Reduced antibody response to the repetitive sequence of the Plasmodium falciparum circumsporozoite protein in mice infected with Plasmodium yoelii blood forms

The immunogenicity of the carrier-free synthetic peptide, (NANP)₄₀, from the repetitive region of the Plasmodium falciparum circumsporozoite (CS) protein was investigated in genetically responder mice (C57BL/6, H-2^b) acutely infected with blood forms of the non-lethal murine malaria parasite, P. yoelii. As compared to non-infected mice, P. yoelii-infected C57BL/6 mice produced significantly lower titers of anti-(NANP)₄₀ IgG antibodies. This decrease in the anti-(NANP)₄₀ antibody response peaked with the peak of parasitemia, and involved all the IgG subclasses. Interestingly, this P. yoelii-mediated effect was evident both on the development of the antibody response to the (NANP)₄₀ peptide, and on an already established anti-(NANP)₄₀ antibody titer, as seen in mice immunized with the peptide 1 month before the infection. Since (NANP)_n-based constructs are strongly envisaged as potential vaccines against falciparum malaria, these results might be important in the evaluation of the efficacy of these vaccine candidates, when they will be used in individuals living in endemic areas.     

Monoclonal antibody identifies circumsporozoite protein of Plasmodium malariae and detects a common epitope on Plasmodium brasilianum sporozoites.

We produced a hybridoma secreting an immunoglobulin G1 monoclonal antibody against the circumsporozoite protein of the human malaria parasite Plasmodium malariae (Uganda 1/CDC). The monoclonal antibody produces a circumsporozoite precipitation reaction when incubated with viable sporozoites of P. malariae and reacts at high titers with heat-fixed sporozoites in an indirect immunofluorescent antibody test. Using the purified monoclonal antibody and Western blot analysis, we identified two polypeptides with apparent molecular weights of 60,000 (Pm 60) and 48,000 (Pm 48) in extracts of P. malariae sporozoites. Two-dimensional electrophoretic analysis of Pm 60 and the circumsporozoite protein Pm 48 indicated their isoelectric points to be acidic, with values of 5.3 and 4.7 to 5.0, respectively. A two-site immunoradiometric assay showed that the circumsporozoite protein recognized by the monoclonal antibody contains a repetitive epitope. P. malariae monoclonal antibody also reacted strongly with sporozoites of the simian parasite Plasmodium brasilianum, indicating a shared epitope on sporozoites of the two species. The P. malariae antibody did not bind sporozoite antigens of any other primate malarias, including Plasmodium falciparum, Plasmodium vivax, and Plasmodium ovale.     

Lack of H-2 restriction of the Plasmodium falciparum (NANP) sequence as multiple antigen peptide

The major surface antigen of malaria sporozoites, the circumsporozoite protein, contains a region of tandem amino acid repeats, which in the case of the human malaria parasite Plasmodium falciparum, consist of four amino acids Asn-Ala-Asn-Pro (NANP) repeated up to about 40 times. This repetitive sequence has been considered as the basis for the development of subunit vaccines against P. falciparum malaria. We and others had previously shown that synthetic and recombinant NANP peptides were immunogenic only in H-2b mice. In the present report we show that, when mice with different H-2 haplotypes are immunized with the repetitive NANP sequence incorporated in a synthetic branching multiple antigen peptide (MAP), all except one of the mouse strains tested mounted an anti-peptide antibody response. Such a response does not appear to be due to the peculiar assembly of the NANP sequence. In fact, MAP containing repetitive sequences from circumsporozoite proteins of other malaria parasites did not overcome the genetic restriction of the immune response to the linear peptides. These data show that in the case of the P. falciparum NANP repeats, their immunogenicity can be dramatically changed and increased when these peptides are assembled as MAP. This unexpected finding may be of interest in the design of synthetic candidate malaria vaccines.     

