Impact of recombinant adenovirus serotype 35 priming versus boosting of a Plasmodium falciparum protein: characterization of T- and B-cell responses to liver-stage antigen 1

Prime-boost vaccination regimens with heterologous antigen delivery systems have indicated that redirection of the immune response is feasible. We showed earlier that T-cell responses to circumsporozoite (CS) protein improved significantly when the protein is primed with recombinant adenovirus serotype 35 coding for CS (rAd35.CS). The current study was designed to answer the question whether such an effect can be extended to liver-stage antigens (LSA) of Plasmodium falciparum such as LSA-1. Studies with mice have demonstrated that the LSA-1 protein induces strong antibody response but a weak T-cell immunity. We first identified T-cell epitopes in LSA-1 by use of intracellular gamma interferon (IFN-gamma) staining and confirmed these epitopes by means of enzyme-linked immunospot assay and pentamer staining. We show that a single immunization with rAd35.LSA-1 induced a strong antigen-specific IFN-gamma CD8(+) T-cell response but no measurable antibody response. In contrast, vaccinations with the adjuvanted recombinant LSA-1 protein induced remarkably low cellular responses but strong antibody responses. Finally, both priming and boosting of the adjuvanted protein by rAd35 resulted in enhanced T-cell responses without impairing the level of antibody responses induced by the protein immunizations alone. Furthermore, the incorporation of rAd35 in the vaccination schedule led to a skewing of LSA-1-specific antibody responses toward a Th1-type immune response. Our results show the ability of rAd35 to induce potent T-cell immunity in combination with protein in a prime-boost schedule without impairing the B-cell response.     

Characterization of conserved T- and B-cell epitopes in Plasmodium falciparum major merozoite surface protein 1

Vaccines for P. falciparum will need to contain both T- and B-cell epitopes. Conserved epitopes are the most desirable, but they are often poorly immunogenic. The major merozoite surface protein 1 (MSP-1) is currently a leading vaccine candidate antigen. In this study, six peptides from conserved or partly conserved regions of MSP-1 were evaluated for immunogenicity in B10 congenic mice. Following immunization with the peptides, murine T cells were tested for the ability to proliferate in vitro and antibody responses to MSP-1 were evaluated in vivo. The results showed that one highly conserved sequence (MSP-1#1, VTHESYQELVKKLEALEDAV; located at amino acid positions 20 to 39) and one partly conserved sequence (MSP-1#23, GLFHKEKMILNEEEITTKGA; located at positions 44 to 63) contained both T- and B-cell epitopes. Immunization of mice with these peptides resulted in T-cell proliferation and enhanced production of antibody to MSP-1 upon exposure to merozoites. MSP-1#1 stimulated T-cell responses in three of the six strains of mice evaluated, whereas MSP-1#23 was immunogenic in only one strain. Immunization with the other four peptides resulted in T-cell responses to the peptides, but none of the resulting peptide-specific T cells recognized native MSP-1. These results demonstrate that two sequences located in the N terminus of MSP-1 can induce T- and B-cell responses following immunization in a murine model. Clearly, these sequences merit further consideration for inclusion in a vaccine for malaria.     

Human antibodies against Plasmodium falciparum liver-stage antigen 3 cross-react with Plasmodium yoelii preerythrocytic-stage epitopes and inhibit sporozoite invasion in vitro and in vivo

The Plasmodium falciparum liver-stage antigen 3 (LSA3), a recently identified preerythrocytic antigen, induces protection against malaria in chimpanzees. Using antibodies from individuals with hyperimmunity to malaria affinity purified on recombinant or synthetic polypeptides of LSA3, we identified four non-cross-reactive B-cell epitopes in Plasmodium yoelii preerythrocytic stages. On sporozoites the P. yoelii protein detected has a molecular mass similar to that of LSA3. T-cell epitopes cross-reacting with P. yoelii were also demonstrated using peripheral blood lymphocytes from LSA3-immunized chimpanzees. In contrast, no cross-reactive epitopes were found in Plasmodium berghei. LSA3-specific human antibodies exerted up to 100% inhibition of in vitro invasion of P. yoelii sporozoites into mouse hepatocytes. This strong in vitro activity was reproduced in vivo by passive transfer of LSA3 antibodies. These results indicate that the homologous epitopes may be biologically functional and suggest that P. yoelii could be used as a model to assess the antisporozoite activity of anti-LSA3 antibodies.     

