A number of malarial blood-stage candidate vaccines are currently being tested in human clinical trials, but our understanding of the relationship between clinical immunity and data obtained from in vitro assays remains inadequate. An in vitro assay which could reliably predict protective immunity in vivo would facilitate vaccine development. Merozoite surface protein1 (MSP1) is a leading blood-stage malaria vaccine candidate, and anti-MSP1 antibodies from individuals that are clinically immune to malaria inhibit the invasion of Plasmodium merozoites into erythrocytes in vitro. Using expression in Escherichia coli and subsequent refolding, we have produced two allelic forms of MSP1 42 (FVO and 3D7). Aotus nancymai monkeys were immunized with MSP1 42 -FVO, MSP1 42 -3D7, or a combination of FVO and 3D7 allelic forms, (MSP1 42 -C1) and were subsequently challenged with Plasmodium falciparum FVO parasites. Sera obtained prior to challenge were tested by standardized enzyme-linked immunosorbent assay (ELISA) to determine antibody titer, and immunoglobulin G (IgG) fractions were also obtained from the same sera; the IgG fractions were tested in an in vitro growth inhibition (GI) assay to evaluate biological activity of the antibodies. Regardless of the immunogen used, all monkeys that had >200,000 ELISA units against MSP1 42 -FVO antigen before challenge controlled their infections. By contrast, all monkeys whose purified IgGs gave <60% inhibition activity in an in vitro GI assay with P. falciparum FVO required treatment for high parasitemia after challenge. There is a strong correlation between ELISA units (Spearman rank correlation of greater than 0.75) or GI activity (Spearman rank correlation of greater than 0.70) and protective immunity judged by various parameters (e.g., cumulative parasitemia or day of patency). These data indicate that, in this monkey model, the ELISA and GI assay values can significantly predict protective immunity induced by a blood-stage vaccine, and they support the use of these assays as part of evaluation of human clinical trials of MSP1-based vaccines.

A major challenge facing malaria vaccine development programs is identifying efficacious combinations of antigens. To date, merozoite surface protein 1 (MSP1) is regarded as the leading asexual vaccine candidate. Apical membrane antigen 1 (AMA1) has been identified as another leading candidate for an asexual malaria vaccine, but without any direct in vivo evidence that a recombinant form of Plasmodium falciparum AMA1 would have efficacy. We evaluated the efficacy of a form of P. falciparum AMA1, produced in Pichia pastoris, by vaccinating Aotus vociferans monkeys and then challenging them with P. falciparum parasites. Significant protection from this otherwise lethal challenge with P. falciparum was observed. Five of six animals had delayed patency; two of these remained subpatent for the course of the infection, and two controlled parasite growth at <0.75% of red blood cells parasitized. The protection induced by AMA1 was superior to that obtained with a form of MSP1 used in the same trial. The protection induced by a combination vaccine of AMA1 and MSP1 was not superior to the protection obtained with AMA1 alone, although the immunity generated appeared to operate against both vaccine components.Many malaria vaccine strategies, including our own, depend on including multiple asexual antigens in order (i) to improve coverage of polymorphisms in field isolates, (ii) to overcome individual nonresponsiveness to some antigens, (iii) to improve vaccine efficacy by eliciting immunity to multiple targets, and (iv) to prevent or delay the evolution of escape mutants. If we include two different antigens in a vaccine combination, ideally, synergy in protection will be induced.The most extensive experience in vaccine trials with New World monkeys has been obtained with the C-terminal 42-kDa portion of merozoite surface protein 1 (MSP1 42 ) (3,10,17,18). Recombinant forms of MSP1 42 have been efficacious against homologous parasite challenges, and MSP1 42 is regarded as a leading asexual vaccine candidate.We have now produced a second antigen, Plasmodium falciparum apical membrane antigen 1 (AMA1) (see reference 13a), and in the present study we test the efficacy of this antigen, both alone and in combination with MSP1 42 , in a vaccine trial with Aotus vociferans monkeys.AMA1 is the subject of intensive vaccine research; at least six of the major malaria vaccine research centers have AMA1 programs. This is based on protection against rodent malaria (Plasmodium chabaudi [1,2,6,20] and Plasmodium yoelii [16]) and nonhuman primate malarias (Plasmodium knowlesi [7] and Plasmodium fragile [5]) by use of purified parasite and recombinant antigens and on the generation in rabbits of an in vitro growth-inhibitory antiserum to an Escherichia coli-expressed recombinant P. falciparum AMA1 (12).However, no evidence exists that vaccine-elicited immunity to a recombinant form of P. falciparum AMA1 will be effective against a P. falciparum challenge in vivo, and establishing this prior to human trials is seen by us as essential, especi...

Hookworms digest hemoglobin from erythrocytes via a proteolytic cascade that begins with the aspartic protease, APR-1. Ac-APR-1 from the dog hookworm, Ancylostoma caninum, protects dogs against hookworm infection via antibodies that neutralize enzymatic activity and interrupt blood-feeding. Toward developing a human hookworm vaccine, we expressed both wild-type (Na-APR-1wt) and mutant (Na-APR-1mut—mutagenesis of the catalytic aspartic acids) forms of Na-APR-1 from the human hookworm, Necator americanus. Refolded Na-APR-1wt was catalytically active, and Na-APR-1mut was catalytically inactive but still bound substrates. Vaccination of canines with Na-APR-1mut and heterologous challenge with A. caninum resulted in significantly reduced parasite egg burdens (P=0.034) and weight loss (P=0.022). Vaccinated dogs also had less gut pathology, fewer adult worms, and reduced blood loss compared to controls but these did not reach statistical significance. Vaccination with Na-APR-1mut induced antibodies that bound the native enzyme in the parasite gut and neutralized enzymatic activity of Na-APR-1wt and APR-1 orthologues from three other hookworm species that infect humans. IgG1 against Na-APR-1mut was the most prominently detected antibody in sera from people resident in high-transmission areas for N. americanus, indicating that natural boosting may occur in exposed humans. Na-APR-1mut is now a lead antigen for the development of an antihematophagy vaccine for human hookworm disease.—Pearson, M. S., Bethony, J. M., Pickering, D. A., de Oliveira, L. M., Jariwala, A., Santiago, H., Miles, A. P., Zhan, B., Jiang, D., Ranjit, N., Mulvenna, J., Tribolet, L., Plieskatt, J., Smith, T., Bottazzi, M. E., Jones, K., Keegan, B., Hotez, P. J., Loukas, A. An enzymatically inactivated hemoglobinase from Necator americanus induces neutralizing antibodies against multiple hookworm species and protects dogs against heterologous hookworm infection.