GP96NTD-CSP重组DNA疟疾疫苗诱导小鼠产生保护性免疫的研究

目的探讨pFLAG CMV8 gp96NTD-CSP重组DNA疟疾疫苗免疫能否诱导小鼠产生保护性免疫及其效应机制。方法以pFLAG CMV8质粒为载体,构建免疫用重组质粒,按照DNA疫苗免疫方法免疫小鼠;野生子孢子进行攻击后,采用Real-time PCR和吉氏染色观察被攻击小鼠的肝脏虫荷和原虫血症,即免疫小鼠抵御野生子孢子攻击的能力;并通过ELISA和ELISPOT方法探讨免疫小鼠保护性免疫的可能机制。结果核酸疫苗pFLAG CMV8 gp96NTD-CSP免疫小鼠能显著抵御野生子孢子的攻击,并且能诱导小鼠产生较高的抗体水平和较高的CSP特异的CD8+T细胞频率。结论 pFLAG CMV8 gp96NTD-CSP重组DNA疫苗可能通过诱导小鼠CSP特异抗体和CSP特异的CD8+T细胞的产生,一定程度上抵御野生子孢子的攻击。     

NYVAC-Pf7: a poxvirus-vectored, multiantigen, multistage vaccine candidate for Plasmodium falciparum malaria.

The highly attenuated NYVAC vaccinia virus strain has been utilized to develop a multiantigen, multistage vaccine candidate for malaria, a disease that remains a serious global health problem and for which no highly effective vaccine exists. Genes encoding seven Plasmodium falciparum antigens derived from the sporozoite (circumsporozoite protein and sporozoite surface protein 2), liver (liver stage antigen 1), blood (merozoite surface protein 1, serine repeat antigen, and apical membrane antigen 1), and sexual (25-kDa sexual-stage antigen) stages of the parasite life cycle were inserted into a single NYVAC genome to generate NYVAC-Pf7. Each of the seven antigens was expressed in NYVAC-Pf7-infected culture cells, and the genotypic and phenotypic stability of the recombinant virus was demonstrated. When inoculated into rhesus monkeys, NYVAC-Pf7 was safe and well tolerated. Antibodies that recognize sporozoites, liver, blood, and sexual stages of P. falciparum were elicited. Specific antibody responses against four of the P.falciparum antigens (circumsporozoite protein, sporozoite surface protein 2, merozoite surface protein 1, and 25-kDa sexual-stage antigen) were characterized. The results demonstrate that NYVAC-Pf7 is an appropriate candidate vaccine for further evaluation in human clinical trials.     

Identification, cloning, expression, and characterization of the gene for Plasmodium knowlesi surface protein containing an altered thrombospondin repeat domain

Proteins present on the surface of malaria parasites that participate in the process of invasion and adhesion to host cells are considered attractive vaccine targets. Aided by the availability of the partially completed genome sequence of the simian malaria parasite Plasmodium knowlesi, we have identified a 786-bp DNA sequence that encodes a 262-amino-acid-long protein, containing an altered version of the thrombospondin type I repeat domain (SPATR). Thrombospondin type 1 repeat domains participate in biologically diverse functions, such as cell attachment, mobility, proliferation, and extracellular protease activities. The SPATR from P. knowlesi (PkSPATR) shares 61% and 58% sequence identity with its Plasmodium falciparum and Plasmodium yoelii orthologs, respectively. By immunofluorescence analysis, we determined that PkSPATR is a multistage antigen that is expressed on the surface of P. knowlesi sporozoite and erythrocytic stage parasites. Recombinant PkSPATR produced in Escherichia coli binds to a human hepatoma cell line, HepG2, suggesting that PkSPATR is a parasite ligand that could be involved in sporozoite invasion of liver cells. Furthermore, recombinant PkSPATR reacted with pooled sera from P. knowlesi-infected rhesus monkeys, indicating that native PkSPATR is immunogenic during infection. Further efficacy evaluation studies in the P. knowlesi-rhesus monkey sporozoite challenge model will help to decide whether the SPATR molecule should be developed as a vaccine against human malarias.     