Cytokine responses to Plasmodium falciparum liver-stage antigen 1 vary in rainy and dry seasons in highland Kenya

Seasonal epidemics of malaria occur in highland areas of western Kenya where transmission intensity varies according to rainfall. This study describes the seasonal changes in cytokine responses to Plasmodium falciparum liver-stage antigen 1 (LSA-1) by children (< or =17 years old) and adults (> or =18 years old) living in such a highland area. Fourteen- to 24-mer peptides corresponding to the N- and C-terminal nonrepeat regions of LSA-1 stimulated production of interleukin-5 (IL-5), interleukin-10 (IL-10), gamma interferon (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha) by peripheral blood mononuclear cells (PBMC) from 17 to 73% of individuals in both age groups in both seasons. IL-10 and TNF-alpha responses were more frequent during the high-transmission, rainy season than during the low-transmission, dry season (73 and 67% versus 17 and 25% response rates, respectively). In contrast, there was no seasonal change in the proportion of LSA-1-driven IFN-gamma and IL-5 responses. Children produced less IFN-gamma than adults, but IL-5, IL-10, and TNF-alpha levels were similar for both age groups. Depletion of CD8(+) cells from PBMC decreased IFN-gamma but increased IL-10 production. Individuals with LSA-1-stimulated IL-10 responses in the dry season were less likely to become reinfected in the subsequent rainy season than those without IL-10 responses (25% versus 49%; P = 0.083). These data support the notion that maintenance of LSA-1-driven IL-10 and TNF-alpha responses requires repeated and sustained exposure to liver-stage P. falciparum. In contrast, IFN-gamma responses increase slowly with age but persist once acquired. CD8(+) T cells are the major source of IFN-gamma but may suppress production or secretion of IL-10.     

Gamma interferon responses to Plasmodium falciparum liver-stage antigen 1 and thrombospondin-related adhesive protein and their relationship to age, transmission intensity, and protection against malaria

Gamma interferon (IFN-gamma) responses to the Plasmodium falciparum antigens liver-stage antigen 1 (LSA-1) and thrombospondin-related adhesive protein (TRAP) are thought to be important in protection against malaria. Optimal methods of testing and the effects of age and transmission intensity on these responses are unknown. IFN-gamma responses to LSA-1 and TRAP peptides were assessed by the enzyme-linked immunospot assay (ELISPOT) and enzyme-linked immunosorbent assay (ELISA) in children and adults from areas of stable and unstable malaria transmission in Kenya. Adults in the areas of stable and unstable transmission had similar frequencies and levels of IFN-gamma responses to LSA-1 and TRAP as determined by ELISPOT and ELISA. In contrast, IFN-gamma responses to the LSA-1 T3 peptide (assessed by ELISPOT) and to any LSA-1 peptide (assessed by ELISA) were less frequent in children in the area of unstable transmission than in children in the area of stable transmission. IFN-gamma responses to LSA-1 were more frequently detected by ELISA than by ELISPOT in the stable-transmission area. IFN-gamma responses detected by ELISA and ELISPOT did not correlate with each other. In children in the stable-transmission area, IFN-gamma responses to LSA-1 peptides assessed by ELISA, but not by ELISPOT, were associated with protection against clinical malaria and anemia. IFN-gamma responses to LSA-1 appear to require repeated P. falciparum exposure and/or increased age and, as measured by ELISA, are associated with protection against clinical malaria and anemia.     

Natural antibody responses against the non-repeat-sequence-based B-cell epitopes of the Plasmodium falciparum circumsporozoite protein.

