Growth-inhibitory antibodies are not necessary for protective immunity to malaria infection

The absence of a validated surrogate marker for the immune state has complicated the design of a subunit vaccine against asexual stages of Plasmodium falciparum. In particular, it is not known whether the capacity to induce antibodies that inhibit parasite growth in vitro is an important criterion for selection of P. falciparum proteins to be assessed in human vaccine trials. We examined this issue in the Plasmodium yoelii rodent malaria model using the 19-kDa C-terminal fragment of merozoite surface protein 1 (MSP1(19)). To examine the relationship between inhibitory antibodies in immunized mice and the immune state, as indicated by resistance to a blood-stage challenge, we used an allelic replacement strategy to generate a transgenic P. falciparum line that expresses MSP1(19) from P. yoelii. We show that MSP1(19) is functionally conserved across these two divergent Plasmodium species, and replacing PfMSP1(19) with PyMSP1(19) has no detectable effect on parasite growth in vitro. By comparing growth rates of this transgenic line with a matched transgenic line that expresses the endogenous PfMSP1(19), we developed an assay to measure the specific growth-inhibitory activity directed exclusively to the PyMSP1(19) protein in the sera from vaccinated animals. To validate this assay, sera from rabbits immunized with recombinant PyMSP1(19) were tested and showed specific inhibitory activity in a concentration-dependent manner. In mice that were immunized with recombinant PyMSP1(19), the levels of PyMSP1(19)-specific inhibitory activity did not correlate with the total antibody levels measured by enzyme-linked immunosorbent assay. Furthermore, they did not correlate with resistance to subsequent blood-stage infection, and some mice with complete protection showed no detectable inhibitory activity in their prechallenge sera. These data indicated that growth-inhibitory activity measured in vitro was not a reliable predictor of immune status in vivo, and the reliance on this criterion to select vaccine candidates for human clinical trials may be misplaced. The transgenic lines further offer useful tools for comparing the efficacy of MSP1(19)-based vaccines that utilize different immunization regimens and antigen formulations.