紅內期瘧疾疫苗的研究進展

陳琳,黃復生

(第三軍醫大學基礎部病原生物學教研室,重慶 400038)

Background

Malaria remains one of the global devastating infectious disease,and results in 300-500 million clinical cases and nearly one million deaths annually worldwide[1,2].There are five species of Plasmodium parasites transmitted to humans:P.falciparum,P.vivax,P.ovale,P.malariae and P.knowlesi.P.falciparum is associated with the great majority of morbidity and mortality in human parasite,whereas P.vivax causes considerable symptomatic disease.Malaria is a mosquito-borne disease,and the parasite＇s sporozoites(SPZ)invade blood vessels and are transported to the liver after bitten by infected Anopheles mosquitoes.Here they infect hepatocytes,replicate and form a liver stage(LS)that grows asymptomatically.The blood-stage of the lifecycle commences when merozoites are released from the individual hepatocyte and infect erythrocytes.Red blood cell invasion is thought to involve multi-step process that several proteins at the parasite and erythrocyte surface receptor-ligand interaction[3].Once inside the red blood cell,parasites are partially hidden from immune recognition because erythrocytes lack a nucleus and major histocompatibility complex molecules.The parasites express proteins on the surface of infected erythrocyte and exposed to the immune system.

Human repeated exposure to malaria parasites can naturally acquire immune and predominantly target the blood-stage parasites[4,5].Humoral-and cell-mediated immune responses play crucial roles in protective immunity against malaria parasites.But the immune effector mechanisms and the correlates of protection are poorly understood.It is suggested by passive immunoglobulin-transfer studies that antibodies are crucial components in blood-stage protective immunity,which may play a role in preventing merozoites invasion,antibody-dependent cell-mediated cytotoxity and greater clearance of infected red blood cell[6].With the development of resistance of malaria parasites to drugs and resistance of mosquitoes to insecticides,a cheap,broadly protective malaria vaccine is urgently required.A high level of antigenic diversity,redundancy of parasite invasion pathways and mechanisms of immune evasion pose significant challenges for vaccine development.The purpose of this review is to focus on the development of blood-stage vaccines,the advantages and challenges of this approach,potential target antigens expressed in the blood-stage and future directions.

1 Potential targets

The greatest challenge for developing effective blood-stage vaccine is the antigen diversity and identification of potential targets[7,8].At present,Merozoite surface proteins,as initial attachment antigens,and whole blood-stage parasites are the most promising candidates in blood-stage vaccine.

1.1 Merozoite surface protein 1(MSP1)

MSP1 is GPI(glycosylphosphatidylinositol)anchored to the merozoite surface and a 200 kDa polymorphic protein which undergoes proteolytic processing into four fragments p83,p30,p38,and p42.MSP1-42 undergoes secondary processing into two fragments p19 and p33.MSP1,which is important for the invasion process of erythrocytes by merozoite,is considered a prime candidate antigen for a blood stage vaccine.Several lines of evidence indicated thatthe C-terminal region of MSP1(MSP1-19 and-42)demonstrated the high antigenicity,and the naturally acquired antibodies to the C-terminus can inhibit the erythrocyte invasion and MSP processing[9,10].Therefore,both MSP1-42 and MSP1-19 fragments are thought to be the most advanced candidates for MSP1 vaccine development.But anti-MSP1-42 sera demonstrated strain-specific protection for P.falciparum-Aotus challenge model,and a PhaseⅡb trial of MSP1-42 in Kenyan found no protective effect[11].Antigen diversity was thought to a be major reason of failure of the vaccine.The study of genetic diversity in coding regions of PvMSP1-42 showed that 19kDa fragment at amino acid level was highly conserved in Sri Lankan isolates,whereas 33kDa fragment demonstrated extensive genetic diversity[12].But the opposite pattern was observed in P.falciparum[13].These observations indicate that the immunological interference between epitopes of PvMSP1-19 and PvMSP1-33 may affect the protection of PvMSP1-42 vaccine and a vaccine based on PvMSP1-19 alone could be effective against P.vivax infections in Sri Lankan.However,the analysis of antibody responses to PfMSP1-19 variant forms suggested the presence of cross-reactive antibody responses and non-variant specific antibodies to PfMSP1-19 in I-ranian individual infected by P.falciparum.One variant of this antigen,especially Q/KNG/L,may be sufficient for developing a PfMSP1-19-based vaccine[14].A study by Woehlbier showed that antibody elicited by entire MSP molecule can efficiently inhibit parasite multiplication and RBC invasion.Moreover,anti-MSP-1D antibodies would provide cross-protection for FCB-1 strain,a representative of K1 prototype.This study may provide the possibility of developing a vaccine based on the fullsize MSP1[15].