Immunogenicity of a non-repetitive sequence of Plasmodium falciparum circumsporozoite protein in man and mice

In the present work, the hypothesis that individuals naturally exposed to Plasmodium falciparum malaria infection in endemic areas produce antibodies directed against non-repetitive epitopes of the circumsporozoite protein was investigated. Using a synthetic peptide reproducing the non-repetitive group-conserved region I sequence, we have shown that specific anti-region I antibodies are detectable in sera from endemic countries. Of these sera, 87% also had antibodies against the immunodominant repetitive epitope (Asn-Ala-Asn-Pro, NANP) of P. falciparum. In order to study the immunogenicity of this non-repetitive epitope, a synthetic peptide consisting of both region I and three (NANP) repeats [RI-(NANP)3] was used to immunize inbred strains of mice. H-2b mice produced antibodies against both the repetitive and the non-repetitive epitope. These antibodies were specific for each epitope, recognized P. falciparum sporozoites in immunofluorescence, and inhibited sporozoite penetration into human liver cells in vitro. Non-H-2b mice were completely unresponsive. Lymph node cells from H-2b mice immunized with RI-(NANP)3 peptide proliferated in the presence of RI-(NANP)3 and of (NANP)4 peptide, but never in the presence of RI peptide alone. These findings demonstrate that in the configuration used (i) the non-repetitive epitope region I does not carry T-helper epitopes; (ii) the (NANP) repetitive epitope may act as a carrier for the immune response to region I in mice; and (iii) therefore, immune response to region I in man probably depends on the recognition of T-cell epitopes similar to those involved in the anti-NANP response: i.e. such a T epitope may be NANP itself in responding individuals or another, not yet recognized, sporozoite T-cell epitope.     

Characterization of murine monoclonal antibodies against a repetitive synthetic peptide from the circumsporozoite protein of the human malaria parasite, Plasmodium falciparum

Monoclonal antibodies (mAbs) were raised in mice against the synthetic peptide (NANP)40, consisting of 40 (NANP) repeats of the circumsporozoite (CS) protein of the human malaria parasite, Plasmodium falciparum, and characterized. (i) Five of these mAbs recognized the P. falciparum CS protein in western blot experiments and in immunofluorescence assays using different preparations of sporozoites. The remaining two mAbs (CT3.2 and CT3.3, both IgG1) gave negative results by both techniques. (ii) When the anti-(NANP)40 peptide mAbs were functionally tested in vitro to assess their ability to inhibit the attachment and penetration of the parasites into cultured human liver cells, six of them exhibited inhibitory activities ranging between 66 and 90%. CT3.2 mAbs, also, inhibited sporozoite attachment and penetration, despite the negative results by immunofluorescence and western blot experiments. However, when immunofluorescence was repeated in the presence of calcium, CT3.2 did reveal a positive recognition of P. falciparum sporozoites, suggesting that this mAb could recognize the (NANP) sequence when calcium was bound to the repetitive peptide. (iii) Furthermore, the binding of an anti-(NANP)40 IgM mAb (CT1) to the solid-phase peptide was not inhibited by preincubation of the peptide with a mAb against the P. falciparum CS protein. (iv) Finally, one anti-(NANP)40 IgG1 mAb (CT3.1) was unable to bind to the shorter (NANP)3 peptide, although it recognized the (NANP)40 peptide and the P. falciparum CS protein. The results presented here suggest that heterogeneous antibody populations are produced upon immunization of mice with (NANP)40 synthetic peptide and that epitopes different from those simply related to the linear (NANP) amino acid sequence are likely to be present in long (NANP)n constructs as well as in the repetitive domain of the P. falciparum CS protein.     

