Protection of Rhesus Macaques against LethalPlasmodium knowlesiMalaria by a Heterologous DNA Priming and Poxvirus Boosting Immunization Regimen

Abstract

We tested a cytokine-enhanced, multiantigen, DNA priming and poxvirus boosting vaccine regimen for prevention of malaria in the Plasmodium knowlesi-rhesus macaque model system. Animals were primed with a mixture of DNA plasmids encoding two preerythrocytic-stage proteins and two erythrocytic-stage proteins from P. knowlesi and combinations of the cytokines granulocyte-macrophage colony-stimulating factor, interleukin-4, and tumor necrosis factor alpha and were boosted with a mixture of four recombinant, attenuated vaccinia virus strains encoding the four P. knowlesi antigens. Two weeks after boosting, the geometric mean immunofluorescence titers in the immunized groups against sporozoites and infected erythrocytes ranged from 160 to 8,096 and from 1,810 to 5,120, respectively. The geometric mean anti-P. knowlesi circumsporozoite protein (PkCSP) titers ranged from 1,761 to 24,242. Peripheral blood mononuclear cells (PBMC) from the immunized monkeys produced gamma interferon (IFN-␥) in response to incubation with pooled peptides from the PkCSP at frequencies of 10 to 571 spot-forming cells/10 6 PBMC. Following challenge with 100 infectious P. knowlesi sporozoites, 2 of 11 immunized monkeys were sterilely protected, and 7 of the 9 infected monkeys resolved their parasitemias spontaneously. In contrast, all four controls became infected and required treatment for overwhelming parasitemia. Early protection was strongly associated with IFN-␥ responses against a pool of peptides from the preerythrocytic-stage antigen, PkCSP. These findings demonstrate that a multistage, multiantigen, DNA priming and poxvirus boosting vaccine regimen can protect nonhuman primates from an otherwise lethal malaria sporozoite challenge.Each year, malaria parasites infect 270 to 350 million people and kill 1.5 to 2.7 million people, mostly children in subSaharan Africa (29); drug resistance is spreading rapidly, and there is currently no licensed vaccine. In a mammalian host Plasmodium sporozoites injected by a mosquito move within minutes to hepatocytes, in which they develop during several days before emerging to infect circulating erythrocytes. Two models suggest that immune control of malaria is possible. First, in mice (15), monkeys (10), and humans (3), immunization with radiation-attenuated sporozoites can provide sterile protection against sporozoite challenge, mediated by CD8 ϩ T cells and gamma interferon (IFN-␥) directed at the intrahepatocytic stage of the parasite (6). Adults in areas where malaria is endemic develop partial clinical immunity, which is largely mediated by antibodies directed against blood-stage antigens (19,21). An effective malaria vaccine will likely need to induce both T-cell responses against infected hepatocytes and antibodies against blood-stage parasites. While DNA vaccines represent a flexible vaccine technology, well adapted to simultaneous delivery of multiple antigens, they have been less than optimally immunogenic in human trials, inducing modest Tcell responses and small amounts of antibodies or no...

A Review of Plasmodium coatneyi–Macaque Models of Severe Malaria

“…This is evidenced by the incompatibility of P. knowlesi and the rhesus macaque to cohabitate the same range of forest because P. knowlesi causes a rapidly fatal course of disease in this monkey, making malaria persistence impossible. 92,106 This degree of pathogenicity may be the result of recent evolutionary collision between the host and pathogen in which the malaria parasite maintained a high level of virulence due to insufficient evolutionary time to adapt to the host. This particular point is potentially important in selection and interpretation of a model, as differing species of macaques from diverse geographic origins may display differing susceptibility to various plasmodia.…”

Successful Induction of CD8 T Cell-Dependent Protection Against Malaria by Sequential Immunization with DNA and Recombinant Poxvirus of Neonatal Mice Born to Immune Mothers

“…falciparum DNA vaccines have been shown to be immunogenic in humans (2,3), and strategies in which DNA vaccines are boosted with recombinant poxvirus expressing the same proteins have been shown to be protective in mice (4 -6) and nonhuman primates (7). Vaccines based on this strategy have now entered human clinical trials (8).…”

Molecular Mechanism of Host Specificity in Plasmodium falciparum Infection

“…Cloning, expression, and purification of recombinant CS protein of P. falciparum (16) and P. knowlesi (17) has been described previously. DNA encoding P. gallinaceum mature CS protein was amplified as a NdeI-EcoRI fragment by PCR and cloned in pET11a, a T7 promoterbased Escherichia coli expression vector.…”

“…DNA encoding PfCSP, PkCSP, and PgCSP were in pET11a, a T7 promoter-based E. coli expression vector. Both PfCSP and PkCSP were secreted into the periplasm, which was used as a source to obtain the purified protein, as previously described (16,17). Both the proteins were initially purified on a heparin-Sepharose affinity column followed by gel filtration chromatography, to obtain the purified protein (see online supplemental data).…”