Synthetic peptides and human serum or plasma samples from regions of Brazil, Papua New Guinea, and Kenya in which malaria is endemic were used to identify B-cell epitopes localized outside the repeat region of the circumsporozoite (CS) protein of the human malaria parasite Plasmodium falciparum. In agreement with recent observations, our results confirm the presence of two non-repeat-region-based B-cell epitopes of the CS protein. Of these two epitopes, only the region I epitope (KPKHKKLKQPGDGNP) was previously shown to be recognized by human sera. In this study, we show that human immune sera from malarious regions recognize another B-cell epitope, ENANANNAV, that resides carboxyl to the repeat region. The present study reveals that (i) the repeat-sequence (NANP)-based B-cell epitope of the CS protein is an immunogenic but not immunodominant epitope; (ii) the natural expression of antibody responses to the two non-repeat-region-based B-cell epitopes of the CS protein varies in different populations in which malaria is endemic; (iii) although the host immune responses to the non-repeat-region-based B-cell epitopes increase as a function of host age, this increase is not statistically significant for the region I epitope but is significant for the other epitope; and (iv) the Th1R T-cell site but not the Th2R or Th3R T-cell site induces an antibody response in the human host. This study confirms the immunogenic potential of non-repeat-region-based B-cell epitopes and suggests that antibody pressures may also contribute to the maintenance of the antigenic diversity of the CS protein.     

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.     

Process development and analysis of liver-stage antigen 1, a preerythrocyte-stage protein-based vaccine for Plasmodium falciparum

Plasmodium falciparum liver-stage antigen 1 (LSA-1) is expressed solely in infected hepatocytes and is thought to have a role in liver schizogony and merozoite release. Specific humoral, cellular, and cytokine immune responses to LSA-1 are well documented, with epitopes identified that correlate with antibody production, proliferative T-cell responses, or cytokine induction. With the goal of developing a vaccine against this preerythrocyte-stage protein, we undertook the good manufacturing practices (GMP) manufacture of a recombinant LSA-1 construct, LSA-NRC, incorporating the N- and C-terminal regions of the protein and two of the centrally placed 17-amino-acid repeats. To improve the protein yield, a method of codon harmonization was employed to reengineer the gene sequence for expression in Escherichia coli. A 300-liter GMP fermentation produced 8 kg of bacterial cell paste, and a three-step column chromatographic method yielded 8 mg of purified antigen per g of paste. The final bulk protein was >98% pure, demonstrated long-term stability, and contained <0.005 endotoxin units per 50 microg of protein. To accomplish the initial stages of evaluation of this protein as a human-use vaccine against malaria, we immunized rabbits and mice with LSA-NRC in Montanide ISA 720. New Zealand White rabbits and A/J (H-2K) mice produced high-titer antibodies that recognized liver-stage parasites in infected cultured human hepatocytes. Gamma interferon-producing cells, which have been associated with LSA-1-mediated protection, were detected in splenocytes harvested from immunized mice. Finally, sera taken from people living in a region where malaria is holoendemic recognized LSA-NRC by Western blotting.     

Analysis of antibodies directed against merozoite surface protein 1 of the human malaria parasite Plasmodium falciparum

The 190-kDa merozoite surface protein 1 (MSP-1) of Plasmodium falciparum, an essential component in the parasite's life cycle, is a primary candidate for a malaria vaccine. Rabbit antibodies elicited by the heterologously produced MSP-1 processing products p83, p30, p38, and p42, derived from strain 3D7, were analyzed for the potential to inhibit in vitro erythrocyte invasion by the parasite and parasite growth. Our data show that (i) epitopes recognized by antibodies, which inhibit parasite replication, are distributed throughout the entire MSP-1 molecule; (ii) when combined, antibodies specific for different regions of MSP-1 inhibit in a strictly additive manner; (iii) anti-MSP-1 antibodies interfere with erythrocyte invasion as well as with the intraerythrocytic growth of the parasite; and (iv) antibodies raised against MSP-1 of strain 3D7 strongly cross-inhibit replication of the heterologous strain FCB-1. Accordingly, anti-MSP-1 antibodies appear to be capable of interfering with parasite multiplication at more than one level. Since the overall immunogenicity profile of MSP-1 in rabbits closely resembles that found in sera of Aotus monkeys immunized with parasite-derived MSP-1 and of humans semi-immune to malaria from whom highly inhibiting antigen-specific antibodies were recovered, we consider the findings reported here to be relevant for the development of MSP-1-based vaccines against malaria.     

