A Hybrid Multistage Protein Vaccine Induces Protective Immunity against Murine Malaria

Abstract

We have previously reported the design and expression of chimeric recombinant proteins as an effective platform to deliver malaria vaccines. The erythrocytic and exoerythrocytic protein chimeras described included autologous T helper epitopes genetically linked to defined B cell epitopes. Proof-of-principle studies using vaccine constructs based on the Plasmodium yoelii circumsporozoite protein (CSP) and P. yoelii merozoite surface protein-1 (MSP-1) showed encouraging results when tested individually in this mouse malaria model. To evaluate the potential synergistic or additive effect of combining these chimeric antigens, we constructed a synthetic gene encoding a hybrid protein that combined both polypeptides in a single immunogen. The multistage vaccine was expressed in soluble form in Escherichia coli at high yield. Here we report that the multistage protein induced robust immune responses to individual components, with no evidence of vaccine interference. Passive immunization using purified IgG from rabbits immunized with the hybrid protein conferred more robust protection against the experimental challenge with P. yoelii sporozoites than passive immunization with purified IgG from rabbits immunized with the individual proteins. High antibody titers and high frequencies of CD4 ؉ -and CD8 ؉ -specific cytokine-secreting T cells were elicited by vaccination. T cells were multifunctional and able to simultaneously produce interleukin-2 (IL-2), gamma interferon (IFN-␥), and tumor necrosis factor alpha (TNF-␣). The mechanism of vaccine-induced protection involved neutralizing antibodies and effector CD4؉ T cells and resulted in the control of hyperparasitemia and protection against malarial anemia. These data support our strategy of using an array of autologous T helper epitopes to maximize the response to multistage malaria vaccines. M alaria remains a major public health problem, even though the implementation of control measures has significantly reduced the overall transmission in the past few years (32). Parasites of the genus Plasmodium are responsible for an estimated 216 million clinical cases and over a half million deaths annually worldwide (32). The spread of multidrug-resistant strains of parasites has emphasized the need for developing novel intervention measures. Several vaccine candidates mainly focused on Plasmodium falciparum are in different phases of clinical development. Among them, RTS,S/AS02, an adjuvanted fusion protein based on the circumsporozoite protein, has reached phase 3 clinical trials (4). However, the prospect of developing a highly effective multistage vaccine that includes more than a single antigen has not been pursued vigorously.The multistage life cycle of Plasmodium and the intricate hostparasite interactions during the course of malaria infection support the idea of targeting several antigens simultaneously for vaccine development. We have developed several chimeric recombinant proteins for proof-of-principle studies to test the feasibility of developing effective multistage...

Full-Length Plasmodium falciparum Circumsporozoite Protein Administered with Long-Chain Poly(I·C) or the Toll-Like Receptor 4 Agonist Glucopyranosyl Lipid Adjuvant-Stable Emulsion Elicits Potent Antibody and CD4+T Cell Immunity and Protection in Mice

“…The protective role of CD4 ϩ effector cells against malaria was further substantiated in other murine and human malaria models (22,63,64). In data not shown, antibody depletion of CD4 ϩ T cells in mice immunized with CSP1 and poly(I·C)LC at the time of sporozoite challenge resulted in only modestly reduced protection.…”

A Recombinant Chimeric Ad5/3 Vector Expressing a Multistage Plasmodium Antigen Induces Protective Immunity in Mice Using Heterologous Prime-Boost Immunization Regimens

“…Experiments have shown that these chimeric proteins, engineered to contain several autologous promiscuous T cell epitopes, have superior efficacy compared to a non-chimeric vaccine constructs (2). The potential synergistic effect of combining these novel antigens was subsequently investigated by comparing the immune responses after administration of the proteins formulated as a mixture or delivered as a single fusion protein (P. yoelii LPC/RMC [PyLPC/RMC]) (4). We confirmed that both approaches were effective in inducing multi-stage immune responses.…”

Induction of Multifunctional Broadly Reactive T Cell Responses by a Plasmodium vivax Circumsporozoite Protein Recombinant Chimera

“…We previously designed and expressed chimeric recombinant proteins based on well-characterized vaccine candidates that contain several cognate T cell epitopes arrayed in tandem conformation and genetically linked to immunodominant B cell epitopes. Proof of concept studies conducted in mice using P. yoelii sequences have shown enhanced immunogenicity and efficacy of this type of construct (23,29). Here we report the design, expression, and characterization of a P. vivax CSP chimeric protein (PvRMC-CSP) expressed in E. coli that incorporates similar features.…”

“…For Western blots, following electrophoresis, the proteins were blotted onto nitrocellulose membranes following standard procedures as described previously (23,29 (Fig. 1C).…”