1.2 Merozoite surface protein 2

Merozoite surface protein 2(MSP-2)is a highly polymorphic 45-53 kDa protein,which like MSP-1,is also GPI anchored to the merozoite surface.High IgG3 antibody titers to MSP2 suggested that the molecule might be involved in protective immunity against P.falciparum[16,17].The N-and C-terminal domains of two allelic families,3D7 and FC27,are highly conserved with a variable region composed of central repeat region.MSP-2 was an important component in Combination B,including full-length 3D7 MSP2,block 2,3 of MSP1 and ring-infected erythrocyte surface antigen(RESA)formulated with the Montanide ISA720[18].A phase I/IIb field trial involving 120 children living in an area of Papua New Guinea reported that parasite densities were significantly decreased(62%)in the vaccine recipients[19].Furthermore,the activity of MSP-2 subunit included in the vaccine couldn＇t pro-tect against MSP-2 allelic family(FC27),suggesting that the major component of protective effect in Combination B was MSP-2[20].T he success of Combination B makes it possible that MSP-2 may be the most promising candidate antigen.Inclusion of both allelic variant in MSP-based vaccine could give the higher protective efficacy.Flueck found that two MSP-2 long synthetic peptides(LSP),comprising the semi-conserved family-specific domain plus the C-terminal domain of the two allelic families,could yield high titer antibody responses,and the response was associated with protective effect[21].However,MSP-2 is an intrinsically unstructured protein,which contains a single intramolecular disulphide bond and lacks hydrophobic residues.The lack of the structure knowledge of MSP-2 is the major hurdle to presume that an MSP-2 fragment will be as effective as the fulllength protein to induce a high antibody response.

1.3 Merozoite surface protein 3

Merozoite surface protein 3(MSP-3)is associated with merozoite surface through the non-covalent linkages and has been suggested to be involved in erythrocyte binding,although its function is unknown[22].Structurally,the C-terminal domain containing leucine zipper sequence is entirely conserved,whereas N-terminal region comprised of three blocks of heptad repeats is relatively polymorphic,which define two allele classes termed 3D7 and K1[23].The naturally acquired MSP-specific IgG3 antibody has strong antiparasitic effect in P.falciparum infected children.The cytophilic antibodies inhibited intra-erythrocytic parasite growth in a monocyte-dependent manner[24].In ADCI assays,amino acid residues 194 to 257 in C-terminal domain have been shown to exert a strong inhibition activity against P.falciparum parasite growth.MSP-3,as a long synthetic peptide,underwent a Phase Ib clinical trial among Tanzanian children and Burkina Faso adult males.The result showed that the MSP3-LSP vaccine with two adjuvants,Montanide ISA and aluminium hydroxide,was safe and immunogenic even at low dose in a malaria-na?ve population.Further studies are to define the true protective effects through Phase II clinical trial[25,26].However,recent studies demonstrated that the N-terminal domain of PfMSP3 is significantly more immunogenic than the C-terminal region.Antibody responses to N-terminal domain were largely allele specific and no been found allele specific within each allele class.These data raise the possibility for development of any PfMSP3 N-terminal domain-based vaccine[27,28].

1.4 Apical membrane antigen-1

AMA1 is thought to play an essential role in erythrocyte invasion.However,it is highly polymorphic and allelic-specific antibodies are not cross-protective[29].AMA1-C1 contains sequences from the FVO and 3D7 allelic form,adjuvanted with alhydrogel,and has progressed though Phase I/II trial in Mali.But the antibody levels were not maintained.Adding CPG 7909 to the AMA1/Alhydrogel formulation can improve immunogenicity[30].A vaccine containing the FVO form with three different adjuvants,including Alhydrogel,Montanide ISA 720 and ASO2A,Phase I clinical trail is ongoing and the highest antibody response were induced by ASO2A formulations[31].

