Safety and enhanced immunogenicity of a hepatitis B core particle Plasmodium falciparum malaria vaccine formulated in adjuvant Montanide ISA 720 in a phase I trial

Highly purified subunit vaccines require potent adjuvants in order to elicit optimal immune responses. In a previous phase I trial, an alum formulation of ICC-1132, a malaria vaccine candidate comprising hepatitis B core (HBc) virus-like particle containing Plasmodium falciparum circumsporozoite (CS) protein epitopes, was shown to elicit Plasmodium falciparum-specific antibody and cellular responses. The present study was designed as a single-blind, escalating-dose phase I trial to evaluate the safety and immunogenicity of single intramuscular doses of ICC-1132 formulated in the more potent water-in-oil adjuvant Montanide ISA 720 (ICC-1132/ISA 720). The vaccine was safe and well tolerated, with transient injection site pain as the most frequent complaint. All vaccinees that received either 20 mug or 50 mug of ICC-1132/ISA 720 developed antiimmunogen and anti-HBc antibodies. The majority of volunteers in these two groups developed sporozoite-specific antibodies, predominantly of opsonizing immunoglobulin G subtypes. Peak titers and persistence of parasite-specific antibody following a single injection of the ISA 720 formulated vaccine were comparable to those obtained following two to three immunizations with alum-adsorbed ICC-1132. Peripheral blood mononuclear cells of ICC-1132/ISA 720 vaccinees proliferated and released cytokines (interleukin 2 and gamma interferon) when stimulated with recombinant P. falciparum CS protein, and CS-specific CD4(+) T-cell lines were established from volunteers with high levels of antibodies to the repeat region. The promising results obtained with a single dose of ICC-1132 formulated in Montanide ISA 720 encourage further clinical development of this malaria vaccine candidate.     

A modified hepatitis B virus core particle containing multiple epitopes of the Plasmodium falciparum circumsporozoite protein provides a highly immunogenic malaria vaccine in preclinical analyses in rodent and primate hosts

Despite extensive public health efforts, there are presently 200 to 400 million malaria infections and 1 to 2 million deaths each year due to the Plasmodium parasite. A prime target for malaria vaccine development is the circumsporozoite (CS) protein, which is expressed on the extracellular sporozoite and the intracellular hepatic stages of the parasite. Previous studies in rodent malaria models have shown that CS repeat B-cell epitopes expressed in a recombinant hepatitis B virus core (HBc) protein can elicit protective immunity. To design a vaccine for human use, a series of recombinant HBc proteins containing epitopes of Plasmodium falciparum CS protein were assayed for immunogenicity in mice [A. Birkett, B. Thornton, D. Milich, G. A. Oliveira, A. Siddique, R. Nussenzweig, J. M. Calvo-Calle, and E. H. Nardin, abstract from the 50th Annual Meeting of the American Society of Tropical Medicine and Hygiene 2001, Am. J. Trop. Med. Hyg. 65(Suppl. 3):258, 2001; D. R. Milich, J. Hughes, J. Jones, M. Sallberg, and T. R. Phillips, Vaccine 20:771-788, 2001]. The present paper summarizes preclinical analyses of the optimal P. falciparum HBc vaccine candidate, termed ICC-1132, which contains T- and B-cell epitopes from the repeat region and a universal T-cell epitope from the C terminus of the CS protein. The vaccine was highly immunogenic in mice and in Macaca fascicularis (cynomolgus) monkeys. When formulated in adjuvants suitable for human use, the vaccine elicited antisporozoite antibody titers that were logs higher than those obtained in previous studies. Human malaria-specific CD4(+)-T-cell clones and T cells of ICC-1132-immunized mice specifically recognized malaria T-cell epitopes contained in the vaccine. In addition to inducing strong malaria-specific immune responses in na?ve hosts, ICC-1132 elicited potent anamnestic antibody responses in mice primed with P. falciparum sporozoites, suggesting potential efficacy in enhancing the sporozoite-primed immune responses of individuals living in areas where malaria is endemic.     

