Plasmodium falciparum: restricted polymorphism of T cell epitopes of the circumsporozoite protein in Brazil.

We examined the extent of variation of the 3' region of the circumsporozoite gene among Plasmodium falciparum isolates through amplification of a selected DNA fragment followed by DNA sequencing. A total of 32 isolates were analyzed, of which 24 were from Amazon endemic areas in Brazil and 8 from widely separated geographical regions in the world. Among Brazilian isolates only 2 variants were detected: 19 displayed the same sequence of strain 7G8 whereas the 4 remaining isolates differed from the 7G8 strain at five nucleotide positions which also led to amino acid changes. Variation was restricted to one of the T-helper epitopes while the sequence identified as a cytotoxic T cell epitope was conserved in all Brazilian isolates. P. falciparum samples from other geographical regions in the world showed sequences distinct from those of Brazilian isolates. However, some constancy could be observed within that variation. For instance, the most frequent nucleotide substitutions, from A and C at nucleotide positions 1015 and 1024, were the same in all isolates.     

An immunologically cryptic epitope of Plasmodium falciparum circumsporozoite protein facilitates liver cell recognition and induces protective antibodies that block liver cell invasion

Circumsporozoite, a predominant surface protein, is involved in invasion of liver cells by Plasmodium sporozoites, which leads to malaria. We have previously reported that the amino terminus region (amino acids 27-117) of P. falciparum circumsporozoite protein plays a critical role in the invasion of liver cells by the parasite. Here we show that invasion-blocking antibodies are induced by a polypeptide encoding these 91 amino acids, only when it is presented in the absence of the rest of the protein. This suggests that when present in the whole protein, the amino terminus remains immunologically cryptic. A single reactive epitope was identified and mapped to a stretch of 21 amino acids from position 93 to 113. The epitope is configurational in nature, since its recognition was affected by deleting as little as 3 amino acids from either end of the 21-residue peptide. Lysine 104, the only known polymorphic position in the epitope, affected its recognition by the antibodies, and its conversion to leucine in the protein led to a substantial loss of binding activity of the protein to the hepatocytes. This indicated that in the protein, the epitope serves as a binding ligand and facilitates the interaction between sporozoite and hepatic cells. When considered along with the observation that in its native state this motif is immunologically unresponsive, we suggest that hiding functional moieties of the protein from the immune system is an evasion strategy to preserve liver cell binding function and may be of importance in designing anti-sporozoite vaccines.     

Plasmodium falciparum: glycosylation status of Plasmodium falciparum circumsporozoite protein expressed in the baculovirus system

We expressed the main surface antigen of Plasmodium falciparum sporozoites, the circumsporozoite protein (CSP), in High Five (Trichoplusia ni) insect cells using the baculovirus system. Significant amounts of the recombinant protein could be obtained, as judged by SDS-PAGE, Western blot, and immunofluorescence analysis. The cellular localization for recombinant CSP was determined by immunofluorescence. The high fluorescence signal of the permeabilized cells, relative to that of fixed nonpermeabilized cells, revealed a clear intracellular localization of this surface antigen. Analysis of possible posttranslational modifications of CSP showed that this recombinant protein is only N-glycosylated in the baculovirus system. Although DNA-sequence analysis revealed a GPI-cleavage/attachment site, no GPI anchor could be demonstrated. These analyses show that the glycosylation status of this recombinant protein may not reflect its native form in P. falciparum. The impact of these findings on vaccine development will be discussed.     

Two-step chromatographic purification of recombinant Plasmodium falciparum circumsporozoite protein from Escherichia coli

The Plasmodium falciparum circumsporozoite (PfCS) protein (aa 19–405) has been cloned and expressed in E. coli. The protein was purified in a two-step process that was rapid and reproducible. E. coli cells were grown to a high density before induction for 1 h. Cells were disrupted by high pressure microfluidization and the total bacterial protein solubilized in 6 M Gu-HCl. The protein was refolded while bound to Ni–NTA agarose by exchange of 6 M Gu-HCl for 8 M urea and then slow removal of the urea. The eluted protein was further purified on Q Sepharose Fast Flow using conditions developed to remove E. coli proteins and reduce endotoxin (to 10 EU/50 μg). Yield was 20 mg of PfCS protein from 10 g of wet cell paste. The final protein product bound to HepG2 liver cells in culture and inhibited the invasion of those cells by sporozoites in an ISI assay greater than 80% over control cultures when used at 10 μg/ml.     

Human T cells recognize polymorphic and non-polymorphic regions of the Plasmodium falciparum circumsporozoite protein.

