Immune responses and protection of Aotus monkeys immunized with irradiated Plasmodium vivax sporozoites

Abstract

A non-human primate model for the induction of protective immunity against the pre-erythrocytic stages of Plasmodium vivax malaria using radiation-attenuated P. vivax sporozoites may help to characterize protective immune mechanisms and identify novel malaria vaccine candidates. Immune responses and protective efficacy induced by vaccination with irradiated P. vivax sporozoites were evaluated in malaria-naive Aotus monkeys. Three groups of six monkeys received two, five, or ten intravenous inoculations, respectively, of 100,000 irradiated P. vivax sporozoites; control groups received either 10 doses of uninfected salivary gland extract or no inoculations. Immunization resulted in the production low levels of antibodies that specifically recognized P. vivax sporozoites and the circumsporozoite protein. Additionally, immunization induced low levels of antigen-specific IFN-γ responses. Intravenous challenge with viable sporozoites resulted in partial protection in a dose-dependent manner. These findings suggest that the Aotus monkey model may be able to play a role in preclinical development of P. vivax pre-erythrocytic stage vaccines.

Figure 1.

Immunization schedule. Aotus monkeys were immunized by 10, 5, and 2 intravenous inoculation of Plasmodium vivax irrad-spz every 2 weeks (Groups Ia, Ib, and Ic). Two groups (II and III) were used as a mock-immunized and life sporozoites control of infection. All monkeys were challenge with 7 × 10 P. vivax life sporozoite on Day 150.

Figure 2.

Recognition of native protein by antisera from individual monkeys in the group that received 10 immunizations. Antibodies against Plasmodium vivax sporozoites evaluated by the immunofluorescent antibody test (IFAT) in samples collected every 2 weeks from the monkeys (N = 6). The titer was the last serum dilution at which fluorescence could be detected. The negative control was a pool of sera taken from six control malaria-naive Aotus monkeys. The arrow indicates the day of the challenge.

Figure 3.

Antibodies to PvCSP peptides in Aotus monkeys immunized with Plasmodium vivax irrad-spz. Antibodies from the ten-immunization group against the long synthetic peptides of the PvCSP, measured by enzyme-linked immunosorbent assay (ELISA) in samples collected every 2 weeks from Day 0 to Day 210. The titer was the last serum dilution at which the optical density at 450 nm was greater than the mean plus 3 SD of pooled sera taken from six controls, malaria-naive Aotus monkeys. The arrow indicates the day of the challenge.

Figure 4.

IFN-γ production by peripheral blood mononuclear cells (PBMCs) from the group that received 10 immunizations with Plasmodium vivax irrad-spz as compared with the mock-immunized group. Data were from Aotus monkeys (N = 6) immunized 10 times with P. vivax irrad-spz (ten-immunization group) and immunized with salivary gland extracts (N = 6) (mock-immunized group). The number of IFN-γ SFCs/10⁶ cells was evaluated by enzyme-linked immunosorbent spot (ELIspot) using fresh Aotus PBMC cultured for 36 hrs in the absence or in the presence of the N, R-common, and C long peptides, or P. vivax sporozoites. The results are expressed as the number of IFN-γ SFCs/10⁶ cells PBMC of immune monkeys.

Figure 5.

Percent of infection of monkeys immunized with different inoculation (10, 5, and 2 immunizations) of Plasmodium vivax irrad-spz and challenged with 7 × 10 P. vivax live sporozoites. Monkeys were bleed every other week after Day 7 of challenge and parasitemia was detected by polymerase chain reaction (PCR).