Evaluation of naturally acquired immune responses against novel pre-erythrocytic Plasmodium vivax proteins in a low endemic malaria population located in the Peruvian Amazon Basin.

BACKGROUND: Plasmodium vivax represents the most geographically widespread human malaria parasite affecting civilian and military populations in endemic areas. Targeting the pre-erythrocytic (PE) stage of the parasite life cycle is especially appealing for developing P. vivax vaccines as it would prevent disease and transmission. Here, naturally acquired immunity to a panel of P. vivax PE antigens was explored, which may facilitate vaccine development and lead to a better understanding of naturally acquired PE immunity. METHODS: Twelve P. vivax PE antigens orthologous to a panel of P. falciparum antigens previously identified as highly immunogenic in protected subjects after immunization with radiation attenuated sporozoites (RAS) were used for evaluation of humoral and cellular immunity by ELISA and IFN-γ ELISpot. Samples from P. vivax infected individuals (n = 76) from a low endemic malaria region in the Peruvian Amazon Basin were used. RESULTS: In those clinical samples, all PE antigens evaluated showed positive IgG antibody reactivity with a variable prevalence of 58-99% in recently P. vivax diagnosed patients. The magnitude of the IgG antibody response against PE antigens was lower compared with blood stage antigens MSP1 and DBP-II, although antibody levels persisted better for PE antigens (average decrease of 6% for PE antigens and 43% for MSP1, p < 0.05). Higher IgG antibodies was associated with one or more previous malaria episodes only for blood stage antigens (p < 0.001). High IgG responders across PE and blood stage antigens showed significantly lower parasitaemia compared to low IgG responders (median 1,921 vs 4,663 par/µl, p < 0.05). In a subgroup of volunteers (n = 17),positive IFN-γ T cell response by ELISPOT was observed in 35% vs 9-35% against blood stage MSP1 and PE antigens, respectively, but no correlation with IgG responses. CONCLUSIONS: These results demonstrate clear humoral and T cell responses against P. vivax PE antigens in individuals naturally infected with P. vivax. These data identify novel attractive PE antigens suitable for use in the potential development and selection of new malaria vaccine candidates which can be used as a part of malaria prevention strategies in civilian and military populations living in P. vivax endemic areas.

Hemozoin-induced IFN-γ production mediates innate immune protection against sporozoite infection.

Hemozoin is a crystal synthesized by Plasmodium parasites during hemoglobin digestion in the erythrocytic stage. The hemozoin released when the parasites egress from the red blood cell, which is complexed with parasite DNA, is cleared from the circulation by circulating and tissue-resident monocytes and macrophages, respectively. Recently, we reported that intravenous administration of purified hemozoin complexed with Plasmodium berghei DNA (HzPbDNA) resulted in an innate immune response that blocked liver stage development of sporozoites that was dose-dependent and time-limited. Here, we further characterize the organismal, cellular, and molecular events associated with this protective innate response in the liver and report that a large proportion of the IV administered HzPbDNA localized to F4/80+ cells in the liver and that the rapid and strong protection against liver-stage development waned quickly such that by 1 week post-HzPbDNA treatment animals were fully susceptible to infection. RNAseq of the liver after IV administration of HzPbDNA demonstrated that the rapid and robust induction of genes associated with the acute phase response, innate immune activation, cellular recruitment, and IFN-γ signaling observed at day 1 was largely absent at day 7. RNAseq analysis implicated NK cells as the major cellular source of IFN-γ. In vivo cell depletion and IFN-γ neutralization experiments supported the hypothesis that tissue-resident macrophages and NK cells are major contributors to the protective response and the NK cell-derived IFN-γ is key to induction of the mechanisms that block sporozoite development in the liver. These findings advance our understanding of the innate immune responses that prevent liver stage malaria infection.

Plasmodium berghei Purified Hemozoin Associated with DNA Strongly Inhibits P. berghei Liver-Stage Development in BALB/c Mice after Intravenous Inoculation.

In the acidic lysosome-like digestive vacuole, Plasmodium parasites crystallize heme from hemoglobin into hemozoin, or malaria pigment. Upon release of progeny merozoites, the residual hemozoin is phagocytized by macrophages principally in the liver and spleen where the heme crystals can persist for months to years, as heme oxygenase does not readily degrade the crystal. Previous studies demonstrated hemozoin modulation of monocytes and macrophages. Hemozoin modulates immune function activity of monocytes/macrophages. Here, we used purified/washed hemozoin (W-Hz) isolated from murine Plasmodium berghei infections and intravenously (i.v.) injected it back into naive mice. We characterized the modulating effect of W-Hz on liver-stage replication. Purified washed hemozoin decreases P. berghei liver levels both at 1 week and 1 month after i.v. injection in a dose and time dependent fashion. The injected hemozoin fully protected in nine out of 10 mice given a 50 sporozoite inoculum, and in 10 out of 10 mice against 2,000 sporozoites when they were infected an hour or a day after hemozoin inoculation. DNase treatment at the hemozoin reversed the observed liver load reduction. The liver load reduction was similar in mature B- and T-cell-deficient RAG-1 knockout (KO) mice suggesting an innate immune protection mechanism. This work indicates a role for residual hemozoin in down modulation of Plasmodium liver stages.