In order to characterize T cell epitopes in the Plasmodium falciparum circumsporozoite (CS) protein sequence, we isolated T cell clones, from non-immune donors, which reacted with synthetic peptides corresponding to two predicted CS protein T cell epitopes. Peptide CS.T3 (corresponding to a non-polymorphic region of the CS protein, residues 378-398) was recognized in association with either DR2 or DRw9 restriction elements. T cell clones recognizing CS.T3 also reacted with the sporozoite-derived CS protein. Peptide CS.T2 corresponds to a polymorphic region (residues 325-341) of the CS protein. Unlike the CS.T3-specific clones, the CS.T2-specific clones did not recognize the CS protein. Since the CS.T2 peptide includes residues which are polymorphic in different P. falciparum isolates, we investigated whether these residues were critical for recognition of the peptide. We show here that a single amino acid substitution at a position of the CS protein which shows genetic polymorphism affects recognition of the sequence by human T cells. The implications of these data for malaria vaccine development are discussed.     

Naturally acquired CD8+ cytotoxic T lymphocytes against the Plasmodium falciparum circumsporozoite protein.

In rodent malaria model systems, protective immunity induced by immunization with irradiated sporozoites is eliminated by in vivo depletion of CD8+ T cells, and adoptive transfer of CTL clones against the circumsporozoite protein protects against malaria. We recently demonstrated that volunteers immunized with irradiated Plasmodium falciparum sporozoites produce CTL against peptide 368-390 of the P. falciparum circumsporozoite protein. To determine whether natural exposure to malaria induced similar CTL, we studied 11 adult, male, life-long residents of a highly malarious area of Kenya, who were selected because their lymphocytes had been shown to proliferate after stimulation with peptides 361-380, 371-390, or 368-390 and because nine had been resistant to malaria in previous studies. In four of the 11 individuals there was peptide-specific, genetically restricted, CTL activity. In all four individuals, this activity was unaffected by depletion of CD4+ T cells. In three volunteers the activity was eliminated or reduced by depletion of CD8+ T cells; in the fourth volunteer the CD8+ T cell depletion was uninterpretable. This first demonstration of CD8+ T cell, genetically restricted, Ag-specific CTL against a malaria protein among individuals exposed to endemic malaria provides a foundation for studying the relationship between circulating CTL and resistance to malaria infection.     

Cell-mediated immune responses to vaccine peptides derived from the circumsporozoite protein of Plasmodium falciparum

T cell epitopes residing within vaccine candidate peptides have been identified by delayed-type hypersensitivity (DTH) responses in mice. The recombinant sporozoite vaccine candidate, R32tet32, contains at least two T epitopes, one located within the repeat region and another in the tet tail. When C57BL/6 (H-2b) and BALB/c (H-2d) mice were sensitized intradermally with R32tet32 or the truncated protein R32LR emulsified in CFA and challenged 5 days later with R32tet32, only H-2b mice recognized a T epitope located within the major repeat sequence (NANP) and encoded by four or less repeats. H-2d mice responded solely to the T epitope located on the tet tail. Ear swelling was maximal at 48 h and revealed a histologic pattern characteristic of DTH. CD4+ T cell lines derived from immunized animals demonstrated the ability to mediate local DTH, proliferate, and secrete lymphokines in response to stimulation with Ag. High dose i.v. administration of R32tet32 in C57BL/6 and BALB/c mice before intradermal sensitization with R32tet32 revealed that DTH responses were suppressed only in BALB/c mice. Further experiments localized the suppressive determinant to the tet tail. Collectively, these data indicate that DTH may prove to be a useful method to characterize the biologic activity of T epitopes, furthermore they suggest that candidate vaccine peptides should be tested for suppressive activity before inclusion in a vaccine.     

Human recognition of T cell epitopes on the Plasmodium vivax circumsporozoite protein.

In order to identify T cell epitopes recognized by human in the Plasmodium vivax circumsporozoite protein, 28 overlapping synthetic peptides spanning the entire circumsporozoite protein were tested for their ability to stimulate proliferation of PBMC from 22 adults living in a malaria-endemic area of the Colombian Pacific Coast and from four individuals who never had a history of malaria infection. In addition, BALB/c mice were immunized with pools of peptides, and their lymph node cells were stimulated in vitro with individual peptides. Four epitopes were recognized by human lymphocytes but not all of them by mice. One of the epitopes was located inside the central repetitive B cell immunodominant domain. Several of the variants of the repeats were recognized by about one-third of the studied individuals. Another T cell epitope was located in the amino terminus and the other two in the carboxyl region. Peptides were recognized by both immune and nonimmune donors. Some of them were frequently recognized suggesting a lack of genetic restriction, whereas some others were recognized by only a few individuals but induced strong proliferation. These epitopes may be of potential value for a malaria subunit vaccine.     