Enhanced immunogenicity of modified hepatitis B virus core particle fused with multiepitopes of foot-and-mouth disease virus

Hepatitis B virus core (HBc) particles, self-assemble into capsid particles and are extremely immunogenic, hold promise as an immune-enhancing vaccine carrier for heterologous antigens. However, formation of virus-like particles (VLP) can be restricted by size and structure of heterlogous antigens. In the study, we investigated formation of VLP by modified HBc fused with specified foot-and-mouth disease virus (FMDV) multiepitopes and evaluated their immune effects. Firstly, three HBc display vectors (pHBc1, pHBc2 and pHBc3) were constructed by deletions of different lengths within the HBc c/e1 region: 75-78 amino acid (aa), 75-80 aa and 75-82 aa respectively. Secondly, we inserted different compositions of FMDV multiepitopes, BT [VP1(141-160)-VP4(21-40)] and BTB [VP1(141-160)-VP4(21-40)-VP1(141-160)], into modified regions. As a result, only plasmid pHBc3-BTB of six recombinant vectors was expressed as soluble protein, which resulted in the formation of complete VLP confirmed by electron microscopy. Recombinant VLP could be taken up by cells and presented in vitro and in vivo. Furthermore, the modified VLP displayed a significantly stronger immunogenicity than other five recombinant proteins and GST-BTB with a higher titer of peptide-specific and virus-specific antibody, elevated IFN-gamma and interleukin-4 production, especially enhanced lymphocyte proliferation. The results encourage further work towards the development of FMDV vaccines using hepatitis B virus core particles fused with FMDV epitopes.     

Virus-like particles: a new family of delivery systems

The development of antiviral vaccines has almost exclusively been based on live attenuated vaccines up until now. However, the efficacy of HBsAg particles as an antiHBV vaccine has clearly demonstrated that protective antiviral immunity can be achieved by other strategies. Virus-like particles formed by structural proteins were proven to be highly immunogenic and capable of inducing protective immunity against various viral infections in preclinical studies. Clinical trials using virus-like particles confirmed their safety and immunogenicity. Moreover, chimeric virus-like particles carrying foreign peptidic sequences were shown to elicit potent B- and T-cell responses. Virus-like particles formed by a fusion protein between the HBsAg and the circumsporozo?te surface protein are safe and immunogenic in volunteers and induce a partial protection against natural Plasmodium falciparum infection.     

Safety, immunogenicity, and efficacy of a Plasmodium falciparum vaccine comprising a circumsporozoite protein repeat region peptide conjugated to Pseudomonas aeruginosa toxin A.

Twenty-one malaria-naive volunteers were immunized with a vaccine consisting of a 22-kDa recombinant peptide (R32LR), derived from the repeat region of Plasmodium falciparum circumsporozoite (CS) protein, covalently coupled to detoxified Pseudomonas aeruginosa toxin A. Nineteen volunteers received a second dose of vaccine at 8 weeks, and eighteen received a third dose at 8 to 12 months. The vaccine was well tolerated, with only one volunteer developing local discomfort and induration at the site of injection which limited function for 48 h. The geometric mean anti-CS immunoglobulin G antibody concentration 2 weeks after the second dose of vaccine was 10.6 micrograms/ml (standard deviation = 3.0 micrograms/ml). Eleven volunteers (52%) developed anti-CS antibody levels of greater than 9.8 micrograms/ml, the level measured in the one volunteer protected against P. falciparum challenge after immunization with the alum-adjuvanted recombinant protein R32tet32 in a prior study. Three separate experimental challenges were conducted with 10 volunteers 2 to 4 weeks after the third dose of vaccine. The four best responders, on the basis of antibody levels (6 to 26 micrograms/ml), were challenged with two infected-mosquito bites, but only one of four immunized volunteers and one of three malaria-naive controls became parasitemic. In a second challenge study using five infected-mosquito bites as the challenge dose, three of three malaria-naive control volunteers and two of three immunized volunteers developed malaria. The third vaccine was apparently completely protected. In the third and last challenge, three of three controls and five of five vaccinees became infected. Sera obtained on the days of challenge inhibited sporozoite invasion of hepatocytes variably in vitro (range, 45 to 90% inhibition), but the degree of inhibition did not correlate with protection. Although antibody against the CS repeat region may protect some individuals against experimental challenge, this protection cannot be predicted from antibody levels by current in vitro assays. The functionality and fine specificity of anti-CS antibody are probably critical determinants.     

