Comparison of immunogenicities of recombinant Plasmodium vivax merozoite surface protein 1 19- and 42-kiloDalton fragments expressed in Escherichia coli

Abstract

The 42- and 19-kDa C-terminal fragments of merozoite surface protein 1 (MSP-1(42) and MSP-1(19), respectively) are both promising blood-stage vaccine candidate antigens. At present, it is not clear which of the two antigens will be more suitable for inclusion in a cocktail malaria vaccine. In the present study, we expressed the two C-terminal fragments of Plasmodium vivax MSP-1 (PvMSP-1) in an Escherichia coli expression system and purified them by using a rapid two-step protocol. Both of the products were recognized by monoclonal antibodies against PvMSP-1 as well as by immune sera from several individuals exposed to P. vivax. We analyzed and compared the immunological responses to recombinant PvMSP-1(19) and PvMSP-1(42) in mice by using six different adjuvant formulations. Moderate to high antibody responses were observed with both of the antigens in different adjuvant formulations. Surprisingly, alum, which is generally considered to be a poor adjuvant for recombinant malaria antigens, was found to be as good an adjuvant as Montanide ISA 720, ASO2A, and other adjuvant formulations. Most adjuvant formulations induced high levels of immunoglobulin G1 (IgG1), followed by IgG3 and IgG2. Lymphocytes from animals in the PvMSP-1(42)- and PvMSP-1(19)-immunized groups showed proliferative responses upon stimulation with the respective antigens, and high levels of interleukin-4 (IL-4), IL-5, and gamma interferon were detected in the culture supernatants. Immunodepletion studies with sera from mice immunized with these two antigens showed that while immunization with PvMSP-1(42) does produce a PvMSP-1(19)-specific response, a substantial portion is also focused on structures in PvMSP-1(42) not represented by the epidermal growth factor-like domains of PvMSP-1(19). These findings may have implications for the design of MSP-1-based vaccine constructs.

FIG. 1.

(A and B) Coomassie blue-stained SDS-polyacrylamide gel of purified PvMSP-1₁₉ (A) and PvMSP-1₄₂ (B). Lanes: 1, nonreduced; 2, reduced. Arrows indicate the positions of the proteins. (C) Reverse-phase HPLC profiles of PvMSP-1₄₂ (broken line) and PvMSP-1₁₉ (solid line).

FIG. 2.

Reactivity of PvMSP-1₁₉ (A) and PvMSP-1₄₂ (B) antigens in an ELISA with sera collected from P. vivax-infected patients and used at a dilution of 1:200. Error bars indicate SDs.

FIG. 3.

Immune responses in BALB/c mice immunized with PvMSP-1₁₉ (A) and PvMSP-1₄₂ (B) formulated in CFA and incomplete Freund's adjuvant, Montanide ISA 720, alum, ASO2A, QS21, and MF59 at a dilution of 1:25,000. Endpoint titers for each adjuvant at day 56 are shown next to each curve. Error bars indicate SDs.

FIG. 4.

(A) IgG isotype-specific antibody levels in mice immunized with PvMSP-1₄₂ antigen formulated in different adjuvants. Similar patterns were observed for PvMSP-1₁₉-immunized mice (data not shown). (B) IgG subclass responses to PvMSP-1₁₉ and PvMSP-1₄₂ in pooled sera from P. vivax-infected sera. Error bars indicate SDs.

FIG. 5.

Immunodepletion assay showing relative contributions of PvMSP-1₄₂- and PvMSP-1₁₉-specific epitopes in pooled sera from P. vivax-infected patients. Error bars indicate SDs.