posttranslational modification of recombinant Plasmodium falciparum apical membrane antigen 1: impact on functional immune responses to a malaria vaccine candidate

Recombinant apical membrane antigen 1 (AMA1) is a leading vaccine candidate for Plasmodium falciparum malaria, as antibodies against recombinant P. falciparum AMA1 (PfAMA1) interrupt merozoite invasion into erythrocytes. In order to investigate the role of posttranslational modification in modulating the functional immune response to recombinant AMA1, two separate alleles of PfAMA1 (FVO and 3D7), in which native N-glycosylation sites have been mutated, were produced using Escherichia coli and a Pichia pastoris expression system. Recombinant Pichia pastoris AMA1-FVO (PpAMA1-FVO) and PpAMA1-3D7 are O-linked glycosylated, and 45% of PpAMA1-3D7 is nicked, though all four recombinant molecules react with conformation-specific monoclonal antibodies. To address the immunological effect of O-linked glycosylation, we compared the immunogenicity of E. coli AMA1-FVO (EcAMA1-FVO) and PpAMA1-FVO antigens, since both molecules are intact. The effect of antigen nicking was then investigated by comparing the immunogenicity of EcAMA1-3D7 and PpAMA1-3D7. Our data demonstrate that there is no significant difference in the rabbit antibody titer elicited towards EcAMA1-FVO and PpAMA1-FVO or to EcAMA1-3D7 and PpAMA1-3D7. Furthermore, we have demonstrated that recombinant AMA1 (FVO or 3D7), whether expressed and refolded from E. coli or produced from the Pichia expression system, is equivalent and mimics the functionality of the native protein in in vitro growth inhibition assay experiments. We conclude that in the case of recombinant AMA1, the E. coli- and P. pastoris-derived antigens are immunologically and functionally equivalent and are unaffected by the posttranslational modification resulting from expression in these two systems.     

Allele specificity of naturally acquired antibody responses against Plasmodium falciparum apical membrane antigen 1

Antibody responses against proteins located on the surface or in the apical organelles of merozoites are presumed to be important components of naturally acquired protective immune responses against the malaria parasite Plasmodium falciparum. However, many merozoite antigens are highly polymorphic, and antibodies induced against one particular allelic form might not be effective in controlling growth of parasites expressing alternative forms. The apical membrane antigen 1 (AMA1) is a polymorphic merozoite protein that is a target of naturally acquired invasion-inhibitory antibodies and is a leading asexual-stage vaccine candidate. We characterized the antibody responses against AMA1 in 262 individuals from Papua New Guinea exposed to malaria by using different allelic forms of the full AMA1 ectodomain and some individual subdomains. The majority of individuals had very high levels of antibodies against AMA1. The prevalence and titer of these antibodies increased with age. Although antibodies against conserved regions of the molecule were predominant in the majority of individuals, most plasma samples also contained antibodies directed against polymorphic regions of the antigen. In a few individuals, predominantly from younger age groups, the majority of antibodies against AMA1 were directed against polymorphic epitopes. The D10 allelic form of AMA1 apparently contains most if not all of the epitopes present in the other allelic forms tested, which might argue for its inclusion in future AMA1-based vaccines to be tested. Some important epitopes in AMA1 involved residues located in domain II or III but depended on more than one domain.     

Vaccination of monkeys with recombinant Plasmodium falciparum apical membrane antigen 1 confers protection against blood-stage malaria

A major challenge facing malaria vaccine development programs is identifying efficacious combinations of antigens. To date, merozoite surface protein 1 (MSP1) is regarded as the leading asexual vaccine candidate. Apical membrane antigen 1 (AMA1) has been identified as another leading candidate for an asexual malaria vaccine, but without any direct in vivo evidence that a recombinant form of Plasmodium falciparum AMA1 would have efficacy. We evaluated the efficacy of a form of P. falciparum AMA1, produced in Pichia pastoris, by vaccinating Aotus vociferans monkeys and then challenging them with P. falciparum parasites. Significant protection from this otherwise lethal challenge with P. falciparum was observed. Five of six animals had delayed patency; two of these remained subpatent for the course of the infection, and two controlled parasite growth at <0.75% of red blood cells parasitized. The protection induced by AMA1 was superior to that obtained with a form of MSP1 used in the same trial. The protection induced by a combination vaccine of AMA1 and MSP1 was not superior to the protection obtained with AMA1 alone, although the immunity generated appeared to operate against both vaccine components.     