1.5 P.falciparum erythrocyte membrane protein-1

P.falciparum erythrocyte membrane protein-1(PfEMP1)family is expressed on the surface of infected erythrocytes.PfEMP1 is encoded by 60 or more var genes.And different var genes encode PfEMP1 variants with different antigenic properties[32].The extreme variability of this antigen is a challenge for the development of a highly effective vaccine.The success of a subunit vaccine depended on its ability to elicit cross-reactive responses to different variants.A specific variant of PfEMP1,VAR2CSA,is thought to mediate parasite sequestration in the placenta and less antigenically diverse than other PfEMP1 variants.T he antibody raised against VAR2CSA is cross-reactive with different placental-binding isolates,showing that the development of an effective vaccine based on the conversed epitopes of VAR2CSA is possible[33].

2 Whole blood-stage parasites approach

Owing to the success of radiation-attenuated sporozoite studies,the whole-parasite vaccine has regained attention.There is evidence that na?ve adult human volunteers infected with a low dose of live P.falciparum parastes can develop cell-mediated immune response and ultra-low doses of irradiated or CpG adjuvanted killed parasites can induce cellular immunity against both heterologous and homologous parasites in animal models[34,35].The whole-parasite vaccine is likely to involve a vast array antigens,thus reducing the impact of antigenic polymorphisms.More recently,study has suggested that crude extract from Pf whole-parasite could elicit parasite antigen-specific immune response via Toll-like receptor(T LR)9,by adjuvanted with the malaria heme-detoxification byproduct,hemozoin(HZ).A synthetic HZ could be used as an adjuvant to improve immunogenicity of whole-parasite vaccines[36].However,the safety and large-scale production of whole-parasite vaccine is an important challenge for the widespread use.

3 Challenges and future directions

There are many evidences supporting the development of a vaccine based on blood-stage parasite antigens.However,the recent two Phase II clinical trials show no protective efficacy in African children.One possible hurdle for development of the blood-stage vaccine is the difference in parasitespecific,immune and pathogenic responses to malaria parasites between mice and humans.Recently,efforts are also going toward"humanize"the mouse model that a valid approach to evaluate blood-stage vaccine,but further studies are needed to evaluate their relevance.However,the greatest challenge for blood-stage vaccine development is perhaps highly antigenic polymorphism.Multiple antigens or allelic forms are possible required in a single vaccine to overcome the challenge.More knowledge on identification of potential antigens as blood-stage vaccine candidate isneeded.Highthroughput serological screening of whole plasmodium proteomes might guide vaccine and diagnostic antigen discovery[37].

4 Conclusion

In this review,we have highlighted the development and challenge of leading blood-stage candidate vaccine.Many target antigens expressed on the surface of merozoites have been demonstrated that antibodies to these proteins can confer protection against homologous parasite challenge.However,so far,no recombinant protein has been proved sufficiently efficacious in human test.Effective blood-stage vaccines will almost certainly need to choose several different antigens or several allelic types to overcome antigenic diversity.Therefore,there is a strong need for identification of a multitude of new antigens from genomic and proteomic insights.Recently,efforts are also underway to reexamine whole blood-stage parasite vaccines.We remain optimistic about the development of bloodstage vaccines against malaria and a highly effective vaccine is possible.

[1]Snow RW,Guerra CA,Noor AM,et al.The g lobal distribution of clinical episodes of Plasmodium falciparum malaria[J].Nature,2005,434(7030):214-217.

[2]Guerra CA,Gikandi PW,T atem AJ,et al.The limits and intensity of Plasmodium falciparum transmission:implications for malaria control and elimination worldwide[J].PLoS Med,2008,5(2):38.

[3]Gilson PR,Crabb BS.Morphology and kinetics of the three distinct phases of red blood cell invasion by Plasmodium falciparum merozoites[J].International Journal for Parasitology,2009,39(1):91-96.