A CD4(+) T-cell immune response to a conserved epitope in the circumsporozoite protein correlates with protection from natural Plasmodium falciparum infection and disease

Many human T-cell responses specific for epitopes in Plasmodium falciparum have been described, but none has yet been shown to be predictive of protection against natural malaria infection. Here we report a peptide-specific T-cell assay that is strongly associated with protection of humans in The Gambia, West Africa, from both malaria infection and disease. The assay detects interferon-gamma-secreting CD4(+) T cells specific for a conserved sequence from the circumsporozoite protein, which binds to many human leukocyte antigen (HLA)-DR types. The correlation was observed using a cultured, rather than an ex vivo, ELISPOT assay that measures central memory-'type T cells rather than activated effector T cells. These findings provide direct evidence for a protective role for CD4(+) T cells in humans, and a precise target for the design of improved vaccines against P. falciparum.     

Structural aspects of a protein epitope and their role in the major histocompatibility complex control of T cell responsiveness.

We have previously shown that immunization of C57BL/10 (H-2b) mice with the tobacco mosaic virus protein (TMVP) or with its tryptic peptide number 8, representing residues 93-112 of TMVP, induces T cells which proliferate in vitro in response to TMVP and peptide 8. In contrast, immunization of congenic B10.BR (H-2k) mice with either TMVP or with peptide 8 induces T cells which respond in vitro to the homologous but not the heterologous antigen. The capacity to exhibit cross-reactivity between TMVP and peptide 8 on the T cell level has been shown to be under major histocompatibility complex (MHC)-linked genetic control. The lack of cross-reactivity has been attributed to the inability of the H-2k APC to present the appropriate epitope to T cells. In the present paper, we report results of a comparative analysis of the role of structural aspects of the epitope on the proliferative T cell responses from TMVP and peptide 8-immune C57BL/10 (H-2b) and B10.BR (H-2k) mice. Utilizing a panel of synthetic peptides representing portions of peptide 8 and a panel of peptide-protein conjugates, we have determined that peptide 8-immune T cells of the H-2k strain appear to recognize a single epitope within peptide 8, located at its N-terminus. In contrast, in the H-2b strain, both TMVP and peptide 8-immune T cells appear to recognize two overlapping epitopes within peptide 8; one located in the middle region and the other toward the N-terminus. Experiments with H-2b T cells revealed that random amino acids added to the carboxyl or amino-terminus of nonstimulatory peptides can confer activity to these peptides, demonstrating limited specificity of interaction between antigen and Iab. Results of experiments dealing with fixation of antigen-presenting cells suggest that TMVP requires processing in order to be recognized by peptide 8-immune H-2b proliferative T cells whereas peptide 8 does not. Taken together the results suggest that the T cell responsiveness to TMVP and peptide 8 exhibited by these two congenic strains H-2b and H-2k is not only controlled by the strains MHC but is also influenced by antigen processing. Antigen processing may eliminate a potential epitope for the primary induction and the secondary stimulation of B10.BR T cells.     

Ultrastructural localization of Plasmodium falciparum circumsporozoite protein in newly invaded hepatoma cells

The fate and disposition of the circumsporozoite (CS) protein of Plasmodium falciparum was investigated during hepatoma cell invasion with several sera raised against defined CS peptides, including both repeat and nonrepeat regions spanning approximately 60% of the P. falciparum CS gene product. Distribution of the protein, as revealed by immunoelectron microscopy, was limited to the surface of the sporozoite both before and after invasion. In particular, no CS protein antigen was detected in association with either the parasitophorous vacuole membrane or the host cell surface.     

Imaging effector functions of human cytotoxic CD4+ T cells specific for Plasmodium falciparum circumsporozoite protein

