彭小红(综述),黄复生(审校)

(第三军医大学基础部病原生物学教研室,重庆 400038)

In troduction

Malaria,one of the human infectious diseases with high incidence and fatality rate,is transmitted through the bite of infected Anopheles mosquito.There were about 225 millions malaria cases with 0.781 million deaths reported in 2009(WHO 2010).Currently,drug treatment of infected individuals,preventive drug treatment of populations at high risk of disease,and insecticide-treated bed nets and indoor-insecticide spraying for mosquito control constitute the main weapons to contro lmalaria[1].Nevertheless,the emergence of drug-resistant Plasmodium strain and insecticide resistance of mosquitoes has a major impact on the treatment and prevention of malaria.Effective and long lasting vaccine is urgently needed to control the morbidity and the prevalence of the malaria.However,in the past decades,thousands of scientists devoted to the study on the malaria vaccine,butmade limited success.

Most of malaria vaccine researches focus on the pre-erythrocytic stage and the erythrocytic stage.Current blood-stage preventive vaccine efforts are high ly focused on three proteins,merozoite surface protein 1(MSP1),apicalm embrane antigen 1(AMA 1)and the P.vivax Duffy binding protein(PvDBP).A ll the three proteins participate inthe merozoites invasion[2-4].Antibodies against all the three proteins can inhibit parasite invasion.However,antigen polymorphism and redundancy of invasion pathways significantly challenges for vaccine development.The most advanced MSP1 vaccine candidate has been demonstrated to conferred only strain-specific protection[5].

Pre-erythrocytic stage is the first stage of Plasmodium to invade a human host.Interrupting the proliferation of parasite during the pre-erythrocytic stage can block the development and transmission of malaria parasites.Malaria vaccines for pre-ery throcy tic stage include who le-cell vaccines(irradiation-attenuated sporozoites and genetically attenuated sporozoites)and subunit vaccines.In this review,we discuss the development of these vaccines and difficultiesencountered in the present study.

1 Irradiation-attenuated sporozoites(irrSPZs)

U sing P.bergheimodel,Ruth Nussenzweig et.al firstly found that irradiation-attenuated sporozoites can induce completely protection in mice[6].When sporozoites(SPZs)were irradiated,DNAs of the SPZs have random mutations and breaks.But it＇s hard to control the safety and efficacy of irrSPZs as it is dependent on a precise irradiation dose.Meanwhile,producing large amount of irrSPZs for vaccination is difficult.Although Stephen Hoffman and his company Sanaria have developed a manufacturing process for the aseptic production of irrSPZs inmosquitoes,there are stillmany preclinical issues that need to be resolved at present.Considering these difficulties,m any scientists have been using the irrSPZs model to investigate the mechanism of immunoreaction induce by irrSPZs,expecting to understand the comp lex pre-erythrocytic stage specific immune protection mechanism for design novel protective and long-lasting vaccine.

1.1 Antigen p resent cells(APCs)

When a mosquito bites a host,the SPZs pass though the subcutaneousand then reach liver cells for developm ent.In the p rocess,many immune cells or non-immune cells may involve in the activation of the immune response.So the accurate site for T cell priming is a preliminary problems needed to solve.

To infect hepatocytes,SPZs must cross the layer of sinusoidal cells com posed of specialized,highly fenestrated sinusoidal endothelia interspersed with Kupffer cells(KC)[7],and then pass through several liver cells before settle in a final liver cell.Som e studies suggest that CD8+Tcell may be activated by the antigens presented on the infected liver cell surface by MHC-Ⅰmolecule[8-10].But this view is limited by the classic theory of antigen-presenting.Although liver cells as a non-specific APC can express MHC-Ⅰmolecules to provide the first signal for Tcell activation,the limited expression of co-stimulatorym olecules CD80,CD40 is not able to provide the necessary second signal[11].M ean while,invaded SPZs can decrease the KCs function as APCs by inhibition the expression of MHC-Ⅰmolecules on KC[12].Recent live im aging techniques found thataftermosquito bite or subcutaneous injection the SPZs not only reached directly into the capillaries but also into the lymphatic vessels.Parts of the SPZs into the lymphatic vessels can be captured by dendritic cells(DCs),and a small number of SPZs are able to live and develop in DCs[13].It provides important evidence for the issue that immune cells in local lym ph nodes p lay an important role in the activation of pre-erythrocytic stage specific imm une response.According to these results,Chak ravarty and his colleagues found that the DCs in local lymph nodes first initiate CD8+T cells response,and then activated CD8+Tcellsmigrate from the lymphatic to other tissues such as liver and spleen.Blocking the local lymphocytes flow to the secondary lymph nodes,or early activation of DCs,which can inhibit the function of DCs to capture SPZs,can comp letely interrupt the activation of CD8+T cells and clearance of parasite in the liver[14].So DCs in the draining lymph node may play a core role in the activation of pre-erythrocy tic stage specific CD8+T cells,but further investigation need to carry out.