Broadly distributed T cell reactivity, with no immunodominant loci, to the pre-erythrocytic antigen thrombospondin-related adhesive protein of Plasmodium falciparum in West Africans.

Protective immunity to malaria has been achieved in human volunteers utilizing the pre-erythrocytic Plasmodium falciparum antigen, the circumsporozoite protein (CS). However, T cell reactivity to CS is focused on several highly polymorphic T cell epitope regions, potentially limiting the efficacy of any vaccine to specific malaria strains. Another important pre-erythrocytic malaria antigen, the thrombospondin-related adhesive protein (TRAP), can induce protection in animal models of malaria, but knowledge of human T cell responses is limited to the identification of CD8 T cell epitopes, with no CD4 epitopes identified to date. This comprehensive study assessed reactivity to overlapping peptides spanning almost the whole of P. falciparum TRAP (PfTRAP), as well as peptides selected on the basis of HLA class II-binding motifs. A total of 50 naturally exposed Gambian adults were assessed to define 26 T cell epitopes in PfTRAP capable of inducing rapid IFN-gamma or IL-4 production, as assessed by enzyme-linked immunospot assays. In contrast to the CS protein, this reactivity was broadly distributed along the length of TRAP. Moreover, of the 26 epitopes identified, 10 were found to be conserved in West Africa.     

Major population differences in T cell response to a malaria sporozoite vaccine candidate.

Using a complete series of overlapping peptides, we have identified the T cell epitopes of a malaria vaccine candidate, the circumsporozoite (CS) protein, that are recognized by sporozoite-exposed residents of a non-endemic country. This protein and subunits from it are being considered as malaria sporozoite vaccine candidates, as CS-specific antibodies and cytotoxic T lymphocytes have been shown to have a role in protection. The rationale for developing an antibody-based vaccine is that in Plasmodium falciparum the immunodominant B cell epitope of the protein, (Asn-Ala-Asn-Pro)n [(NANP)n], is invariant. However, the ideal vaccine must contain CS protein-derived T cell antigenic epitopes to allow natural boosting of the antibody response following sporozoite exposure. Here, we show that major differences occur between the CS-specific T cell responses of non-endemic Caucasians and an endemic African population. HLA differences between the populations are, in part, responsible. Subunit malaria vaccines for one population may be ineffective in a different population.     

A linear peptide containing minimal T- and B-cell epitopes of Plasmodium falciparum circumsporozoite protein elicits protection against transgenic sporozoite challenge

An effective malaria vaccine is needed to address the public health tragedy resulting from the high levels of morbidity and mortality caused by Plasmodium parasites. The first protective immune mechanism identified in the irradiated sporozoite vaccine, the "gold standard" for malaria preerythrocytic vaccines, was sporozoite-neutralizing antibody specific for the repeat region of the surface circumsporozoite (CS) protein. Previous phase I studies demonstrated that a branched peptide containing minimal T- and B-cell epitopes of Plasmodium falciparum CS protein elicited antirepeat antibody and CD4(+)-T-cell responses comparable to those observed in volunteers immunized with irradiated P. falciparum sporozoites. The current study compares the immunogenicity of linear versus tetrabranched peptides containing the same minimal T- and B-cell epitopes, T1BT*, comprised of a CS-derived universal Th epitope (T*) synthesized in tandem with the T1 and B repeats of P. falciparum CS protein. A simple 48-mer linear synthetic peptide was found to elicit antisporozoite antibody and gamma interferon-secreting T-cell responses comparable to the more complex tetrabranched peptides in inbred strains of mice. The linear peptide was also immunogenic in outbred nonhuman primates (Aotus nancymaae), eliciting antibody titers equivalent to those induced by tetrabranched peptides. Importantly, the 48-mer linear peptide administered in adjuvants suitable for human use elicited antibody-mediated protection against challenge with rodent malaria transgenic sporozoites expressing P. falciparum CS repeats. These findings support further evaluation of linear peptides as economical, safe, and readily produced malaria vaccines for the one-third of the world's population at risk of malaria infection.     