Determining liver stage parasite burden by real time quantitative PCR as a method for evaluating pre-erythrocytic malaria vaccine efficacy.

The detection and quantitation of blood stage parasitaemia is typically used as a surrogate endpoint for estimating the efficacy of vaccines targeted against the hepatic stage, as well as the erythrocytic stage, of the parasite. However, this does not provide an adequate means of evaluating the efficacy of vaccines, which may be only partially effective at the liver-stage. This is a particular concern for effective evaluation of immune enhancement strategies for candidate pre-erythrocytic stage vaccines. Here, we have developed and validated a method for detecting and quantitating liver stage parasites, using the TaqMan fluorescent real-time quantitative PCR system (PE Applied Biosystems). This method uses TaqMan primers designed to the Plasmodium yoelii 18S rRNA gene and rodent GAPDH to amplify products from infected mouse liver cDNA. The technique is highly reproducible as demonstrated with plasmid controls and capable of efficiently quantitating liver-stage parasite burden following a range of sporozoite challenge doses in strains of mice, which differ in their susceptibility to sporozoite infection. We have further demonstrated the capacity of this technique to evaluate the efficacy of a range of pre-erythrocytic stage vaccines. Our data establish this quantitative real-time PCR assay to be a fast and reproducible way of accurately assessing liver stage parasite burden and vaccine efficacy in rodent malaria models.     

Cloning of Plasmodium yoelii genes expressing three different sporozoite-specific antigens

Genomic DNA isolated from Plasmodium yoelii (strain #17XNL) was prepared by partial digestion and cloned in Escherichia coli TB-1 with pUC18 plasmid. Antigen-producing recombinants were detected by a battery of monoclonal antibodies against antigens of the sporozoite stages. Four clones producing stage-specific sporozoite antigens were identified. One produced P. yoelii circumsporozoite protein, and three produced other P. yoelii sporozoite antigens.     

The Plasmodium falciparum knob-associated PfEMP3 antigen is also expressed at pre-erythrocytic stages and induces antibodies which inhibit sporozoite invasion

The expression of the pfemp3 gene and the corresponding PfEMP3 knob-associated protein in the pre-erythrocytic stages of Plasmodium falciparum was demonstrated by RT-PCR, Western blots, IFAT and IEM. The antigen was found on the surface of the sporozoite and in the cytoplasm of mature hepatic stage parasites. Immunological cross-reactivity was observed with sporozoites from the rodent malaria parasites Plasmodium yoelii yoelii and Plasmodium berghei and was exploited to assess a potential role of this protein at the pre-erythrocytic stages. Specific antibodies from immune individuals were found to inhibit P. yoelii yoelii and P. berghei sporozoite invasion of primary hepatocyte cultures. PfEMP3 should now be added to the small list of proteins expressed at the pre-erythrocytic stages of P. falciparum, and its vaccine potential now deserves to be investigated.     

Boosting of DNA vaccine-elicited gamma interferon responses in humans by exposure to malaria parasites

A mixture of DNA plasmids expressing five Plasmodium falciparum pre-erythrocyte-stage antigens was administered with or without a DNA plasmid encoding human granulocyte-macrophage colony-stimulating factor (hGM-CSF) as an immune enhancer. After DNA immunization, antigen-specific gamma interferon (IFN-gamma) responses were detected by ELISPOT in 15/31 volunteers to multiple class I- and/or class II-restricted T-cell epitopes derived from all five antigens. Responses to multiple epitopes (相似文献   

Enhanced protection against malaria by a chimeric merozoite surface protein vaccine