[4]Marsh K,Snow RW.Host-parasite interaction and morbidity in malaria endemic areas[J].Philosophical Transactions of the Royal Society of London Series B Biological Sciences,1997,352(1359):1385-1394.

[5]Doolan DL,Dobano C,Baird JK.Acquired immunity to malaria[J].Clin Microbiol Rev,2009,22(1):13-36.

[6]Langhorne J,Ndungu FM,Sponaas AM,et al.Immunity to malaria:more questions than answers[J].Nat Immunol,2008,9(7):725-732.

[7]Volkman SK,Hartl DL,Wirth DF,et al.Ex cess polymorphisms in genes for membrane proteins in Plasmodium falciparum[J].Science,2002,298(5591):216-218.

[8]Polley SD,Conway DJ.Strong diversifying selection on domains of the Plasmodium falciparum apical membrane antigen 1 gene[J].Genetics,2001,158(4):1505-1512.

[9]John CC,O＇Donnell RA,Sumba PO,et al.Evidence that invasion-inhibitory antibodies specific for the 19-kDa fragment of merozoite surface protein-1(MSP-1 19)can play a protective role against blood-stage Plasmodium falciparum infection in individuals in a malaria endemic area of Africa[J].J Immunol,2004,173(1):666-72.

[10]O＇Donnell RA,de Koning-Ward TF,Burt RA,et al.A ntibodies against merozoite surface protein(M SP)-19 are a major component of the invasion-inhibitory response in individuals immune to malaria[J].J.Ex p.Med,2001,193(12):1403-1412.

[11]Ogutu BR,Apollo OJ,Mckinney D,et al.Blood stage malaria vaccine eliciting high antigen-specific antibody concentrations confers no protection to young children in Western Kenya[J].P LoS One,2009,4(3):4708.

[12]Dias S,Longacre S,Escalante AA,et al.Genetic diversity and recombination at the C-terminal fragment of the merozoite surface protein-1 of Plasmodium vivax(PvMSP-1)in Sri Lanka[J].Infect Genet Evol,2011,11(1):145-156.

[13]Pacheco MA,Poe AC,Collins WE,et al.A comparative study of the genetic diversity of the 42 kDa fragment of the merozoite surface protein-1 in Plasmodium falciparum and P.vivax[J].Infect.Genet.Evol,2007,7(2):180-187.

[14]Zakeri S,Mehrizi AA,Zoghi S,et al.Non-variant specific antibody responses to the C-terminal region of merozoite surface protein-1 of Plasmodium falciparum(PfMSP-1(19))in Iranians ex posed to unstable malaria transmission[J].Malar J,2010,9(9):257-263.

[15]Woehlbier U,Epp C,Kauth CW,et al.Analysis of Antibodies Directed against Merozoite Surface Protein 1 of the Human M alaria Parasite Plasmodium falciparum[J].Infec Immun,2006,74(2):1313-1322.

[16]M etzger WG,Okenu DM,Cavanagh DR,et al.Serum IgG3 to the Plasmodium falciparum merozoite surface protein 2 is strongly associated with a reduced prospective risk of malaria[J].Parasite Immunol,2003,25(6):307-312.

[17]Stanisic DI,Richards JS,McCallum FJ,et al.IgG subclassspecific responses against Plasmodium falciparum merozoite antigens are associated with control of parasitemia and protection from symptomatic illness[J].Infect Immun,2009,77(3):1165-1174.

[18]Genton B,Al-Yaman F,Betuela I,et al.Safety and immunogenicity ofa three-componentblood-stage malaria vaccine(MSP1,MSP2,RESA)against Plasmodium falciparum in Papua New Guinean children[J].Vaccine,2003,22(1):30-41.

[19]Genton B,Betuela I,Felger I,et al.A recombinant blood-stage malaria vaccine reduces Plasmodium falciparum density and exerts selective pressure on parasite populations in a phase 1-2b trial in Papua New Guinea[J].J Infect Dis,2002,185(6):820-827.

[20]Fluck C,Schopflin S,Smith T,et al.Effect of the malaria vaccine Combination B on merozoite surface antigen 2 diversity[J].Infect Genet Evol,2007,7(1):44-51.