Cell-mediated immune responses to Plasmodium falciparum purified soluble antigens in sickle-cell trait subjects

To determine the possible differences in the immune response to Plasmodium falciparum between sickle-cell trait (Hb AS) and normal haemoglobin (Hb AA) individuals, we examined 35 Hb AS and 24 Hb AA subjects matched for age and microenvironment. Their age was 2-55 years and all lived in a malaria endemic area 300 km south of Khartoum. Antibodies to ring-infected erythrocyte surface antigen (Pf155/RESA) and to circumsporozoite (CS) protein (anti-NANP40) indicated equal exposure to falciparum malaria. Peripheral blood mononuclear cells (BMNCs) from 20/35 (57%) Hb AS subjects compared with 10/24 (42%) Hb AA subjects, responded to affinity-purified P. falciparum soluble antigens (SPAg). Of those responding to SPAg, 9 (26%) Hb AS subjects and only two (8%) Hb AA subjects had high responses. The mean proliferative response to SPAg of BMNCs from Hb AS individuals was significantly higher than in Hb AA individuals (P less than 0.025). Responses of BMNCs to PPD and PHA were also higher among Hb AS individuals and correlated positively with responses to SPAg. These findings support the hypotheses that the sickle-cell trait protects individuals from P. falciparum infections, at least in part, by modulating the immune response.     

Protective immune responses to apical membrane antigen 1 of Plasmodium chabaudi involve recognition of strain-specific epitopes.

Apical membrane antigen 1 (AMA-1), an asexual blood-stage antigen of Plasmodium falciparum, is an important candidate for testing as a component of a malaria vaccine. This study investigates the nature of diversity in the Plasmodium chabaudi adami homolog of AMA-1 and the impact of that diversity on the efficacy of the recombinant antigen as a vaccine against challenge with a heterologous strain of P. chabaudi. The nucleotide sequence of the AMA-1 gene from strain DS differs from the published 556KA sequence at 79 sites. The large number of mutations, the nonrandom distribution of both synonymous and nonsynonymous mutations, and the nature of both the codon changes and the resulting amino acid substitutions suggest that positive selection operates on the AMA-1 gene in regions coding for antigenic sites. Protective immune responses induced by AMA-1 were strain specific. Immunization of mice with the refolded ectodomain of DS AMA-1 provided partial protection against challenge with virulent DS (homologous) parasites but failed to protect against challenge with avirulent 556KA (heterologous) parasites. Passive immunization of mice with rabbit antibodies raised against the same antigen had little effect on heterologous challenge but provided significant protection against the homologous DS parasites.     

Development of two monoclonal antibodies against Plasmodium falciparum sporozoite surface protein 2 and mapping of B-cell epitopes.

