Identification Of Pre-Erythrocytic Malaria Antigen That Contribute To Protection Elicited By Genetically-attenuated Parasites Vaccination

Malaria remains a daunting public health challenge and causes627,000people deathsper year. Malaria infections in the mammalian host can be broadly separated into two stages:the asymptomatic pre-erythrocytic (PE) stage and the blood stage (BS), during which allclinical symptoms occur.Since all clinical symptoms derive from the BS, malaria vaccinesthat block parasite development during PE stages prevent all human disease symptoms.Withup to100%efficacy in human trials, live attenuated whole parasite vaccines have been mosteffective to date, and include sporozoites that have been radiation-, drug-, or geneticallyattenuated. All of these can invade hepatocytes but subsequently arrest at different pointsduring the liver stage(LS) or early in the BS of the life cycle of the parasite, whilesimultaneously inducing immune responses that protect against subsequent challenge withwild type sporozoites (wt spz). In spite of their promise, whole-parasite malaria vaccines aretechnically complex, and currently require inoculation via multiple bites of live infectedmosquitoes, or intravenous administration of high doses of purified, cryopreservedsporozoites. Subunit vaccines constitute an alternative to whole parasite vaccines that cancircumvent the logistical constraints of developing and administering live attenuatedparasites. However, the most advanced subunit vaccine, the CSP-based RTS,S, exhibited a32-50%anti-disease (but not anti-infection) efficacy among African children in Phase IIIbtrials. Importantly, several studies have indicated that PE antigens other than CSP are able toinduce sterile protection in mice. This warrants the need for further research aimed atidentifying non-CSP candidate antigens that could be used alone or in combination withCSP to develop subunit vaccines that can provide substantially better protection than thecurrent RTS,S formulation. However, only a few non-CSP PE antigens have been identifiedand evaluated as vaccine candidates to date. Although antibodies have been shown to play arole in the inhibition of hepatocyte invasion by sporozoites, studies in rodent andnon-human primates malaria models, as well as clinical trials in humans have shown that CD8+T cells play a major role in mediating protection induced by whole parasitevaccination. It is important to identify antigens that are the target of CD8+T cells involvedin the elimination of infected hepatocytes, as well as the mechanisms by whichmalaria-infected cells are destroyed by CD8+T cells. Our study determinedthat novelantigen P.yoelii Tmp21can induce a protective response in vivo by a DNA vaccine, andoptimized a novel technical approach thatcharacterizes the ability of antigen-specificCD8+T cells toeliminate hepatocytes in vivo following immunization with whole parasitevaccines as a means to validate potential vaccine candidates. This method combines the useof Hydrodynamic Tail Vein Injection (HTVI) to deliver naked DNA encoding luciferasetagged malaria LS antigens directly to the liver with an in vivo imaging system (IVIS) thatallows real-time monitoring of the abundance of the luciferase-tagged antigens in the liver.We next determined whether the optimized HTVI/IVIS technique can be used to validateother PE antigens as contributing to protection elicited by whole parasite vaccines byevaluating the ability of PyTmp21to induce hepatocyte killing in vivo. Results as below:Identification of PyTmp21as a potential protective antigen that reduces liver stageparasite burden and confer sterile protection against sporozoites challenge. To determine ifthis antigen can induce a protective response in vivo, we generated a DNA vaccine bycloning the PyTmp21into the gWIZ vector. The results shows that immunization with aPyTmp21DNA vaccine triggers an immune response that significantly reduces LS parasiteburden and confer60%sterile protection against P. yoelii17XNL wt spz.To determine whether the HTVI/IVIS method could be used as a tool to validate novelPE antigens that are the target of CD8+T cells immune responses induced by Py fabb/f-whole parasite vaccine.1. To evaluate the expression of PyCSP-Luc fusion protein in vivo, we injected groupsof BALB/cJ mice by HTVI with recombinant plasmid DNA PyCSP-Luc. The result showsthat the PyCSP-luciferase fusion protein is expressed in a stable and persistent manner inthe liver of mice injected with plasmid DNA through HTVI, and that by measuringwhole-body bioluminescence in vivo, IVIS allows the real-time monitoring of theabundance of luciferase-fused tagged proteins. Using flow cytometry, we confirmed thathepatocytes correspond to the major liver cell type expressing PyCSP-Luc after HTVIadministration. 2. Mice were immunized by intravenous injection with two doses of50,000Pyfabb/f-sporozoitesto induced protective immune response and then challenged with PyCSP-lucplasmid DNA by HTVI. Quantification of bioluminescence kinetics in the liver wasmeasured using IVIS after challenge. However, the luciferase signal was dramaticallydecreased in Pyfabb/f-immunized mice challenged with PyCSP-Luc plasmid DNA, ascompared to mock-immunized mice. We used monoclonal antibodies to deplete CD8+andCD4+T cells in vivo and confirmed that the elimination of hepatocytes are mediated byCD8+T cells. We also showed an increase in the level of total and IFN-γ producingCSP-specific CD8+T cells in the liver of mice immunized with Pyfabb/f-and challengedwith PyCSP-Luc. Our results showed that immunization with whole parasites caninduce aCSP-specific immune response that is able to eliminate those hepatocytes that present thisantigen on their surface. A new technique combined HTVI with IVIS can be used to identifynovel LS antigens that are the target of CD8+T cells responses.HTVI/IVIS method was used to identify novel LS antigens that associate with theprotective immunity induced by whole parasite vaccination. To determine whether theHTVI/IVIS method optimized with PyCSP can be used to identify novel antigens ascontributing to protection elicited by whole parasite vaccines, we used it to test PyTmp21.However, we were initially unable to confirm PyTmp21as an antigen that contributes to theprotection elicited by Pyfabb/f-sporozoites using the immunization strategy that worked forPyCSP. Next, we explored different heterologous DNA/spz or spz/DNA priming-boostimmunization strategies. These two immunization strategies resulted in a reduction ofluciferase signal upon challenge with PyTmp21-Luc plasmid DNA. These data suggest thatPyTmp21is expressed by Pyfabb/f-parasites, and that it can induce immune response todestroy hepatocytes presenting specific antigens. Our results showed that the immuneresponses induced by attenuated whole parasites are mainly directed against theimmunodominant CSP. Subsequent boosts with attenuated whole parasites result both inlower levels of LS parasite development and in the inhibition of the boosting of T cellresponses against lower frequency, non-CSP antigens. We confirmed that immunizationwith whole parasites can induce a PyTmp21-specific T cell immune response byheterologous prime/boosting immunization strategy. PyTmp21as a potential PE antigencontribute to the protective immune response upon immunization with attenuated whole parasites.In conclusion, using CSP as a model, we were able to confirm that the HTVI/IVISmethod enables the detection of hepatocytes that are killed as a consequence of presentingspecific parasite antigens, and that this killing depends on CD8+T cells. Furthermore, thedata presented herein show that the use of aheterologous immunization strategy coupledwith the HTVI/IVIS method constitutes a powerful tool to validate pre-erythrocytic antigensthat contribute to the protection elicited by whole parasite vaccines. In particular, weconfirmed that PyTmp21, which we previously identified as a novel pre-erythrocyticantigen, contributes to the protective immunity elicited by whole parasite vaccinations.Ultimately, the method described herein can be used to validate new malaria vaccinecandidates and increase our understanding of how whole parasite immunization protectsagainst malaria, thus paving the way for intelligent vaccine design.