Transformed Toxoplasma gondii tachyzoites expressing the circumsporozoite protein of Plasmodium knowlesi elicit a specific immune response in rhesus monkeys

Toxoplasma gondii tachyzoites were transformed with the coding sequence of the circumsporozoite (CS) protein of the primate malaria parasite Plasmodium knowlesi. A single inoculation of live transformed tachyzoites elicited an antibody response directed against the immunodominant repeat epitope (EQPAAGAGG)2 of the P. knowlesi CS protein in rhesus monkeys. Notably, these animals failed to show a positive serum conversion against T. gondii. Antibodies against Toxoplasma antigens were detected only after a second inoculation with a higher number of transformed tachyzoites. This boost induced an increased antibody response against the P. knowlesi CS protein associated with immunoglobulin class switching, thus demonstrating the establishment of immunological memory. These results indicate that the Toxoplasma-derived CS protein is efficiently recognized by the monkey immune system and represents an immunodominant antigen in transformed parasites.     

A conserved peptide sequence of the Plasmodium falciparum circumsporozoite protein and antipeptide antibodies inhibit Plasmodium berghei sporozoite invasion of Hep-G2 cells and protect immunized mice against P. berghei sporozoite challenge.

Minutes after injection into the circulation, malaria sporozoites enter hepatocytes. The speed and specificity of the invasion process suggest that it is receptor mediated. The region II sequence of Plasmodium falciparum circumsporozoite (CS) protein includes a nonapeptide (WSPCSVTCG) which is highly conserved in all of the CS proteins sequenced to data, including the one from Plasmodium berghei. We have found that two peptides based on the P. falciparum region II sequence, P18 (EWSPCSVTCGNGIQVRIK) and P32 (IEQYLKKIKNS ISTEWSPCSVTCGNGIQVRIK), significantly inhibited P. berghei sporozoite invasion into Hep-G2 cells in vitro. This inhibition was enhanced if either peptide was preincubated with Hep-G2 cells prior to sporozoite invasion. We confirm that region II is a sporozoite ligand for the hepatocyte receptor; moreover, despite the few differences between P. falciparum and P. berghei region II sequences around the nonapeptide sequence (66% homology), the functional characteristics of the motif sequences are not affected. Since the conserved motifs represent a crucial sequence involved in Plasmodium sporozoite invasion of hepatocytes, antibodies to region II should inhibit sporozite invasion into hepatocytes. Indeed, we found that polyclonal antibodies generated to the P. falciparum-based peptide P32 inhibited P. berghei sporozoite invasion of Hep-G2 cells. Furthermore, inbred mice (C57BL/6) immunized with P32 were protected against a lethal challenge of P. berghei sporozoites. Our results suggest that the conserved region II of the CS protein contains crucial B- and T-cell epitopes, that such peptide sequences from the human malaria parasite P. falciparum can be screened in the P. berghei rodent model, and, finally, that region II can be considered useful as one of the components of a malaria vaccine.     

Genetic restriction of the murine humoral response to a recombinant Plasmodium vivax circumsporozoite protein

The antibody response of inbred and congenic strains of mice immunized with a recombinant Plasmodium vivax circumsporozoite protein was investigated. Only mice of the H-2k and H-2a haplotypes developed antibodies that react with P. vivax sporozoite in an indirect immunofluorescence assay and with the recombinant protein in enzyme-linked immunosorbent assay. Congenic recombinant strains were used to map Ir gene control to the I-Ak locus. A similar pattern of genetic restriction was found when congenic strains were immunized with a carrier-free synthetic peptide corresponding to the repeat region of the P. vivax circumsporozoite protein. The results suggest that the ability of T helper cells to recognize a T cell epitope(s) in the repeat region of the P. vivax circumsporozoite protein is genetically restricted.     

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.     

Disruption of disulfide linkages of the Plasmodium falciparum circumsporozoite protein: effects on cytotoxic and antibody responses in mice.

