Safety and Efficacy of Plasmodium berghei Expressing Mouse Interferon Gamma in Inducing Protection against Virulent Wild type P. berghei in a Rodent Model

Abstract

To complement existing control strategies, malaria vaccine is of high priority. During Plasmodium infection, CD4+ T-cells produce interferon gamma (IFN-γ) which in turn induces Th1 responses to up-regulate its production together with other cytokines leading to enhanced protection. With the advancement in transfection technology for malaria parasites, mouse IFN-γ expressing P. berghei ANKA has recently been generated. A single vaccination of this transgenic parasite has been shown to transiently immunomodulate wild-type Plasmodium berghei in mice. However, whether repeated vaccination could induce substantial immune responses is yet to be investigated. This study was therefore designed to evaluate the safety and efficacy of transgenic P. berghei (TfPbA) expressing mIFN-γ in inducing protection against virulent rodent malaria parasites. The parasites were propagated and soluble lysate antigen prepared from in vitro culture. The levels of mouse IFN-γ in lysate was determined by Enzyme-linked immunosorbent assay. To determine the safety and appropriate route of IFN-γ-containing lysate vaccination, two groups of five Balb/C mice each were vaccinated with 75pg/ml IFN-γ in soluble lysate by either intravenous or intraperitoneal route. Control groups were vaccinated or not with soluble lysate of uninfected red blood cells. Clinical parameters were monitored daily for 7 days. At day 7 post-vaccination mice were euthanized with CO2 and blood collected. Haematological parameters, liver and kidney functioning parameters and plasma levels of tumor necrosis factor alpha (TNF-α) were determined. To determine efficacy, a total of 120 BALB/c mice was divided into six groups (three experimental and three controls) of 20 mice each. Two experimental xvi groups were primed using lysate with 75pg/ml IFN-γ or 150pg/ml IFN-γ. At day 14 post priming, the mice were boosted weekly for two weeks. A third group was primed with live TfPbA parasites and at 4% to 6% parasitemia, the mice were orally cured with COARTEM™ at a dose of 450mg/kg. At day 14 post treatment, the mice were boosted weekly for two weeks using lysate with 35pg/ml IFN-γ. The culturing media RPMI 1640, lysate from uninfected red blood cell, and wild-type P. berghei (WtPbA) were used in the control groups. After the last boost, five mice from each group were challenged with WtPbA virulent parasites one week. At the time of challenge, a naϊve control was introduced. Parasitemia and survivorship were monitored for 21 days. Sampling was done at day 0 prior to priming for baseline measurement and at three other individual sampling points (day 14, day 21 and day 28). Blood and spleen were collected and prepared for serum and splenocyte cultures respectively. Total IgG in serum and cytokines (IFN-γ and TNF-α) in culture supernatant were measured by ELISA. Clinical, biochemical and haematological (except platelet count) parameters were not significantly different (p>0.05) among the groups. Compared to unvaccinated control group, TNF-α levels in TfPbA lysate (i.v) group was higher (p=0.01). There was significant effect of vaccination on IFN-γ production between the groups at day 21 (p= 0.035) and day 28 (p< 0.001). Production of TNF-α among the groups was only significant at day 21 post-vaccination (p= 0.037). There was a delay (1-4 days) in onset of parasitemia in IFN-γ vaccinated groups compared to the naive control. Mean patent parasitemia was lowest in mice vaccinated with 75g/ml IFN-γ lysate and highest in WtPbA control (p= 0.633). Mice vaccinated with 75pg/ml IFN-γ lysate survived wild type challenge infection 5 days longer xvii than naive control group (p=0.013). The study has demonstrated that, vaccination of mice with transgenic P. berghei lysate with 75pg/ml IFN-γ may be safe in mice and repeated vaccination has significant effect on IFN-γ and TNF-α production by Th 1 cells leading to enhanced malaria protection. The study recommends combination of cytokine expressing parasites with soluble lysate in a priming-boosting manner in future vaccination experiments.