Malaria vaccines, comprised of irradiated Plasmodium falciparum sporozoites or a synthetic peptide containing T and B cell epitopes of the circumsporozoite protein (CSP), elicit multifunctional cytotoxic and non-cytotoxic CD4⁺ T cells in immunised volunteers. Both lytic and non-lytic CD4⁺T cell clones recognised a series of overlapping epitopes within a ‘universal’ T cell epitope EYLNKIQNSLSTEWSPCSVT of CSP (NF54 isolate) that was presented in the context of multiple DR molecules. Lytic activity directly correlated with T cell receptor (TCR) functional avidity as measured by stimulation indices and recognition of naturally occurring variant peptides. CD4⁺ T cell-mediated cytotoxicity was contact-dependent and did not require de novo synthesis of cytotoxic mediators, suggesting a granule-mediated mechanism. Live cell imaging of the interaction of effector and target cells demonstrated that CD4⁺ cytotoxic T cells recognise target cells with their leading edge, reorient their cytotoxic granules towards the zone of contact, and form a stable immunological synapse. CTL attacks induced chromatin condensation, nuclear fragmentation and formation of apoptotic bodies in target cells. Together, these findings suggest that CD4⁺ CTLs trigger target cell apoptosis via classical perforin/granzyme-mediated cytotoxicity, similar to CD8⁺ CTLs, and these multifunctional sporozoite- and peptide-induced CD4⁺ T cells have the potential to play a direct role as effector cells in targeting the exoerythrocytic forms within the liver.     

Plasmodium falciparum: immunogenicity of circumsporozoite protein constructs produced in Escherichia coli

The immunogenicity of Plasmodium falciparum recombinant circumsporozoite protein constructs R16tet32, R32tet32, and R48tet32 in mice was examined by measuring antibody responses by enzyme linked immunosorbent assay, immunofluorescence, circumsporozoite precipitation, and inhibition of sporozoite invasion. All three constructs were found to be immunogenic when administered alone, but antibody responses were greater for the larger constructs, R32tet32 and R48tet32. Increased dose, boosting, and the use of adjuvants further augmented antibody responses. R32tet32 was found to be the most immunogenic of the three constructs, and high levels of protective antibodies were found to persist for at least 44 weeks when the construct was given with alum. Clinical trials with alum adjuvanted R32tet32 have now begun.     

Multiple display of foreign peptides on a filamentous bacteriophage. Peptides from Plasmodium falciparum circumsporozoite protein as antigens

We describe here two systems for encoding foreign amino acid sequences in the exposed N-terminal segment of the major coat protein of bacteriophage fd. Small peptides can be encoded directly; larger peptides are encoded in hybrid bacteriophage particles, in which the capsid is formed from a mixture of wild-type and modified coat proteins. In both cases, the peptides are present in multiple copies per phage particle. Peptides that represent the circumsporozoite protein, the major surface antigen of the sporozoites of the malaria parasite, Plasmodium falciparum, were inserted in this way and found to be highly immunogenic. These systems should prove to be valuable in displaying specific or random peptides as antigens, and could lead to cheap and effective vaccines. They will also allow rapid screening of peptides as potential agents of other biological effects, with important applications in biomolecular design.     

Immunogold determination of Plasmodium falciparum circumsporozoite protein in Anopheles stephensi salivary gland cells

The distribution of circumsporozoite (CS) proteins of Plasmodium falciparum sporozoites was observed during the passage of mature sporozoites in the hemocoel of Anopheles stephensi and during their entrance and sojourn in the salivary gland cells (SGC). The CS protein was visualized using a monoclonal antibody (3SP2) and immunogold labeling on ultrathin cryosections. In the hemocoel the sporozoites cease synthesizing CS protein, and some of it is shedded resulting in a patchy labeling pattern on the outer pellicular membrane. No internal labeling was observed. The sporozoites enter the SGC by puncturing the basal or lateral membrane. Inside the SGC, CS protein synthesis is turned on again; the Golgi system, nuclear envelope and all 3 pellicular membranes contain CS immunoreactivity. In the last phase of maturation, micronemes display abundant CS immunoreactivity. Rhoptries also show some immunogold labeling, but not as much as the micronemes.     

Protective effects of combining monoclonal antibodies and vaccines against the Plasmodium falciparum circumsporozoite protein

Combinations of monoclonal antibodies (mAbs) against different epitopes on the same antigen synergistically neutralize many viruses. However, there are limited studies assessing whether combining human mAbs against distinct regions of the Plasmodium falciparum (Pf) circumsporozoite protein (CSP) enhances in vivo protection against malaria compared to each mAb alone or whether passive transfer of PfCSP mAbs would improve protection following vaccination against PfCSP. Here, we isolated a panel of human mAbs against the subdominant C-terminal domain of PfCSP (C-CSP) from a volunteer immunized with radiation-attenuated Pf sporozoites. These C-CSP-specific mAbs had limited binding to sporozoites in vitro that was increased by combination with neutralizing human “repeat” mAbs against the NPDP/NVDP/NANP tetrapeptides in the central repeat region of PfCSP. Nevertheless, passive transfer of repeat- and C-CSP-specific mAb combinations did not provide enhanced protection against in vivo sporozoite challenge compared to repeat mAbs alone. Furthermore, combining potent repeat-specific mAbs (CIS43, L9, and 317) that respectively target the three tetrapeptides (NPDP/NVDP/NANP) did not provide additional protection against in vivo sporozoite challenge. However, administration of either CIS43, L9, or 317 (but not C-CSP-specific mAbs) to mice that had been immunized with R21, a PfCSP-based virus-like particle vaccine that induces polyclonal antibodies against the repeat region and C-CSP, provided enhanced protection against sporozoite challenge when compared to vaccine or mAbs alone. Collectively, this study shows that while combining mAbs against the repeat and C-terminal regions of PfCSP provide no additional protection in vivo, repeat mAbs do provide increased protection when combined with vaccine-induced polyclonal antibodies. These data should inform the implementation of PfCSP human mAbs alone or following vaccination to prevent malaria infection.     

