High Affinity Mimotope of the Polysaccharide Capsule of Cryptococcus neoformans Identified from an Evolutionary Phage Peptide Library

Abstract

Cryptococcus neoformans causes a life-threatening meningoencephalitis in a significant percentage of AIDS patients. Mice immunized with a glycoconjugate vaccine composed of the glucuronoxylomannan (GXM) component of the cryptococcal capsular polysaccharide conjugated to tetanus toxoid (TT) produce Abs that, based on the epitope recognized, can be either protective or nonprotective. Since nonprotective Abs block the efficacy of protective Abs, we are interested in developing a vaccine that would focus the immune response specifically to protective epitopes. Previously, we screened a phage display library with 2H1, a protective anti-GXM mAb, and isolated PA1, a representative peptide that had a Kd of 295 nM for 2H1. Mice immunized with PA1 conjugated to keyhole limpet hemocyanin developed high anti-peptide (1/13,000), but low anti-GXM (maximum, 1/200) titers. We now report our efforts to improve this vaccine by screening a sublibrary with six random amino acids added to either end of the PA1 motif to identify higher affinity peptides. P206.1, a peptide isolated from this sublibrary, had 80-fold higher affinity for 2H1 (Kd = 3.7 nM) than PA1. P206.1 bound protective, but not nonprotective, anti-GXM Abs. Mice immunized with P206.1 conjugated to various carriers did not mount an Ab response to GXM despite developing high anti-peptide titers. However, mice primed with GXM-TT and boosted with P206.1-TT developed significant anti-GXM titers (maximum, 1/180,000). This latter immunization scheme focused the immune response on protective epitopes, since only 2–5% of these titers were directed against nonprotective de-O-acetylated GXM epitopes compared with 20–60% in animals primed and boosted with GXM-TT.

Structural basis of peptide–carbohydrate mimicry in an antibody-combining site

“…In addition, the absence of structural mimicry might not preclude use of the peptide in a vaccine protocol. Thus, although it is possible that, by itself, the peptide conjugate will not elicit an antipolysaccharide response, it could still be used in a prime͞boost strategy to enhance the immune response to an already-present carbohydrate epitope, as demonstrated recently for the case of a peptide mimic of the capsular polysaccharide of Cryptococcus neoformans (36).…”

NMR Studies of Carbohydrates and Carbohydrate-mimetic Peptides Recognized by an Anti-Group B Streptococcus Antibody

“…Further, we note that the level of structural mimicry required for effective immunogenicity is still unknown. The success of a cross-priming strategy in vaccine development against tumor-associated antigens (55,56) and against Cryptococcus neoformans (57) has been demonstrated. The level of structural mimicry in these systems is unknown; however, the crystal structure of an anti-Cryptococcus mAb-peptide complex showed less than perfect complementarity, leaving open a reasonable possibility that the peptide is not a structural mimic (58).…”

Filamentous phage as an immunogenic carrier to elicit focused antibody responses against a synthetic peptide

“…Synthetic peptides have been used in developing immunogens that target the humoral immune response; typically, to elicit antibodies (Abs) that cross-react with a discrete epitope on a protein (and sometimes on carbohydrate (CHO) [1][2][3][4][5][6] or DNA [7][8][9]). Currently, there are no FDAapproved conjugate vaccines like this; however, there is noteworthy research focused on developing peptides as immunogenic B-cell epitopes (BCEs).…”

“…For example, peptides are being used to produce Abs against specific epitopes on tumor Agns that decrease cell proliferation [10][11][12][13]. Peptides are also used as alternatives to CHO Agns because they can elicit a T-cell dependent immune response along with CHO-cross-reactive Abs [1][2][3][4][5][6]. Others have proposed using peptide immunogens to produce HIV-1 neutralizing Abs [14][15][16].…”

“…To do this, vaccine-lead peptides are chemically-conjugated to strongly-immunogenic "carrier" proteins that provide T-cell epitopes (TCEs). These typically include: ovalbumin (OVA) [17][18][19], tetanus toxoid (TT, which is FDA-approved) [2,12,20], BSA [3,18,21], and keyhole limpet hemocyanin (KLH) [5,6,18]. However, these types of carrier protein themselves elicit strong Ab responses such that immundominant BCEs on the carrier may undermine the Ab response against the synthetic peptide, especially if that peptide is less immunogenic than carrier BCEs.…”