Fusion of Two Malaria Vaccine Candidate Antigens Enhances Product Yield, Immunogenicity, and Antibody-Mediated Inhibition of Parasite Growth In Vitro

Abstract

A Plasmodium falciparum chimeric protein 2.9 (PfCP-2.9) was constructed consisting of the C-terminal regions of two leading malaria vaccine candidates, domain III of apical membrane ag-1 (AMA-1) and 19-kDa C-terminal fragment of the merozoite surface protein 1 (MSP1). The PfCP-2.9 was produced by Pichia pastoris in secreted form with a yield of 2600 mg/L and ∼1 g/L of final product was obtained from a three-step purification process. Analysis of conformational properties of the chimeric protein showed that all six conformational mAbs interacted with the recombinant protein were reduction-sensitive, indicating that fusion of the two cysteine-rich proteins retains critical conformational epitopes. PfCP-2.9 was found to be highly immunogenic in rabbits as well as in rhesus monkeys (Macaca mulatta). The chimeric protein induced both anti-MSP1–19 and anti-AMA-1(III) Abs at levels 11- and 18-fold higher, respectively, than individual components did. Anti-PfCP-2.9 sera from both rabbits and rhesus monkeys almost completely inhibited in vitro growth of the P. falciparum FCC1/HN and 3D7 lines when tested at a 6.7-fold dilution. It was shown that the inhibition is dependent on the presence of Abs to the chimeric protein and their disulfide bond-dependent conformations. Moreover, the activity was mediated by a combination of growth-inhibitory Abs generated by the individual MSP1–19 and AMA-1(III) of PfCP-2.9. The combination of the extremely high yield of the protein and enhancement of its immune response provides a basis to develop an effective and affordable malaria vaccine.

Safety and immunogenicity of multi-antigen AMA1-based vaccines formulated with CoVaccine HT™ and Montanide ISA 51 in rhesus macaques

“…The AM/ISA vaccine also induced appreciable levels of anti-MSP1 19 IgG, especially in the two animals that also had the highest anti-AMA1 IgGs in this group. Vaccine design studies with MSP1 19 fusion proteins have shown that IgG responses induced against the MSP1 19 component increase several fold compared to IgG responses to formulations with MSP1 19 alone [35,53], suggesting that fusion enhances antibody formation against the MSP1 19 component.…”

Phase I dose escalation safety and immunogenicity trial of Plasmodium falciparum apical membrane protein (AMA-1) FMP2.1, adjuvanted with AS02A, in malaria-naïve adults at the Walter Reed Army Institute of Research

“…Sero-epidemiologic evidence [35], and the limited GIA data presented here, suggest a PfAMA-1-based vaccine may elicit allele-specific antibodies. Consequently, in addition to monovalent PfAMA-1 vaccines [14][15][16][17]21] there are also bivalent PfAMA-1 vaccines in development in an effort to broaden protective immune responses [18,19,21]. With FMP2.1/AS02A, we intend to determine if high titer antibodies elicited against sporozoite and asexual stages, as well as potent anti-PfAMA-1 cellular responses, might act against diverse alleles.…”

“…Immunization of New World monkeys with recombinant PfAMA-1 formulated with Freund's adjuvant has conferred significant protection against homologous P. falciparum challenge [12,13], but limited protection when formulated with adjuvants intended for human use such as Montanide or AS02A [Barnwell, unpublished]. Several PfAMA-1 vaccines are in development, but none have been tested for clinical efficacy [14][15][16][17][18][19][20]. A recent Phase I clinical trial of a recombinant PfAMA-1 antigen adjuvanted with alhydrogel showed that this formulation elicited functional antibodies that, after affinity purification, exhibited growth inhibition of P. falciparum in vitro [21].…”

Malaria Vaccine Development: A Challenge for Africa

“…The two candidates (AMA-1 and MSP-1) enhance the product yield and immunogenicity of the individual components when combined. The protective immune responses induced by either AMA-1 or MSP1-19 are dependent on protein conformation (38).…”