Major histocompatibility complex gene products on macrophages influence T cell activation.

Antigen-pulsed macrophages were used to sensitize or elicit sensitivity from mice of different strains to a variety of antigens. The results indicate that sensitization is directed, not to antigen as such, but to a complex structure on the macrophage surface determined partly by the antigen, and partly by a product coded by the major histocompatibility complex. Delayed type hypersensitivity could be provoked by antigen in responder (R) mice and in the F1 between responder and low responder (LR) strains, but not in LR mice unless pretreated by cyclophosphamide. Sensitivity could be transferred to naive LR-strain mice by lymph node cells taken 5 days after sensitization of cyclophosphamide-pretreated LR mice but not of F1 hybrids between LR and R strains. Sensitivity from these could be transferred only to naive F1 or R-strain mice. The results suggest that low responsiveness cannot be accounted for solely in terms of the operation of a cyclophosphamide-sensitive suppressor mechanism. It is postulated that antigen is less immunogenic when presented by LR-strain cells than by R-strain cells.     

Conformational analysis of the immunodominant epitopes of the circumsporozoite protein of Plasmodium falciparum and knowlesi

All of the coat proteins of the sporozoite and merozoite stages of Plasmodium, determined to date, contain tandem repeats and most of these contain at least one proline residue. These tandemly repeated segments of the circumsporozite (CS) proteins of P. falciparum and P. knowlesi have been shown to constitute an immunodominant epitope. Antibodies to these peptide segments have been shown to be protective and cause the shedding of the CS protein, known as the CSP reaction. In this study, four synthetic peptides were prepared by solid-phase peptide synthesis. The first peptide corresponds to the tetrapeptide tandem repeat in the CS protein of P. falciparum, repeated eight times, (NANP)₈. The second peptide is an analogue of the first in which glycine is substituted for proline, (NANG)₈. The third peptide corresponds to the tandem repeat of P. knowlesi, PK(1–24), which is repeated twice (QAQGDGANAGQP)₂. The fourth peptide is a tetrapeptide repeat, corresponding to the C-terminal tetrapeptide of PK(1–24) and is repeated eight times, (AGQP)₈. It is shown by CD measurements that the presence of proline in these repeats induces an increase in β-sheet (β-turn) content in the (NANP)₈ peptide relative to the repeat of (NANG)₈ and PK(1–24) peptide in aqueous media. The (AGQP)₈ peptide has the highest β-sheet (β-turn) content in the synthetic peptides. It is concluded that this increase in defined structure correlates well with and hence may contribute to the increased antigenicity in these repeats.     

Human T cell recognition of polymorphic epitopes from malaria circumsporozoite protein

Lymphocytes obtained from forty individuals living in a malaria endemic area of West Africa were tested for in vitro proliferative responses to peptides representing variant regions of the immunodominant T cell domain of the circumsporozoite protein (amino acids 326 to 345, referred to as Th2R, and 361 to 380, referred to as Th3R) from three distinct strains of Plasmodium falciparum. A total of 83% of the individuals responded to at least one of the six peptides tested, confirming that these epitopes are immunodominant. A much greater number of individuals than expected by chance (32% of the responders to Th2R and 27% of the responders to Th3R) reacted to all three of the variant peptides for that epitope, indicating interdependency of the T cell responses, suggestive of cross-reactivity. Nevertheless, some subjects' T cells were clearly able to distinguish each variant peptide from the others. Using EBV transformed B cells, lymphocytes from 10 of the individuals were HLA typed. In this small group, HLA DRw13 was associated with a positive response to any of the peptides, whereas there was a negative association between DQw3 and response to any of the peptides. These results, although limited by the small sample size, suggest that recognition of T epitopes may be Ir gene linked. Our findings suggest that it may be possible to broaden the immunogenicity of an anti-sporozoite malaria vaccine.