The Plasmodium yoelii sporozoite surface protein 2 (PySSP2) is the target of protective cellular immunity. Cytotoxic T cells specific for the Plasmodium falciparum analog PfSSP2, also known as thrombospondin-related anonymous protein (TRAP), are induced in human volunteers immunized with irradiated sporozoites. PfSSP2 is an important candidate antigen for a multicomponent malaria vaccine. We generated and characterized three monoclonal antibodies (MAbs) specific for PfSSP2/TRAP. The MAbs PfSSP2.1 (immunoglobulin G1 [IgG1]), PfSSP2.2 (IgG2a), and PfSSP2.3 (IgM) were species specific and identified three distinct B-cell epitopes containing sequences DRYI, CHPSDGKC, and TRPHGR, respectively. PfSSP2.1 partially inhibited P. falciparum liver-stage parasite development in human hepatocyte cultures (42 and 86% in two experiments at 100 microg/ml). Mice immunized with vaccinia virus expressing full-length PfSSP2 protein produced antibodies to (DRYIPYSP)3, and humans living in malaria-endemic areas (Indonesia and Kenya), who have lifelong exposure and partial clinical immunity to malaria, had antibodies to both (DRYIPYSP)3 and (CHPSDGKCN)2. Mice immunized with multiple antigen peptides MAP4 (DRYIPYSP)3P2P30 and MAP4 (CHPSDGKCN)3P2P30 in TiterMax developed antibodies to sporozoites that partially inhibited sporozoite invasion of human hepatoma cells (39 to 71% at a serum dilution of 1:50 in three different experiments). The modest inhibitory activities of the MAbs and the polyclonal antibodies to PfSSP2/TRAP epitopes do not suggest that a single-component vaccine designed to induce antibodies against PfSSP2/TRAP will be protective. Nonetheless, the MAbs directed against PfSSP2, and the peptides recognized by these MAbs, will be essential reagents in the development of PfSSP2/TRAP as a component of a multivalent P. falciparum human malaria vaccine.     

Cellular immune responses to Plasmodium falciparum antigens in Gambian children during and after an acute attack of falciparum malaria.

Peripheral blood mononuclear cells from 63 Gambian children with acute Plasmodium falciparum malaria were examined for lymphoproliferation and interferon-gamma (IFN) production in response to stimulation by mitogens, malaria antigens and other soluble antigens. Mitogen or Candida-induced proliferation was not depressed during acute infection but was enhanced 2 to 4 weeks after treatment. Responses to partially purified soluble P. falciparum antigens were minimal or absent in all children in the acute phase but approximately 50% of the children responded by proliferation or IFN-gamma production during the 2 to 8 week convalescent period. These proliferative responses were severely depressed in the presence of the patient's own serum. Nine children with significant convalescent phase proliferative responses were re-examined several months after acute infection. Of these, four remained responsive for at least 8 months in the probable absence of reinfection.     

Associations between responses to the rhoptry-associated membrane antigen of Plasmodium falciparum and immunity to malaria infection

Rhoptry proteins participate in the invasion of red blood cells by merozoites during the malaria parasite's asexual-stage cycle. Interference with the rhoptry protein function has been shown to prevent invasion, and three rhoptry proteins have been suggested as potential components of a vaccine against malaria. Rhoptry-associated membrane antigen (RAMA) is a 170-kDa protein of Plasmodium falciparum which is processed to a 60-kDa mature form in the rhoptries. p60/RAMA is discharged from rhoptries of free merozoites and binds to the red-cell membrane before being internalized to form part of the parasitophorous vacuole of the newly developing ring. We examined the range of anti-RAMA responses in individuals living in an area of endemicity for malaria and determined its association with clinical immunity. RAMA is immunogenic during infections, and at least three epitopes within RAMA are recognized by hyperimmune sera in immunoblots. Sera from individuals living in a region of Vietnam where malaria is endemic possessed strong antibody responses toward two C-terminal regions of RAMA. Cytophilic antibody isotypes (immunoglobulin G1 [IgG1] and IgG3) predominated in humoral responses to both C-terminal epitopes. Acute episodes of P. falciparum infection result in significant boosting of levels of antibody to an epitope at the extreme C terminus of RAMA that harbors the red-cell-binding domain. Immunity to P. falciparum infection was linked to elevated levels of IgG3 responses to this functional domain of RAMA, suggesting that the region may contain a protective epitope useful for inclusion in a multiepitope vaccine against malaria.     

A Plasmodium falciparum malaria vaccine candidate which contains epitopes from the circumsporozoite protein and a blood stage antigen, 5.1.