1.2 Protectivemechanism

Up to date,CD8+T cells have been considered having a critical role in the protection ofm ice and human immunized w ith irrSPZs[15],whereas interleukin-4(IL-4)-secreting CD4+Tcellsare essential for the development of CD8+T cells responses to LS parasites.It has been presumed the p rotection to bemediated directly by CD 8+CTL or indirectly by IFN-γreleased from CD8+T cells for a number of years.This view was turnover by Doolan in 1999.They found that the sporozoite-induced protective immunity is still existed after perforin knockout and Fas ligand-deficient mice immunized with irrSPZs[16].Meanw hile,IFN-γasa critical mediator of sporozoite-induced protective immunity was confirmed using IFN-γKOmice.Because wildtype mice were solidly protected against challenge sporozoites,whereas wild type mice depleted of IFN-γand IFN-γKO mice were not protected.Inducible NOS(iNOS)is considered as amajormediator of cytotoxicity against intracellular pathogens.In vivo and in vitro study both demonstrated IFN-γ contributes to iNOS production by hepatocy tes as well asother cells following infection with Plasmodium[17,18].Previously,Seguin etal.also demonstrated that protection in mice immunized with irradiated P.berghei sporozoites was dependent upon the inducible,but not constitutive,NO pathw ay.And that induction of iNOS in the liver was dependent on CD8+T cells and IFN-γ[19].Therefore,IFN-γ induction of the iNOS pathway,and subsequent elimination of infected hepatocytesor hepatic schizonts within those cells,is anecessary component for CD8+T cells dependent protection in irrSPZs immunized mice.Maintenance of the protection needs generation of them em ory CD8+T cells.Activation of the CD8+T cells reached the peak after 8 hours with irrSPZs immunization.But it began to decline approximately 48 hours after immunization[20].Antigen specific CD8+T cells proliferation occurred at 48～72 hours after immunization and achieved peak at 4～5 days.The amount of the cells declined at 6～7 days asapoptosis occurred in 70～80%of the cells.After this initial phase,antigen specificmemory CD8+T cells will produce in liver and spleen and maintain approxim ately 6 months[21].The quantity of memory CD8+T cellshasgreat relationship with the sporozoites immunization dose.H igh doze at the first vaccination produces large quantities ofmemory CD8+T cells[22].However,one large dose of antigen at the first time may lead to immune tolerance,and repeated low doses of sporozoiteimm unity can better maintain the memory CD8+T cells survival[23].

2 Genetically attenuated sporozoites

The advanced genetic engineering techniques,the availability of genome sequences for a number of Plasm odium species and the generation of stagespecific gene expression data enabled the search for genes that p lay essential roles for parasite survival at distinct points during the life cycle.Genetically attenuated parasites(GAPs)are the use of gene knockout app roach to the parasite purpose gene to achieve the effect of attenuated.In 2005,using rodentmalaria parasite species P.berghei,Kappe and colleagues firstly knocked out pre-ery throcy tic stage-expressed genes known as UIS3(Upregulated in Infectious Sporozoites).Moreover,immunization with UIS3-deficient sporozoites confers comp lete protection against infectious sporozoite challenge in a rodent malaria model[24].This was a landmark finding that open the new era of GAPs.Then,so many genes had been investigated by this method,such as UIS4[25],P52[26],P36[27],SAP1[28]and fabb/f[29].A ll these genes are expressed at liver stage and indispensable for the development of liver stage.Parasites w ith these genes knock out can pass though the mosquito but arrest of development at different stage of hepatocyte infection.However,few of the single gene knockout(UIS4,P52,P36)showed occasional breakthrough in fec-tions when high numbers of SPZs were used for immunization[25,26].The advantage of the GAPs as com pared with irrSPZs is that the safety of the vaccine can artificially controlled.The GAPs induced protection wasm ediated mainly by CD8+T cells[30,31].And antibodies also contributed to protection.Mean while,CD8+Tcells mediate killing of infected hepatocytes,which is depend on cells-contacted.IFN-γand perforin may both play crucial role in the protection[32].A lthough rodent malaria GAPs obtained encouraging development,the investigation of GAPs on the human malaria still have a long way to go.Sofar,P.falciparum p52-/p36-dual gene deletion parasites exhibited complete grow th arrest of liver stage in vitro in a hepatocytic cell line and in a humanized mousemodel carrying human hepatocytes.Moreover,it has been selected for advance into phaseⅠ/Ⅱa field trials through the administration of GAP-infected mosquito bites to hum an volunteers[33].We are looking forward to the surprise brought by this vaccine to us.

