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Immunogenicity of self-associated aggregates and chemically cross-linked conjugates of the 42 kDa Plasmodium falciparum merozoite surface protein-1.

Qian F, Reiter K, Zhang Y, Shimp RL, Nguyen V, Aebig JA, Rausch KM, Zhu D, Lambert L, Mullen GE, Martin LB, Long CA, Miller LH, Narum DL - PLoS ONE (2012)

Bottom Line: Self-associated protein aggregates or cross-linked protein conjugates are, in general, more immunogenic than oligomeric or monomeric forms.Unlike observations made for Pfs25, no significant enhancement in MSP1(42) 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Malaria Immunology and Vaccinology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, United States of America.

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 (MSP1(42)) 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. MSP1(42) conjugates were prepared and characterized biochemically and biophysically to determine their molar mass in solution and stoichiometry, when relevant. The immunogenicity of the MSP1(42) 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 MSP1(42) 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.

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Characterization of chemically modified EPA.Analysis of un-modified EPA, EPAAPA batch 1, EPAAPA batch 2 and EPAPEO by RP-HPLC (A), and titration analysis of EPAAPA batch 1, EPAAPA batch 2 and EPAPEO (B) are shown.
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pone-0036996-g003: Characterization of chemically modified EPA.Analysis of un-modified EPA, EPAAPA batch 1, EPAAPA batch 2 and EPAPEO by RP-HPLC (A), and titration analysis of EPAAPA batch 1, EPAAPA batch 2 and EPAPEO (B) are shown.

Mentions: Batches of chemical cross-linked MSP142-FUP-EPA conjugates were prepared using two different maleimide cross-linkers Sulfo-EMCS and NHS-PEO2-maleimide. The composition of each linker indicates that the solubility properties may be different, even though this did not appear to impact the solubility of the conjugates (data not shown). Recombinant EPAAPA and EPAPEO, modified by Sulfo-EMCS and NHS-PEO2-maleimide, respectively, were prepared and the addition of the bifunctional linkers was monitored by RP-HPLC analysis. The retention times as well as the peak shape of the modified EPA shifted with the addition of the maleimide groups onto the protein (Figure 3A). The extent of the shift in retention time appeared dependent on the number and physical properties of the chemical modifiers (Figure 3A and data not shown). The number of maleimide groups added on EPA was assessed by titrating with cysteine and measuring the absorbance at 405 nm (Figure 3B). The titration curves of two batches of EPAAPA and one batch of EPAPEO were similar (Figure 3B). Based on the titration curves and the standard curve of cysteine, the number of maleimide groups added onto the EPA was calculated to be 5.0 and 4.7 for the two batches of EPAAPA and 5.0 for the EPAPEO.


Immunogenicity of self-associated aggregates and chemically cross-linked conjugates of the 42 kDa Plasmodium falciparum merozoite surface protein-1.

Qian F, Reiter K, Zhang Y, Shimp RL, Nguyen V, Aebig JA, Rausch KM, Zhu D, Lambert L, Mullen GE, Martin LB, Long CA, Miller LH, Narum DL - PLoS ONE (2012)

Characterization of chemically modified EPA.Analysis of un-modified EPA, EPAAPA batch 1, EPAAPA batch 2 and EPAPEO by RP-HPLC (A), and titration analysis of EPAAPA batch 1, EPAAPA batch 2 and EPAPEO (B) are shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3366955&req=5

pone-0036996-g003: Characterization of chemically modified EPA.Analysis of un-modified EPA, EPAAPA batch 1, EPAAPA batch 2 and EPAPEO by RP-HPLC (A), and titration analysis of EPAAPA batch 1, EPAAPA batch 2 and EPAPEO (B) are shown.
Mentions: Batches of chemical cross-linked MSP142-FUP-EPA conjugates were prepared using two different maleimide cross-linkers Sulfo-EMCS and NHS-PEO2-maleimide. The composition of each linker indicates that the solubility properties may be different, even though this did not appear to impact the solubility of the conjugates (data not shown). Recombinant EPAAPA and EPAPEO, modified by Sulfo-EMCS and NHS-PEO2-maleimide, respectively, were prepared and the addition of the bifunctional linkers was monitored by RP-HPLC analysis. The retention times as well as the peak shape of the modified EPA shifted with the addition of the maleimide groups onto the protein (Figure 3A). The extent of the shift in retention time appeared dependent on the number and physical properties of the chemical modifiers (Figure 3A and data not shown). The number of maleimide groups added on EPA was assessed by titrating with cysteine and measuring the absorbance at 405 nm (Figure 3B). The titration curves of two batches of EPAAPA and one batch of EPAPEO were similar (Figure 3B). Based on the titration curves and the standard curve of cysteine, the number of maleimide groups added onto the EPA was calculated to be 5.0 and 4.7 for the two batches of EPAAPA and 5.0 for the EPAPEO.

Bottom Line: Self-associated protein aggregates or cross-linked protein conjugates are, in general, more immunogenic than oligomeric or monomeric forms.Unlike observations made for Pfs25, no significant enhancement in MSP1(42) 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Malaria Immunology and Vaccinology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, United States of America.

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 (MSP1(42)) 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. MSP1(42) conjugates were prepared and characterized biochemically and biophysically to determine their molar mass in solution and stoichiometry, when relevant. The immunogenicity of the MSP1(42) 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 MSP1(42) 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.

Show MeSH
Related in: MedlinePlus