The circumsporozoite protein is a predominant surface antigen present on Plasmodium sporozoites. In Plasmodium falciparum circumsporozoite protein (PfCSP), two cysteine residues (396 and 401) are present adjacent to two overlapping cytotoxic T-lymphocyte epitopes of the protein and are involved in the formation of disulfide bridges. We investigated the role of these cysteines on the cellular and antibody responses towards the CS protein because disruption of disulfide linkages and the presence of cysteine residues in the flanking region of an epitope has been shown to significantly alter the immune responses to various proteins. Mice were immunized with variant forms of PfCSP DNA vaccine plasmids where these cysteine residues were individually mutated to alanine. The plasmid vaccines induced antigen specific antibody and cytotoxic T lymphocyte responses. While no alterations of cysteine influenced the CTL responses to P. falciparum CS protein, vaccine pVRCS4, containing an altered cysteine at position 401, dramatically improved the antibody response to the carboxyl-terminal region of the protein. This work indicates that sequence alterations of genes in an anti-malarial vaccine could enhance the response towards the native protein. Given the fact that long term natural immunity to the pathogen has not been documented, it may be important to challenge the immune system with non-native proteins.     

Induction of sporozoite-specific memory cells in mice immunized with a recombinant Plasmodium vivax circumsporozoite protein

A recombinant Plasmodium vivax circumsporozoite (CS) protein (rPvCS-1) has been investigated as a possible malaria sporozoite vaccine candidate. Experiments were carried out to determine whether sporozoite-specific memory cells develop in Swiss Webster mice immunized with rPvCS-1. Challenge of rPvCS-1-immunized mice with P. vivax sporozoites resulted in a 100-fold increase in the mean serum anti-sporozoite antibody titer. The presence of parasite-specific T helper cells was demonstrated using an in vitro assay. Anti-CS antibodies were detected in the culture supernatants of spleen cells of rPvCS-1-immunized mice following in vitro challenge with P. vivax sporozoite extract. Immune spleen cells depleted of T cells did not produce antibodies when challenged with sporozoite extract in vitro. In conclusion, immunization of mice with the rPvCS-1 protein induced memory T cells which recognized native CS antigen and functioned as T helper cells in the production of anti-sporozoite antibodies both in vivo and in vitro.     

H-2b restriction of the immune response to the p126 Plasmodium falciparum antigen.

Inbred BALB/c (H-2d), CBA (H-2k) and C57B1/6 (H-2b) mice immunized with Plasmodium falciparum schizonts or culture supernates develop antibodies of different antigenic specificities. It has been observed that C57B1/6 mice were unable to produce detectable antibodies against the p126 antigen (native molecule and p73 or p50 processed fragments) compared with other inbred mice. Similar results were obtained using BALB congenic mice with a lack of p126 antibody response in H-2b mice, while H-2d and H-2k mice produced antibodies against the p126. Lymphocyte proliferation assays performed by incubation of spleen cells with immunopurified p126 were positive for immunized BALB/c (H-2d) and congenic H-2d or H-2k mice. On the other hand, no lymphocyte stimulation was observed with either C57B1/6 (H-2b) or congenic H-2b mice. These results suggest an MHC restriction of the immune response against the entire p126 (found in schizonts) and its p73 and p50 naturally processed fragments (found in culture supernates).     

Rhoptry-associated protein 1-binding monoclonal antibody raised against a heterologous peptide sequence inhibits Plasmodium falciparum growth in vitro