3 Subunit Vaccine

Although whole-cell vaccines induce complete and long-lasting protection,the difficulties to produce and transport have limited its clinicalap plication.Therefore,subunit vaccine is expected to have an ideal effect of controlling malaria.In the past several decades,studieson the malaria subunit vaccine included antigen screening,ad juvants,immune protective mechanism,verification the clinical effective and so on.But sofar there are no effective vaccine caming out.

3.1 Circum sporozoite protein(CSP)

Most of the malaria subunit vaccines for preerythrocytic stage research were focusing on CSP,which is an immunodomin ant antigen of SPZs and is completely conserved am ong all strains sequenced to date.The most advanced malaria vaccine candidate RTS,Sisbased on CSP.RTS,S consists of part of the CSP fused to hepatitis Bsurface antigen and expressed with free Hep B surface antigen to enable the polypeptides to form virus-like particles[34].Thesewere presented with novel adjuvants(AS series)containing monophosphoryl lipid A(MPLA)and QS21.The vaccine efficacy could be attributed partially to the ad juvants.A recent review of RTS,S summarized the results of all studies and demonstrated that of 214 malaria-naive adults vaccinated with RTS,S with an AS adjuvant,85(39.7%)were protected from malaria follow ing deliberate mosquito challenge[35].A ll RTS,SAS-0X formulations induced high levels of CSP-specific antibody and CD4+T cell responses,but failed to induce CD 8+T cells.And the protection correlates with the higher titer of serum antibodies present in vaccines.While the level of CSP-specific antibody following vaccination does in general correlate with protection,the rapid diminution of antibody levels following vaccination appears to be responsible for the lack of durable protection[36].The reason why antibody levels are not maintained is unknown,but could relate to the inability of sporozoites to naturally boost vaccine-induced antibody responses which in turn could relate to exposure to only lownumbers of sporozoitesor to the polymorphic nature of the T cell epitopes on the CS protein.Nevertheless,it is reasonable to expect that if the antibody response was higher to commence w ith and/or persisted longer then the level and duration of efficacy of the vaccine would be improved.This is a goal of ongoing research.

3.2 Other proteins

Although the CSP-based RTS,S vaccine is the most advanced malaria vaccine candidate,it is not 100%efficacious and will need to be com bined with other antigens.Other pre-erythrocytic stage specific proteins,such as thrombospondin-related anonymous protein(TRAP),Liver-stage antigen 1(LSA-1),are also considered to be targets of antiin fectionimm unity.Although all of them can induce comparable IFN-γand antibody responses in rodent animals,they were no or have little protection in field trials[37,38].Encouragingly,LSA-3,which is expressed by SPZs,liver stage and blood-stage parasites,is confirmed in prim ates has a protective effect[39].

Together,a novel protective and long-lasting subunit vaccine need further study of the protective mechanism,identify conserved vaccine targets,appropriate ad juvants and long-term clinical trails.

4 Conclusion

In this review,the development of the malaria vaccines for pre-erythrocytic stage is discussed.In short,malaria vaccines researches are facing formidable obstacles.Subunit vaccines do not yet give the desired levels of protection,but would be easiest to manufacture and deliver.Whole-cell vaccines are protective,but we do not know how to best manufacture and deliver them.Antigenic diversity,immune protective mechanism needed to be further investigated for an available subunit vaccine.Avaccine must therefore identify novel antigens that they are not normally immunogenic in the whole organism but should be antigenic and recognized by vaccine-induced immune responses.Meanwhile,the whole-cell vaccines are in the progress of human trials.If the human trials with great success,we should pay more attention to its methods of manufacture and delivering.Although we face formidable obstacles on the road ahead,we should be optimistic that we will finally beat it with our unremitting effort.

[1] Greenwood BM.Control to elimination：implications for malaria research[J].Trends Parasitol,2008,24(10)：449-454.

[2] Singh AP,Ozwara H,Kocken CH,et al.Targeted deletion of Plasm odium knowlesiDuffy binding protein confirms its role in junction formation during invasion[J].Mol Microbiol,2005,55(6)：1925-1934.