The 42-kDa processed fragment of Plasmodium falciparum merozoite surface protein 1 (MSP-1(42)) is a prime candidate for a blood-stage malaria vaccine. Merozoite surface protein 8 contains two C-terminal epidermal growth factor (EGF)-like domains that may function similarly to those of MSP-1(42). Immunization with either MSP-1 or MSP-8 induces protection that is mediated primarily by antibodies against conformation-dependent epitopes. In a series of comparative immunogenicity and efficacy studies using the Plasmodium yoelii rodent model, we tested the ability of recombinant P. yoelii MSP-8 (rPyMSP-8) to complement rPyMSP-1-based vaccines. Unlike MSP-1, PyMSP-8-dependent protection required immunization with the full-length protein and was not induced with recombinant antigens that contained only the C-terminal EGF-like domains. Unlike PyMSP-8, the immunogenicity of the PyMSP-1 EGF-like domains was low when present as part of the rPyMSP-1(42) antigen. Immunization with a mixture of rPyMSP-1(42) and rPyMSP-8 further inhibited the antibody response to protective epitopes of rPyMSP-1(42) and did not improve vaccine efficacy. To improve PyMSP-1 immunogenicity, we produced a chimeric antigen containing the EGF-like domains of PyMSP-1 fused to the N terminus of PyMSP-8. Immunization with the chimeric rPyMSP-1/8 antigen induced high and comparable antibody responses against the EGF-like domains of both PyMSP-1 and PyMSP-8. This enhanced MSP-1-specific antibody response and the concurrent targeting of MSP-1 and MSP-8 resulted in improved, nearly complete protection against lethal P. yoelii 17XL malaria. Unexpectedly, immunization with rPyMSP-1/8 failed to protect against challenge infection with reticulocyte-restricted P. yoelii 17X parasites. Overall, these data establish an effective strategy to improve the efficacy of P. falciparum MSP-based vaccines.     

Immunogenicity and protective efficacy of a Plasmodium yoelii Hsp60 DNA vaccine in BALB/c mice

The gene encoding the 60-kDa heat shock protein of Plasmodium yoelii (PyHsp60) was cloned into the VR1012 and VR1020 mammalian expression vectors. Groups of 10 BALB/c mice were immunized intramuscularly at 0, 3, and 9 weeks with 100 microg of PyHsp60 DNA vaccine alone or in combination with 30 microg of pmurGMCSF. Sera from immunized mice but not from vector control groups recognized P. yoelii sporozoites, liver stages, and infected erythrocytes in an indirect fluorescent antibody test. Two weeks after the last immunization, mice were challenged with 50 P. yoelii sporozoites. In one experiment the vaccine pPyHsp60-VR1012 used in combination with pmurGMCSF gave 40% protection (Fisher's exact test; P = 0.03, vaccinated versus control groups). In a second experiment this vaccine did not protect any of the immunized mice but induced a delay in the onset of parasitemia. In neither experiment was there any evidence of a protective effect against the asexual erythrocytic stage of the life cycle. In a third experiment mice were primed with PyHsp60 DNA, were boosted 2 weeks later with 2 x 10(3) irradiated P. yoelii sporozoites, and were challenged several weeks later. The presence of PyHsp60 in the immunization regimen did not lead to reduced blood-stage infection or development of parasites in hepatocytes. PyHsp60 DNA vaccines were immunogenic in BALB/c mice but did not consistently, completely protect against sporozoite challenge. The observation that in some of the PyHsp60 DNA vaccine-immunized mice there was protection against infection or a delay in the onset of parasitemia after sporozoite challenge deserves further evaluation.     

