Immunogenicity of Self-Associated Aggregates and Chemically Cross-Linked Conjugates of the 42 kDa Plasmodium falciparum Merozoite Surface Protein-1

Feng Qian,Vu Nguyen,Lynn Lambert,Kelly M Rausch,Louis H Miller,Karine Reiter,Gregory E D Mullen,Richard L Shimp,Carole A Long,Daming Zhu,Laura B Martin,Yanling Zhang,David L Narum,Joan A Aebig,Kevin K.A Tetteh

doi:10.1371/journal.pone.0036996

Abstract

Self-associated protein aggregates or cross-linked protein conjugates are, in general, more immunogenic than oligomeric or monomeric forms. In particular, the immunogenicity in mice of a recombinant malaria transmission blocking vaccine candidate, the ookinete specific Plasmodium falciparum 25 kDa protein (Pfs25), was increased more than 1000-fold when evaluated as a chemical cross-linked protein-protein conjugate as compared to a formulated monomer. Whether alternative approaches using protein complexes improve the immunogenicity of other recombinant malaria vaccine candidates is worth assessing. In this work, the immunogenicity of the recombinant 42 kDa processed form of the P. falciparum merozoite surface protein 1 (MSP142) was evaluated as a self-associated, non-covalent aggregate and as a chemical cross-linked protein-protein conjugate to ExoProtein A, which is a recombinant detoxified form of Pseudomonas aeruginosa exotoxin A. MSP142 conjugates were prepared and characterized biochemically and biophysically to determine their molar mass in solution and stoichiometry, when relevant. The immunogenicity of the MSP142 self-associated aggregates, cross-linked chemical conjugates and monomers were compared in BALB/c mice after adsorption to aluminum hydroxide adjuvant, and in one instance in association with the TLR9 agonist CPG7909 with an aluminum hydroxide formulation. Antibody titers were assessed by ELISA. Unlike observations made for Pfs25, no significant enhancement in MSP142 specific antibody titers was observed for any conjugate as compared to the formulated monomer or dimer, except for the addition of the TLR9 agonist CPG7909. Clearly, enhancing the immunogenicity of a recombinant protein vaccine candidate by the formation of protein complexes must be established on an empirical basis.

Highlights

Chemical conjugation is widely used to make haptens such as peptides and polysaccharides immunogenic
Conjugation effectively enhance the immunogenicity of the Plasmodium falciparum Pfs25, a transmission blocking malaria vaccine candidate, when recombinant Pfs25 was conjugated either to carrier proteins such as the outer-membrane protein complex of Neisseria meningitidis or ExoProtein A of Pseudomonas aeruginosa, or to itself [5,6,7]
We evaluated whether the immunogenicity of MSP142 in mice is enhanced when presented as 1) a self-associated aggregated protein or 2) chemically conjugated to a carrier protein formulated on Alhydrogel with or without CPG 7909, a synthetic B type CpG-ODN (unmethylated oligodeoxynucleotide containing cytosine-guanosine (CpG) dinucleotide motifs)

Summary

Introduction

Chemical conjugation is widely used to make haptens such as peptides and polysaccharides immunogenic. This is significant for the development of several important human vaccines against polysaccharide moieties such as Hemophilus influenzae type b, Streptococcus pneumoniae, Neisseria meningitidis and Salmonella enterica serovar Typhi [1,2,3,4]. Conjugation effectively enhance the immunogenicity of the Plasmodium falciparum Pfs, a transmission blocking malaria vaccine candidate, when recombinant Pfs was conjugated either to carrier proteins such as the outer-membrane protein complex of Neisseria meningitidis or ExoProtein A of Pseudomonas aeruginosa, (a detoxified form of exotoxin A from P. aeruginosa) or to itself (self-conjugation) [5,6,7]. Similar results were observed when two other malaria antigens of P. falciparum, Pfs and AMA1, were conjugated to the ExoProtein A [7,8]. While the single MSP119 of P. yoelii failed to induce specific antibody responses in mice expressing H-2s major histocompatibility complex haplotype, its conjugate coupled to diphtheria toxoid induced functional antibody responses in these mice [9]

Methods

Results

Conclusion