[3] MitchellGH,Thomas AW,Margos G,et al.Apicalmem brane antigen 1,a major malaria vaccine candidate,mediates the close attachment of invasivemerozoites to host red blood cells[J].Infect Immun,2004,72(1)：154-158.

[4] Goel VK,Li X,Chen H,et al.Band 3 isahost receptor bindingmerozoite surface protein1 during the Plasmodium falciparum invasion of erythrocytes[J].Proc Natl Acad Sci U S A,2003,100(9)：5164-5169.

[5] Lyon JA,Angov E,Fay MP.et al.Protection induced by Plasmodium falciparum MSP1(42)is strain-specific,antigen and adjuvant dependent,and correlates with an tibody responses[J].PLoSOne,2008,3(7)：2830.

[6] Nussenzw eig RS,Vanderberg J,M ost H,et al.Protective immunity produced by the injection of x-irradiated sporozoitesof Plasmodium berghei[J].Natu re,1967.216(5111)：160-162.

[7] Wisse E,De Zanger RB,Charels K,et al.The liver sieve：considerations concerning the structure and function of endothelial fenestrae,the sinusoidal w all and the space of Disse[J].H epatology,1985,5(4)：683-692.

[8] Frevert U,Engelmann S,Zougbede S,et al.Intravital observation of Plasmodium berghei sporozoite infection of the liver[J].PLoS Biol,2005,3(6)：192.

[9] Frevert U,Usynin I,Baer K,et al.Plasmodium sporozoite passage across the sinusoidal cell layer[J].Subcell Biochem,2008,47：182-197.

[10] Leiriao P,M.M.Mota,and A.Rodriguez,A pop totic Plasm odium-infected hepatocytes provide antigens to liver dendritic cells[J].J Infect Dis,2005,191(10)：1576-1581.

[11] Bertolino P,McCaughan GW,Bow en DG.Role of primary intrahepatic T-cell activation in the＇liver tolerance effect＇[J].Immunol.Cell Biol,2002,80(1)：84-92.

[12] Steers N,Schwenk R,Bacon DJ,et al.The immune status of Kupffer cells profoundly influences their responses to infectious Plasmodium berghei sporozoites[J].Eur J Immunol,2005,35(8)：2335-2346.

[13] Amino R,Thiberge S,Martin B,et al.Quantitative imaging of Plasmodium transmission from mosquito tom amm al[J].Nat Med,2006,12(2)：220-224.

[14] Chakravarty S,Cockburn IA,Kuk S,et al.CD8+T lymphocytes protective against malaria liver stages are primed in skin-d raining lymph nodes[J].Nat M ed,2007,13(9)：1035-1041.

[15] Overstreet MG,Cockburn IA,Chen YC,et al.Protective CD8+T cells against Plasm odium liver stages：immunobiology of an＇unnatu ral＇immune response[J].Immunol Rev,2008,225：272-283.

[16] Doolan DL.and SL.Hoffm an,IL-12 and NK cellsare required for antigen-specific adaptive immunity against malaria initiated by CD8+T cells in the Plasmodium yoeliimodel[J].JImmunol,1999,163(2)：884-892.

[17] Mellouk S,Hoffman SL,Liu ZZ,et al.Nitric oxide-mediated antiplasmodial activity in human and mu rine hepatocytes induced by gamma interferon and the parasite itself：enhancemen t by exogenous tetrahydrobiopterin[J].Infect Immun,1994,62(9)：4043-4046.

[18] K lotz FW,Scheller LF,Seguin MC,et al.Co-localization of inducible-nitric oxide synthase and Plasm odium bergheiin hepatocytes from rats immunized with irradiated sporozoites[J].J Immunol,1995,154(7)：3391-3395.

[19] Seguin MC,K lotz FW,Schneider I,et al.Induction of nitric oxide synthase protectsagainst malariainmice exposed to irradiated Plasmodium berghei infected mosquitoes：involvement of interferon gamma and CD 8+T cells[J].JExp M ed,1994,180(1)：353-358.

[20] Hafalla JC,Sano G,Carvalho LH.et al.Short-term an tigen presentation and single clonal burst limit themagnitude of the CD 8+T cell responses to malaria liver stages[J].Proc Natl Acad Sci U S A,2002,99(18)：11819-118124.

[21] Sano G,H afalla JC,Morrot A,et al.Swift development of protective effector functions in naive CD8+T cells against malaria liver stages[J].JExp Med,2001,194(2)：173-180.

