Fig.…”

DNA Vaccine Expressing the Mimotope of GD2 Ganglioside Induces Protective GD2 Cross-reactive Antibody Responses

“…It is also probable that the carrier protein, due to a highly cross-reactive nature and strong immunogenicity, can act as a polyclonal activator causing suppression of protective immune responses. For example, immunization of mice with a glycoconjugate vaccine composed of the glucuronoxylomannan (GXM) component of the cryptococcal capsular polysaccharide conjugated to tetanus toxoid produced antibodies that were both protective and nonprotective (20). Similarly, peptide mimetics of GXM did not elicit a strong immune response to GXM when they were conjugated to KLH.…”

Reactive Immunization Suppresses Advanced Glycation and Mitigates Diabetic Nephropathy

“…Indeed, anti-KLH Abs have been reported to cross-react with a range of carbohydrate structures. 27,28 This is also the proposed basis for the therapeutic efficacy of KLH in clinical applications. 29 Inhibition of L chain adducts by KLH in solution without reduction of titer suggests reactive Abs of moderate affinities, as could be expected in carbohydrate recognition.…”