[21]Flueck C,Frank G,Smith T,et al.Evaluation of two long synthetic merozoite surface protein 2 peptides as malaria vaccine candidates[J].Vaccine,2009,27(20):2653-2661.

[22]Rodriguez LE,Curtidor H,Ocampo M,et al.Identifying Plasmodium falciparum merozoite surface antigen 3(MSP3)protein peptides that bind specifically to erythrocy tes and inhibit merozoite invasion[J].Protein Sci,2005,14(7):1778-1786.

[23]Huber W,Felger I,Matile H,et al.Limited sequence polymo rphism in the Plasmodium falciparum merozoite surface protein 3[J].Mol Biochem Parasitol,1997,87(2):231-234.

[24]Roussilhon C,Oeuvray C,Mǜller-Graf C,et al.Long-term clinical protection from falciparum malaria is strongly associated with IgG3 antibodies to merozoite surface protein 3[J].PLos M ed,2007,4(11):320.

[25]Sirima SB,Nebie I,Ouedraogo A,et al.Safety and immunogenicity of the Plasmodium falciparum merozoite surface protein-3 long synthetic peptide(MSP3-LSP)malaria vaccine in healthy,semi-immune adult males in Burkina Faso,West Africa[J].Vaccine,2007,25(14):2723-2732.

[26]Lusingu JP,et al.Satisfactory safety and immunogenicity of MSP3 malaria vaccine candidate in Tanzanian children aged 12-24 months[J].Malar J,2009,8(6):163-171.

[27]Polley SD,Tetteh KK,Lloyd JM,et al.Plasmodium falciparum merozoite surface protein 3 is a target of allele-specific immunity and alleles are maintained by natural selection[J].J Infect Dis,2007,195(2):279-287.

[28]Jordan SJ,Oliveira AL,Hernandez JN,et al.Malaria Immunoepidemiology in Low Transmission:Correlation of Infecting Genotype and Immune Response to Domains of Plasmodium falciparum M erozoite Surface Protein 3[J].Infect Immun,2011,79(5):2070-2078.

[29]Cortes A,Mellombo M,Mueller I,et al.Geographical structure of diversity and differences between symptomatic and asymptomatic infections for Plasmodium falciparum vaccine candidate AM A1[J].Infect Immun,2003,71(3):1416-1426.

[30]Sagara I,Ellis RD,Dicko A,et al.A randomized and controlled Phase 1 study of the safety and immunogenicity of the AMA1-C1/Alhydrogel+CPG 7909 vaccine for Plasmodium falciparum malaria in semi-immune Malian adults[J].Vaccine,2009,27(52):7292-7298.

[31]Roestenberg M,Remarque E,de Jonge E,et al.Safety and immunogenicity of a recombinant Plasmodium falciparum AM A1 malaria vaccine adjuvanted with Alhydrogel,Montanide ISA 720 or AS02[J].PLoS One,2008,3(12):3960.

[32]Scherf A,Lopez-Rubio JJ,Riviere L.Antigenic variation in Plasmodium falciparum[J].Annu Rev Microbiol,2008,62:445-470.

[33]Elliott SR,Duffy MF,Byrne TJ,et al.Cross-reactive surface epitopes on chondroitin sulfate A-adherent Plasmodium falciparum-infected ery throcytes are associated with transcription of var2csa[J].Infect Immun,2005,73(5):2848-2856.

[34]Pinzon-Charry,A.and Good,M.F.Malaria vaccines:the case for a wholeorganism approach[J].Expert Opin.Biol,2008,8(4):441-448.

[35]Pombo DJ,Lawrence G,Hirunpetcharat C,et al.Immunity to malaria after administration of ultra-low doses of red cells infected with Plasmodium falciparum[J].Lancet,2002,360(9333):610-617.

[36]Coban C,Igari Y,Yagi M,et al.Immunogenicity of wholeparasite vaccines against Plasmodium falciparum involves malarial hemozoin and host T LR9[J].Cell Host Microbe,2010,7(1):50-61.

[37]Davies DH,Liang X,Hernandez JE,et al.Profiling the humoral immune response to infection by using proteome microarray s:high-throughput vaccine and diagnostic antigen discovery[J].Proc Natl Acad Sci U S A,2005,102(3):547-552.