[22] Morrot A.and F.Zavala,Effector and mem ory CD8+T cells as seen in immunity to malaria[J].Immunol Rev,2004,201：291-303.

[23] Good M F.Identification of early cellular immune factors regulating grow th of malaria parasites in humans[J].Immunity,2005,23(3)：241-242.

[24] Mueller AK,Labaied M,Kappe SH,et al.Genetically modified Plasm odium parasitesasa protectiveexperimen tal malaria vaccine[J].Nature,2005,433(7022)：164-167.

[25] M ueller AK,Camargo N,Kaiser K,et al.Plasmodium liver stage developmental arrest by depletion of a protein at the parasite-host interface[J].Proc Natl Acad Sci USA,2005,102(8)：3022-3027.

[26] van Dijk MR,Douradinha B,Franke-Fayard B,et al.Genetically attenuated,P36p-deficient malarial sporozoites induce protective immunity and apoptosis of infected liver cells[J].Proc NatlAcad Sci U S A,2005,102(34)：12194-12199.

[27] Labaied M,H arupa A,Dumpit RF,et al.Plasm odium yoelii sporozoites with simultaneous deletion of P52 and P36 are completely attenuated and confer sterile immunity against infection[J].Infect Immun,2007,75(8)：3758-3768.

[28] A ly AS,M ikolajczak SA,Rivera HS,et al.Targeted deletion of SAP1 abolishes the expression of infectivity factors necessary for successfulmalaria parasite liver infection[J].Mol Microbiol,2008,69(1)：152-163.

[29] Vaughan AM,O＇Neill MT,Tarun AS,et al.TypeII fatty acid synthesis is essential only for malaria parasite late liver stage development[J].Cell Microbiol,2009,11(3)：506-520.

[30] Tarun AS,Dum pit RF,Camargo N,et al.Protracted sterile protection with Plasmodium yoeliipre-erythrocy tic genetically attenuated parasite malaria vaccines is independent of significant liver-stage persistence and is mediated by CD8+T cells[J].J Infect Dis,2007,196(4)：608-616.

[31] Jobe O,Lumsden J,Mueller AK,et al.Genetically attenuated Plasmodiumberghei liver stages induce sterile protracted protection that ismediated by major histocompatibility complex Class I-dependent in terferon-gamm a-producing CD 8+T cells[J].J Infect Dis,2007,196(4)：599-607.

[32] T rimnell A,Takagi A,Gupta M,et al.Genetically attenuated parasite vaccines induce contact-dependent CD8+T cell killing of Plasmodium yoelii liver stage-infected hepatocy tes[J].J Immunol,2009,183(9)：5870-5878.

[33] Van Buskirk KM,O＇Neill M T,De La Vega P,et al.Preery throcytic,live-attenuated Plasmodium falciparum vaccine candidatesby design[J].Proc Natl Acad Sci U S A,2009,106(31)：13004-13009.

[34] Stoute JA,Slaoui M,H ep pner DG,et al.A prelim inary evaluation of a recombinan t circumsporozoite protein vaccine against Plasmodium falciparum malaria.RTS,S M alaria Vaccine Evaluation G roup[J].N Engl J Med,1997,336(2)：86-91.

[35] Casares S,Brumeanu TD,and Richie TL,The RTS,Smalaria vaccine[J].Vaccine,2010,28(31)：4880-4894.

[36] Kester KE,Cumm ings JF,Ofori-Anyinam O,et al.Randomized,double-blind,phase 2a trial of falciparum malaria vaccines RTS,S/AS01B and RTS,S/AS02A in malaria-naive adults：safety,efficacy,and immunologic associates of protection[J].J Infect Dis,2009,200(3)：337-346.

[37] Bejon P,Mwacharo J,Kai O,et al.A phase 2b random ised trial of the candidate malaria vaccines FP9M E-TRAP and MVA ME-TRAP among children in Kenya[J].PLoS Clin T rials,2006,1(6)：29.

[38] Cummings JF,Spring MD,Schwenk RJ,et al.Recombinant Liver Stage Antigen-1(LSA-1)formulated with AS01 or AS02 is safe,elicits high titer antibody and induces IFN-gamma/IL-2 CD4+Tcells but does not protect against experimental Plasmodium falciparum infection[J].Vaccine,2010,28(31)：5135-5144.

[39] Daubersies P,Ollomo B,Sauzet JP,et al.Genetic immunisation by liver stage antigen 3 protects chimpanzees against malaria despite low immune responses[J].PLoS One,2008,3(7)：2659.