The Current Status of Malaria Vaccines

A vaccine against Plasmodium falciparum malaria is needed now more than ever due the resurgence of the parasite and the increase in drug resistance. However, success in developing an effective malaria vaccine has been elusive. Among pre-erythrocytic antigens, the major antigen coating the surface of the sporozoite, the circumsporozoite protein (CS), has been, and continues to be, the major target for vaccine development. Despite initial limited success with CS-based vaccines, the use of new adjuvant formulations has led to the development of a promising candidate (the RTS,S vaccine) which has shown significant efficacy in a preliminary trial. In addition to CS, many other malaria antigens have been identified that play an important role in the parasite life cycle which are being considered for, or are currently undergoing, clinical trials. Among the blood stage antigens, the merozoite surface protein 1 (MSP-1) is the most promising vaccine candidate. New approaches to immunisation against malaria being considered include the use of multistage, multicomponent vaccines in attenuated viral vectors (NYVAC-Pf7), or in a combination DNA vaccine. While there is reason to be optimistic about the prospects for an effective vaccine, many challenges lie ahead that still have to be overcome. Among these are the antigenic polymorphism exhibited by wild parasite strains and the genetic restriction of immune responses.     

Malaria vaccine: a bright prospect for elimination of malaria

Malaria remains one of the few diseases those continue to scourge human civilization despite the significant advances in disease control strategies over the last century. Malaria is responsible for more than 500 million cases and 1-3 million deaths annually. Approximately 85% of these deaths are among children, mostly in Africa, primarily due to P. falciparum. Whole cell vaccines, irradiated sporozoites and genetically attenuated sporozoites have demonstrated long lasting, sterile protection against plasmodium infection in animal and experimental clinical studies. Atypical membrane protein 1 and merozoite surface protein 1 are the two most extensively studied asexual blood stage vaccine candidates. The most promising candidate vaccine under development is RTS, S combined with AS01 adjuvant. Initial results from phase III trials of this candidate vaccine show 50% reduction of malaria in 5-17 mo aged children during the 12 mo after vaccination. WHO anticipates that the RTS,S/AS01 vaccine will be recommended for the 6-14 week age group for co-administration together with other vaccines as part of routine immunization programs in malaria endemic countries. Malaria vaccine could play an important role in elimination and eventual eradication of malaria.     

Comparison of numerous delivery systems for the induction of cytotoxic T lymphocytes by immunization

A variety of vaccine delivery systems including peptides with various adjuvants, recombinant particles, live recombinant viruses and bacteria and plasmid DNA were tested for their ability to induce CD8⁺ cytotoxic T lymphocytes (CTL) against a well-defined epitope (amino acids 252–260) from the circumsporozoite (CS) protein of Plasmodium berghei. We compared routes of immunization that would be applicable for the administration of a malaria vaccine in humans. The majority of these vaccines did not induce high CTL responses in the spleens of immunized mice. However, both a yeast-derived Ty virus-like particle expressing the optimal nine-amino acid epitope SYIPSAEKI from the CS protein (CSP-VLP) and a lipid-tailed peptide of this same sequence induced high levels of the major histocompatibility complex (MHC) class I-restricted CTL with one and three subcutaneous immunizations, respectively. Moreover, these CTL were able to recognize naturally processed antigen expressed by a recombinant vaccinia virus. The levels of CTL induced by CSP-VLP could be augmented by co-immunization with certain cytokines. Target cells pulsed with CSP-VLP were recognized and lysed, showing that the particles were effectively processed and presented through MHC class I presentation pathway. The levels of CTL induced using CSP-VLP and lipopeptides are comparable to those observed after immunization with multiple doses of irradiated sporozoites.     