Genetic vaccination against malaria infection by intradermal and epidermal injections of a plasmid containing the gene encoding the Plasmodium berghei circumsporozoite protein

The circumsporozoite protein (CSP) from the surface of sporozoite stage Plasmodium sp. malaria parasites is among the most important of the malaria vaccine candidates. Gene gun injection of genetic vaccines encoding Plasmodium berghei CSP induces a significant protective effect against sporozoite challenge; however, intramuscular injection does not. In the present study we compared the immune responses and protective effects induced by P. berghei CSP genetic vaccines delivered intradermally with a needle or epidermally with a gene gun. Mice were immunized three times at 4-week intervals and challenged by a single infectious mosquito bite. Although 50 times more DNA was administered by needle than by gene gun, the latter method induced significantly greater protection against infection. Intradermal injection of the CSP genetic vaccine induced a strong Th1-type immune response characterized by a dominant CSP-specific immunoglobulin G2a (IgG2a) humoral response and high levels of gamma interferon produced by splenic T cells. Gene gun injection induced a predominantly Th2-type immune response characterized by a high IgG1/IgG2a ratio and significant IgE production. Neither method generated measurable cytotoxic T lymphocyte activity. The results indicate that a gene gun-mediated CS-specific Th2-type response may be best for protecting against malarial sporozoite infection when the route of parasite entry is via mosquito bite.     

Targeting antigen to MHC Class I and Class II antigen presentation pathways for malaria DNA vaccines

An effective malaria vaccine which protects against all stages of Plasmodium infection may need to elicit robust CD8(+) and CD4(+) T cell and antibody responses. To achieve this, we have investigated strategies designed to improve the immunogenicity of DNA vaccines encoding the Plasmodium yoelii pre-erythrocytic stage antigens PyCSP and PyHEP17, by targeting the encoded proteins to the MHC Classes I and II processing and presentation pathways. For enhancement of CD8(+) T cell responses, we targeted the antigens for degradation by the ubiquitin (Ub)/proteosome pathway following the N-terminal rule. We constructed plasmids containing PyCSP or PyHEP17 genes fused to the Ub gene: plasmids where the N-terminal antigen residues were mutated from the stabilizing amino acid methionine to destabilizing arginine, plasmids where the C-terminal residues of Ub were mutated from glycine to alanine, and plasmids in which the potential hydrophobic leader sequences of the antigens were deleted. For enhancement of CD4(+) T cell and antibody responses, we targeted the antigens for degradation by the endosomal/lysosomal pathway by linking the antigen to the lysosome-associated membrane protein (LAMP). We found that immunization with DNA vaccine encoding PyHEP17 fused to Ub and bearing arginine induced higher IFN-gamma, cytotoxic and proliferative T cell responses than unmodified vaccines. However, no effect was seen for PyCSP using the same targeting strategies. Regarding Class II antigen targeting, fusion to LAMP did not enhance antibody responses to either PyHEP17 or PyCSP, and resulted in a marginal increase in lymphoproliferative CD4(+) T cell responses. Our data highlight the antigen dependence of immune enhancement strategies that target antigen to the MHC Class I and II pathways for vaccine development.     

Dendritic Cells Can Initiate Protective Immune Responses against Malaria

An understanding of the antigen presentation mechanisms that mediate induction of protective immune responses against malaria is essential for the development of successful immunization approaches. Here we show that dendritic cells presenting Plasmodium yoelii sporozoite antigens are able to activate specific CD4(+) and CD8(+) T cells and initiate protective immune responses against malaria in mice.     

DNA immunization with the cysteine-rich interdomain region 1 of the Plasmodium falciparum variant antigen elicits limited cross-reactive antibody responses

The variant surface antigens of Plasmodium falciparum are an important component of naturally acquired immunity and an important vaccine target. However, these proteins appear to elicit primarily variant-specific antibodies. We tested if naked DNA immunization can elicit more cross-reactive antibody responses and allow simultaneous immunization with several variant constructs. Mice immunized with plasmid DNA expressing variant cysteine-rich interdomain region 1 (CIDR1) domains of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) developed antibodies that were reactive to the corresponding PfEMP1s as measured by an enzyme-linked immunosorbent assay, flow cytometry, and agglutination of parasitized erythrocytes (PEs). We observed some cross-reactive immune responses; for example, sera from mice immunized with one domain agglutinated PEs of various lines and recognized heterologous domains expressed on the surface of Chinese hamster ovary (CHO) cells. We found no significant antigenic competition when animals were immunized with a mixture of plasmids or immunized sequentially with individual constructs. Moreover, mixed or sequential immunizations resulted in greater cross-reactive agglutination responses than immunization with a single domain. Recombinant protein (Sc y179) immunization after priming with DNA (prime-boost regimen) increased antibody titers to the homologous domain substantially but seemed to diminish the cross-reactive responses somewhat. The titer of agglutinating antibodies was previously shown to correlate with protection. Surprisingly, the agglutination titers of sera from DNA immunization were high, similar to those of pooled human hyperimmune sera. These sera also appeared to give limited low-titer variant transcending agglutination. Thus, DNA immunization appears to be a very useful tool for developing variant antigen vaccines.     