The previously described Plasmodium falciparum blood stage antigen, 5.1 (also referred to as exp-1) was expressed at a high level in Escherichia coli. Saimiri monkeys immunised with purified recombinant antigen 5.1 were partially protected from P. falciparum blood stage parasite challenge. The gene coding for 5.1 was combined with DNA coding for an (Asn-Ala-Asn-Pro)19 sequence (abbreviated (NANP)19 in the one-letter amino acid code). To facilitate purification of the recombinant protein, DNA coding for a hexahistidine (His6) sequence was introduced at the 5' end of the gene (proteins containing His6 have high affinity for Ni(2+)-chelate columns even in the presence of 6 M guanidine HCl). The recombinant protein, His6-5.1-(NANP)19 with an apparent molecular size of 40 kDa could be highly purified by a combination of 4 steps: (1) release and solubilization of the recombinant fusion protein from E. coli in the presence of 6 M guanidine-HCl; (2) precipitation of over 60% of the bacterial proteins by the addition of ammonium sulphate to 50% saturation; (3) affinity chromatography on a Ni(2+)-chelate column in the presence of 6 M guanidine-HCl; (4) adsorption onto a cation exchange resin in the presence of 6 M urea, and elution with an increasing NaCl gradient. Compared with the previously tested tetanus toxoid-(NANP)3 malaria vaccine, this protein elicits an anti-(NANP)n response which more closely resembles that evoked by native sporozoites. The recombinant vaccine also induces the production of antibodies against the blood stages of the malaria parasite.     

T- and B-cell epitopes in the secreted Mycobacterium bovis antigen MPB70 in mice

MPB70 is a soluble secreted protein highly expressed in Mycobacterium bovis and strains of bacille Calmette-Guérin (BCG); as such, it is a candidate for subunit and DNA vaccines against tuberculosis. MPB70 was screened for T-cell epitopes in four different inbred mouse strains. Major histocompatibility complex (MHC) H-2b-expressing mice (C57BL/6) secreted interferon-gamma (IFN-gamma) after stimulation with peptides from the regions 1-20, 41-50, 81-110, 121-150 and 161-193 of the MPB70 sequence. H-2db mouse (B6D2) splenocytes secreted IFN-gamma after stimulation with some of the same peptides, whereas H-2d mice (BALB/c and DBA/2) did not secrete IFN-gamma upon stimulation with the peptides. Sera from H-2db mice immunized with native MPB70 in incomplete Freund's adjuvant (IFA), mpb70 DNA or live BCG Moreau were found to contain antibodies against the native MPB70 antigen. H-2db mice immunized with native MPB70 in IFA exhibited high titres of peptide-reactive immunoglobulin G1 (IgG1) antibodies, whereas DNA-immunized mice reacted with IgG2a antibodies against some of the same peptides. As some of the epitopes recognized by mouse T and B cells have previously been found to stimulate immune responses in humans, cattle and rabbits, we conclude that these epitopes may be good general epitopes for the stimulation of T- and B-cell responses and candidates for a DNA vaccine with a broad applicability.     

Long-term multiepitopic cytotoxic-T-lymphocyte responses induced in chimpanzees by combinations of Plasmodium falciparum liver-stage peptides and lipopeptides

Preclinical immunogenicity studies of 12 malaria peptides, selected from four Plasmodium falciparum antigens (Ags), namely, LSA1, LSA3, SALSA, and STARP, that are expressed at the pre-erythrocytic (sporozoite and liver) stages of the human parasite were carried out in chimpanzees. To strengthen their immunogenicity, six of these synthetic peptides were modified by the C-terminal addition of a single palmitoyl chain (lipopeptides) and delivered without adjuvant, whereas the remaining six unmodified peptides were emulsified and delivered by using Montanide ISA51 adjuvant. We have previously reported that these peptides and lipopeptides induce high B-cell and CD4(+)-T-helper responses in chimpanzees. In this report, we show their ability to induce multiepitopic and long-lasting antigen-specific CD8(+) cytotoxic-T-lymphocyte (CTL) responses. The magnitude, consistency, and memory of CTL responses generated by LSA3 peptides point to the strong immunogenicity of this liver-stage Ag. These findings support the screening strategy used to select the four P. falciparum pre-erythrocytic Ags and emphasize their valuable immunogenic properties. The successful immunization of nonhuman primates with combinations of corresponding peptides in a mineral oil emulsion (ISA51) and lipopeptides in saline provide a vaccine formulation that can be tested in humans.