Phase 1 clinical trial of apical membrane antigen 1: an asexual blood-stage vaccine for Plasmodium falciparum malaria

Apical membrane antigen 1 (AMA1), a polymorphic merozoite surface protein, is a leading blood-stage malaria vaccine candidate. A phase 1 trial was conducted with 30 malaria-naive volunteers to assess the safety and immunogenicity of the AMA1-C1 malaria vaccine. AMA1-C1 contains an equal mixture of recombinant proteins based on sequences from the FVO and 3D7 clones of Plasmodium falciparum. The proteins were expressed in Pichia pastoris and adsorbed on Alhydrogel. Ten volunteers in each of three dose groups (5 mug, 20 mug, and 80 mug) were vaccinated in an open-label study at 0, 28, and 180 days. The vaccine was well tolerated, with pain at the injection site being the most commonly observed reaction. Anti-AMA1 immunoglobulin G (IgG) was detected by enzyme-linked immunosorbent assay (ELISA) in 15/28 (54%) volunteers after the second immunization and in 23/25 (92%) after the third immunization, with equal reactivity to both AMA1-FVO and AMA1-3D7 vaccine components. A significant dose-response relationship between antigen dose and antibody response by ELISA was observed, and the antibodies were predominantly of the IgG1 isotype. Confocal microscopic evaluation of sera from vaccinated volunteers demonstrated reactivity with P. falciparum schizonts in a pattern similar to native parasite AMA1. Antigen-specific in vitro inhibition of both FVO and 3D7 parasites was achieved with IgG purified from sera of vaccinees, demonstrating biological activity of the antibodies. To our knowledge, this is the first AMA1 vaccine candidate to elicit functional immune responses in malaria-naive humans, and our results support the further development of this vaccine.     

人乳头状瘤病毒6型类病毒颗粒的制备及其中和抗体的检测

目的 利用大肠杆菌表达系统制备人乳头状瘤病毒6型(human papillomavirus type 6,HPV-6)类病毒颗粒(virus-like particles,VLP)并研究其免疫原性.方法 在大肠杆菌ER2566中表达HPV-6 L1蛋白,并以硫酸铵沉淀、离子交换色谱、疏水相互作用色谱等手段对其进行纯化.纯化后的HPV-6 L1经体外组装形成VLP后以动态光散射,透射电镜对其形态进行检测,并以假病毒中和实验评价HPV-6 L1 VLP在实验动物体内所诱导的抗HPV-6/11中和抗体水平.结果 HPV-6 L1蛋白在大肠杆菌中以可溶形式表达,经过纯化后的HPV-6 L1蛋白可以在体外组装为半径25 nm左右的VLP.该VLP可以在山羊及兔体内诱导高滴度的HPV-6/11中和抗体.结论 大肠杆菌表达系统可以简便高效制备具有免疫原性的HPV-6 VLP,可以用于HPV-6疫苗的研究.     

A peptide-based Plasmodium falciparum circumsporozoite assay to test for serum antibody responses to pre-erythrocyte malaria vaccines

Various pre-erythrocyte malaria vaccines are currently in clinical development, and among these is the adenovirus serotype 35-based circumsporozoite (CS) vaccine produced on PER.C6 cells. Although the immunological correlate of protection against malaria remains to be established, the CS antibody titer is a good marker for evaluation of candidate vaccines. Here we describe the validation of an anti-Plasmodium falciparum circumsporozoite antibody enzyme-linked immunosorbent assay (ELISA) based on the binding of antibodies to a peptide antigen mimicking the CS repeat region. The interassay variability was determined to be below a coefficient of variation (CV) of 15%, and sensitivity was sufficient to detect low antibody titers in subjects from endemic regions. Antibody titers were in agreement with total antibody responses to the whole CS protein. Due to its simplicity and high performance, the ELISA is an easy and rapid method for assessment of pre-erythrocyte malaria vaccines based on CS.     

High frequency of malaria-specific T cells in non-exposed humans.