Plasmodium vivax malaria vaccine development.

Plasmodium vivax represents the most widespread malaria parasite worldwide. Although it does not result in as high a mortality rate as P. falciparum, it inflicts debilitating morbidity and consequent economic impact in endemic communities. In addition, the relapsing behavior of this malaria parasite and the recent resistance to anti-malarials contribute to making its control more difficult. Although the biology of P. vivax is different from that of P. falciparum and the human immune response to this parasite species has been rather poorly studied, significant progress is being made to develop a P. vivax-specific vaccine based on the information and experience gained in the search for a P. falciparum vaccine. We have devoted great effort to antigenically characterize the P. vivax CS protein and to test its immunogenicity using the Aotus monkey model. Together with other groups we are also assessing the immunogenicity and protective efficacy of the asexual blood stage vaccine candidates MSP-1 and DBP in the monkey model, as well as the immunogenicity of Pvs25 and Pvs28 ookinete surface proteins. The transmission-blocking efficacy of the responses induced by these latter antigens is being assessed using Anopheles albimanus mosquitoes. The current status of these vaccine candidates and other antigens currently being studied is described.     

Serological cross-reaction between high molecular weight proteins synthesized in blood schizonts of Plasmodium yoelii, Plasmodium chabaudi and Plasmodium falciparum

A 230 000 molecular weight (MW) Plasmodium yoelii protein, a 250 000 MW P. chabaudi protein and a 195 000 MW P. falciparum protein, identified using monoclonal antibodies, have similar characteristics, and have been implicated as protective antigens. In this study the serological relationship between these proteins was investigated by Western transfer analysis. The monoclonal antibodies specific for each of the high molecular weight proteins did not cross-react with antigens of the other two parasites, but a polyvalent mouse serum raised against the purified 230 000 MW P. yoelii protein cross-reacted with the high molecular weight proteins of P. chabaudi and P. falciparum and also with the fragments derived from these proteins. This result indicates that these proteins belong to the same class of malaria parasite antigen.     

Tricomponent immunopotentiating system as a novel molecular design strategy for malaria vaccine development

The creation of subunit vaccines to prevent malaria infection has been hampered by the intrinsically weak immunogenicity of the recombinant antigens. We have developed a novel strategy to increase immune responses by creating genetic fusion proteins to target specific antigen-presenting cells (APCs). The fusion complex was composed of three physically linked molecular entities: (i) a vaccine antigen, (ii) a multimeric α-helical coiled-coil core, and (iii) an APC-targeting ligand linked to the core via a flexible linker. The vaccine efficacy of the tricomponent complex was evaluated using an ookinete surface protein of Plasmodium vivax, Pvs25, and merozoite surface protein-1 of Plasmodium yoelii. Immunization of mice with the tricomponent complex induced a robust antibody response and conferred substantial levels of P. vivax transmission blockade as evaluated by a membrane feed assay, as well as protection from lethal P. yoelii infection. The observed effect was strongly dependent on the presence of all three components physically integrated as a fusion complex. This system, designated the tricomponent immunopotentiating system (TIPS), onto which any recombinant protein antigens or nonproteinaceous substances could be loaded, may be a promising strategy for devising subunit vaccines or adjuvants against various infectious diseases, including malaria.     