Monoclonal antibodies (MAbs) specific for Plasmodium falciparum rhoptry-associated protein 1 (RAP-1) were generated and tested for inhibition of parasite growth in vitro. The majority of indirect immunofluorescence assay (IFA)-positive MAbs raised against recombinant RAP-1 positions 23 to 711 (rRAP-1(23-711)) recognized epitopes located in the immunodominant N-terminal third of RAP-1. MAbs specific for the building block 35.1 of the synthetic peptide malaria vaccine SPf66 also yielded an IFA staining pattern characteristic for rhoptry-associated proteins and reacted specifically with rRAP-1 and parasite-derived RAP-1 molecules p67 and p82. Cross-reactivity with RAP-1 was blocked by the 35.1 peptide. Epitope mapping with truncated rRAP-1 molecules and overlapping peptides identified the linear RAP-1 sequence Y218KYSL222 as a target of the anti-35.1 MAbs. This sequence lacks primary sequence similarity with the 35.1 peptide (YGGPANKKNAG). Cross-reactivity of the anti-35.1 MAbs thus appears to be associated with conformational rather than sequence homology. While the anti-35.1 MAb SP8.18 exhibited parasite growth-inhibitory activity, none of the tested anti-rRAP-1(23-711) MAbs inhibited parasite growth, independently of their fine specificity for the RAP-1 sequences at positions 33 to 42, 213 to 222, 243 to 247, 280 to 287, or 405 to 446. The growth-inhibitory activity of MAb SP8.18 was, however, accelerated by noninhibitory anti-RAP-1 MAbs. Results demonstrate that in addition to fine specificity, other binding parameters are also crucial for the inhibitory potential of an antibody.     

Characterisation of a repetitive DNA sequence from the malaria parasite,Plasmodium falciparum

A repetitive DNA fragment cloned from the malaria parasite, Plasmodium falciparum, has been analysed. It contains a 21 base pair sequence which occurs in multiple tandem repeats. Two clusters of the same repeat are found in opposite orientations on the same DNA fragment. The repetitive DNA provides an additional way to distinguish between different strains of parasite by hybridisation to genomic blots and may serve as a species-specific probe for diagnosis.     

Human antibody response to the major merozoite surface antigen of Plasmodium falciparum is strain specific and short-lived.

The precursor of the major merozoite antigen of Plasmodium falciparum, gp190, is considered a candidate for inclusion in a malaria vaccine. This protein, which consists of conserved, dimorphic, and polymorphic sequences, is very immunogenic in humans. In a longitudinal study carried out with 94 inhabitants of a rural community in Mali, West Africa, we show that in this endemic area naturally acquired gp190-specific antibodies are predominantly directed against the dimorphic parts of one of the main alleles of gp190. The presence of antibodies against these dimorphic regions correlates with the prevalence of the corresponding antigen in the infecting parasite population. Moreover, qualitative as well as quantitative differences were found in the time course of the humoral immune response to the dimorphic regions in adults and children, who differ in their susceptibility to malaria infection.     

The synthetic, oxidized C-terminal fragment of the Plasmodium berghei circumsporozoite protein elicits a high protective response

A polypeptide of 69 amino acids (PbCS 242-310) encompassing the C-terminal region of the circumsporozoite protein of Plasmodium berghei (PbCS) was generated using solid-phase peptide synthesis. The immunological and protective properties of peptide PbCS 242-310 were studied in BALB/c mice (H-2d). Two subcutaneous injections, in the presence of IFA at the base of the tail, generated (i) high titers of anti-peptide antibodies which also recognized the native P. berghei CS protein, (ii) cytolytic T cells specific for the Kd-restricted peptide PbCS 245-253 and (iii) partial CD8+-dependent protection against sporozoite-induced malaria. The same frequencies of peptide PbCS 245-253 specific CD8+ T cells were found by IFN-gamma ELISPOT in the draining lymph nodes of animals immunized with the short optimal CTL peptide 245-253 or with the polypeptide 242-310, indicating that the longer polypeptide can be processed and presented in vivo in the context of MHC class I as efficiently as the short CTL peptide. Interestingly, higher levels of IFN-gamma producing CD8 T cells and protection were observed when the four cysteine residues present in the C-terminal peptide were fully oxidized. These findings underline the potential importance of the chemical nature of the C-terminal fragment on the activation of the immune system and concomitant protection.     

Antibodies to a conserved-motif peptide sequence of the Plasmodium falciparum thrombospondin-related anonymous protein and circumsporozoite protein recognize a 78-kilodalton protein in the asexual blood stages of the parasite and inhibit merozoite invasion in vitro.