A major goal of current candidate malaria vaccines is to stimulate the expansion of clones of malaria-specific lymphocytes. We have examined the in vitro T cell responses of a group of malaria exposed and non-exposed adult Caucasian donors to recombinant circumsporozoite (CS) proteins, one of which is undergoing clinical trials, to blood-stage parasites, and to synthetic peptides copying the CS protein and defined blood-stage proteins. In nearly all individuals tested, CD4 T cell proliferation or lymphokine production occurred in response to whole parasite or CS protein stimulation, and T cells from many individuals responded to synthetic peptides. T cell responses were major histocompatibility complex-restricted, and stimulation of T cells with malaria parasites or CS protein did not appear to expand a population of T cell receptor gamma/delta cells. Malaria-specific responses were independent of prior malaria exposure, and in some cases exceeded the magnitude of response to tetanus toxoid. Specific T cells are present in high frequency in the peripheral blood of many donors who have never been exposed to malaria. Although malaria-specific CD4 T cells play an important role in immunity, these data question whether vaccines need to stimulate such cells, and focus attention on other aspects of malaria immunity which may be more critical to a successful vaccine.     

Characterization of antibodies to sporozoites in Plasmodium falciparum malaria and correlation with protection.

The antibody response to sporozoites of Plasmodium falciparum and the role of these antibodies in protection against malaria have not been systematically investigated. An understanding of antisporozoite antibodies in natural infection is, however, important to the development of a human malaria vaccine. In a prospective study in Thailand, an antibody response to sporozoites was observed only in individuals who developed parasitemia. Antibodies were detected against an epitope in the repeat region of the circumsporozoite (CS) protein. Current candidate sporozoite vaccines are based on CS repeat antigens. The CS antibody response was of low magnitude, peaked after detection of parasitemia, and had a serum half-life of less than 1 month. CS antibody boosting occurred in only 6% of reinfected individuals. These observations suggest that antisporozoite antibody is poorly developed under natural conditions and appears not to protect against development of malaria.     

Amino acid dimorphism and parasite immune evasion: cellular immune responses to a promiscuous epitope of Plasmodium falciparum merozoite surface protein 1 displaying dimorphic amino acid polymorphism are highly constrained

Like most other surface-exposed antigens of Plasmodium falciparum, the leading malaria vaccine candidate merozoite surface protein (MSP)-1 contains a large number of dimorphic amino acid positions. This type of diversity is presumed to be associated with parasite immune evasion and represents one major obstacle to malaria subunit vaccine development. To understand the precise role of antigen dimorphism in immune evasion, we have analyzed the flexibility of CD4 T cell immune responses against a semi-conserved sequence stretch of the N-terminal block of MSP-1. While this sequence contains overlapping promiscuous T cell epitopes and is a target for growth inhibitory antibodies, three dimorphic amino acid positions may limit its suitability as component of a multi-epitope malaria vaccine. We have analyzed the CD4 T cell responses in a group of human volunteers immunized with a synthetic malaria peptide vaccine containing a single MSP-143-53 sequence variant. All human T cell lines and HLA-DR- or -DP-restricted T cell clones studied were exclusively specific for the sequence variant used for immunization. Competition peptide binding assays with affinity-purified HLA-DR molecules indicated that dimorphism does not primarily affect HLA binding. Modeling studies of the dominant restricting HLA-DRB1*0801 molecule showed that the dimorphic amino acids represent potential TCR contact residues. Lack of productive triggering of the TCR by MHC/variant peptide ligand complexes thus seems to be the characteristic feature of parasite immune evasion associated with antigen dimorphism.     