Safety, immunogenicity, and efficacy of prime-boost immunization with recombinant poxvirus FP9 and modified vaccinia virus Ankara encoding the full-length Plasmodium falciparum circumsporozoite protein

Heterologous prime-boost immunization with DNA and various recombinant poxviruses encoding malaria antigens is capable of inducing strong cell-mediated immune responses and partial protection in human sporozoite challenges. Here we report a series of trials assessing recombinant fowlpox virus and modified vaccinia virus Ankara encoding the Plasmodium falciparum circumsporozoite protein in various prime-boost combinations, doses, and application routes. For the first time, these vaccines were administered intramuscularly and at doses of up to 5 x 10(8) PFU. Vaccines containing this antigen proved safe and induced modest immune responses but showed no evidence of efficacy in a sporozoite challenge.     

Protective immune responses to the 42-kilodalton (kDa) region of Plasmodium yoelii merozoite surface protein 1 are induced by the C-terminal 19-kDa region but not by the adjacent 33-kDa region.

Vaccination of mice with the 42-kDa region of Plasmodium yoelii merozoite surface protein 1 (MSP1(42)) or its 19-kDa C-terminal processing product (MSP1(19)) can elicit protective antibody responses in mice. To investigate if the 33-kDa N-terminal fragment (MSP1(33)) of MSP1(42) also induces protection, the gene segment encoding MSP1(33) was expressed as a glutathione S-transferase (GST) fusion protein. C57BL/6 and BALB/c mice were immunized with GST-MSP1(33) and subsequently challenged with the lethal P. yoelii YM blood stage parasite. GST-MSP1(33) failed to induce protection, and all mice developed patent parasitemia at a level similar to that in naive or control (GST-immunized) mice; mice immunized with GST-MSP1(19) were protected, as has been shown previously. Specific prechallenge immunoglobulin G (IgG) antibody responses to MSP1 were analyzed by enzyme-linked immunosorbent assay and immunofluorescence. Despite being unprotected, several mice immunized with MSP1(33) had antibody titers (of all IgG subclasses) that were comparable to or higher than those in mice that were protected following immunization with MSP1(19). The finding that P. yoelii MSP1(33) elicits strong but nonprotective antibody responses may have implications for the design of vaccines for humans based on Plasmodium falciparum or Plasmodium vivax MSP1(42).     

A multiple antigen peptide from the repetitive sequence of the Plasmodium malariae circumsporozoite protein induces a specific antibody response in mice of various H-2 haplotypes

The major repetitive epitopes of the surface circumsporozoite (CS) protein of malaria sporozoites represent candidates for the development of subunit vaccines against malaria. However, previous experimental work has shown that repetitive peptides from the CS proteins of Plasmodium falciparum, P. vivax, P. yoelii and P. berghei are immunogenic only in mice with the H-2b or H-2k haplotype. This led to the conclusion that strong T helper epitopes from the non-repetitive CS sequences were required in the design of sporozoite vaccines. In the present study, we investigated the immunogenicity in mice of a octa-branched multiple antigen peptide (MAP) containing repeats of the CS protein of the human malaria parasite, P. malariae, [MAP8(NAAG)6], and found that mice with an H-2b, H-2d, H-2k, H-2f, H-2q, and H-2s haplotype produced anti-peptide antibodies after immunization and that only H-2r mice were nonresponsive. This antibody response, not induced in athymic H-2b nu/nu mice, was directed against the (NAAG) sequence, but not against the lysine core of the MAP construct. Finally, when covalently linked to a synthetic polymer of the repetitive (NANP) sequence of the P. falciparum CS protein, [MAP8(NAAG)6] behaved as a carrier molecule for the production of anti-(NANP)n antibodies in H-2d and H-2k mice, genetically nonresponder to the (NANP)n sequence. Should this wide immunogenicity of the P. malariae CS (NAAG) repetitive sequence also apply to humans, it might be considered for the design of multivalent subunit malaria vaccines.