Athrombospondin-related anonymous protein (TRAP) of the human malaria parasite Plasmodium falciparum shares highly conserved amino acid sequence motifs with the circumsporozoite protein of all plasmodia sequenced so far, as well as with unrelated proteins like thrombospondin and properdin. Although it was first described as an asexual blood stages protein, there has been some controversy about its expression in these stages. Pursuant to our interest in the conserved sequences within the malaria antigens, we synthesized an 18-residue peptide (18-mer) representing a conserved motif of TRAP and raised polyclonal antibodies against it. In an immunoblot assay in which we probed proteins from the asexual blood stages of the parasite, we found that this antibody recognized predominantly a 78-kDa protein in the whole parasite lysate. Furthermore, in another immunoblot, the recombinant TRAP constructs containing the conserved-motif sequence were distinctly recognized by the antipeptide antibodies, whereas a construct lacking the motif sequence was not, suggesting that the antibodies specifically cross-reacted with a protein which might be a TRAP-like protein present in the asexual blood stages of the parasite. Also, in an immunofluorescence assay, this antibody brightly stained the acetone-fixed trophozoites of the parasite. Most significantly, anti-18-mer immunoglobulin G, as well as antipeptide antibody against a smaller (nonamer) construct representing the most conserved motif within the 18-mer, inhibited the merozoite invasion of erythrocytes in a dose-dependent manner. These results provide evidence of the expression of TRAP or a TRAP-like protein in the asexual blood stages of the parasite and of a possible role of the conserved motifs in the parasite-host cell interaction during the process of invasion.     

Monoclonal antibodies to a synthetic peptide corresponding to a repeated sequence in the Plasmodium falciparum antigen Pf155

Mouse monoclonal antibodies were prepared against a synthetic peptide (EENVEHDA) corresponding to a tandemly repeated sequence in the C-terminus of the Plasmodium falciparum antigen Pf155. One antibody (IgG1) producing hybridoma was studied in detail. The specificity of the antibody was determined by enzyme-linked immunosorbent assays using bovine serum albumin-conjugated or free peptides as solid phase antigens and various synthetic peptides for inhibition. The antibody reacted with Pf155 as detected by immunofluorescence and immunoblotting. It was also an efficient inhibitor of merozoite invasion in P. falciparum in vitro cultures indicating that it defines a biologically important epitope present on the native Pf155 molecule.     

H-2-linked genetic control of antibody response to soluble calf skin collagen in mice

An autosomal dominant immune response gene could be demonstrated in congenic resistant strains of mice which is linked to the H-2 locus and controls the antibody response to soluble calf collagen. High responsiveness was associated with the H-2 alleles, b and f, low responsiveness with the H-2 alleles, d, k, m and r. Studies with calf procollagen, which contains an additional carrier moiety, indicated that these genetic differences might be expressed at the level of T cells.     

Interaction of antigen load and antibody response in determining heterologous protein nephritogenicity in inbred mice

Genetically disparate inbred mouse strains mounting quantitatively different immune responses to a potentially nephritogenic heterologous protein had varied development of glomerulonephritis (GN) dependent upon the interaction between level of antibody response and antigen dose. High responder BALB/c mice given high doses of horse apoferritin developed a severe necrotizing GN. BALB/c mice given low dose horse apoferritin developed less severe GN, or when antibodies were in extreme excess, no GN. Low responder C3H mice given high dose horse apoferritin were in extreme antigen excess and developed no GN. C3H mice given low dose horse apoferritin developed a glomerulopathy characterized by capillary loop immune deposits. Interstrain differences in immune clearance and avidity did not account for this varied nephritogenicity, although avidity did relate to glomerular site of immune deposits in the BALB/c model. In the models under consideration susceptibility to heterologous antigen-induced glomerular injury was a function of the magnitudes of both the antibody response and the antigenic challenge.