含APPβ裂解位点肽及Aβ1-15的嵌合型HBcAg颗粒抗原的制备及其免疫原性分析

目的:构建含APPβ位点裂解肽ABCSP、β-淀粉样肽氨基段15肽(Aβ1-15)及删除了c/e1表位的截短型HBcAg基因的原核表达质粒pET/c-ABCSP-Aβ15-c,并在大肠杆菌中表达,观察融合蛋白C-ABCSP-Aβ15-C形成的病毒样颗粒,检测其免疫原性,为多表位AD基因工程疫苗的研究奠定基础。方法:PCR扩增含APPβ位点裂解肽ABCSP、β-淀粉样肽氨基段15肽(Aβ1-15)的基因,连接于HBcAg的1～71的3’端,再将HBcAg的88～144位氨基酸的基因片断连接于Aβ1-15的基因的3’端,构建重组质粒pUC/c-ABCSP-Aβ15-c,将重组基因亚克隆于原核表达载体pET-28a(+)中,构建表达质粒pET/c-ABCSP-Aβ15-c,IPTG诱导表达。用SDS-PAGE、考马斯亮蓝染色,观察重组基因的表达。透射电镜观察融合蛋白形成的病毒样颗粒。融合蛋白经腹腔注射免疫昆明小鼠,间接ELISA法检测小鼠血清中抗-ABCSP、抗-Aβ抗体的滴度。结果:经酶切鉴定、DNA序列测定证实,重组基因位于表达质粒之中,其大小、序列与理论设计相符。诱导表达后,SDS-PAGE显示,在细菌裂解液的上清和沉淀中均可见到表达蛋白条带,且以沉淀中为多,约占沉淀总蛋白的40%。纯化后的融合蛋白形成电镜下可观察到的病毒样颗粒。昆明小鼠经融合蛋白免疫5次后,其血清中抗-ABCSP抗体的滴度可达1∶5 000,抗-Aβ抗体的滴度可达1∶10 000,检测不到抗-HBc抗体。结论:c-ABCSP-Aβ15-c融合基因在大肠杆菌中可高效表达,表达的融合蛋白具有较强的免疫原性。     

Location of human cytotoxic T cell epitopes within a polymorphic domain of the Plasmodium falciparum circumsporozoite protein.

Studies in mice have shown that cytotoxic T lymphocytes (CTL) specific for epitopes within the circumsporozoite (CS) protein of malaria sporozoites can prevent malaria probably by destroying infected hepatocytes. This has provided a model for the development of a sporozoite vaccine. It has not been shown whether humans can mount a CTL response to this protein nor what determinants on the protein could be considered as target epitopes for such cells and thus merit inclusion in a sporozoite vaccine. We have used a novel technique to study a caucasian population which would benefit from a sporozoite vaccine and have been able to demonstrate that some individuals with a history of sporozoite exposure do contain peripheral blood CTL specific for the Plasmodium falciparum CS protein. The prevalence of CTL among different individuals is low and there is evidence that recent malaria exposure may be a prerequisite for finding such CTL. In three individuals, CTL could be repeatedly found and in all cases the epitopes mapped to one of the two polymorphic C-terminal domains. Using a CTL line, we mapped a recognition site to residues 351-395 of the CS protein, overlapping the region of the protein recognized by murine CTL.     

Synthesis and immunological characterization of 104-mer and 102-mer peptides corresponding to the N- and C-terminal regions of the Plasmodium falciparum CS protein

We investigated the immunogenicity and the conformational properties of the non-repetitive sequences of the Plasmodium falciparum circumsporozoite (CS) protein. Two polypeptides of 104 and 102 amino acids long, covering, respectively, the N- and C-terminal regions of the CS protein, were synthesized using solid phase Fmoc chemistry. The crude polypeptides were purified by a combination of size exclusion chromatography and RP-HPLC. Sera of mice immunized with the free polypeptides emulsified in incomplete Freund's adjuvant strongly reacted with the synthetic polypeptides as well as with native CS protein as judged by ELISA and IFAT assays. Most importantly, these antisera inhibited the sporozoite invasion of hepatoma cells. In addition, sera derived from donors living in a malaria endemic area recognized the CS 104- and 102-mers. Conformational studies of the CS polypeptides were also performed by circular dichroism spectroscopy showing the presence of a weakly ordered structure that can be increased by addition of trifluoroethanol. The obtained results indicate that the synthetic CS polypeptides and the natural CS protein share some common antigenic determinants and probably have similar conformation. The approach used in this study might be useful for the development of a synthetic malaria vaccine.