FcγRI activation of phospholipase Cγ1 and protein kinase C in dibutyryl cAMP-differentiated U937 cells is dependent solely on the tyrosine-kinase activated form of phosphatidylinositol-3-kinase

In the present study, the genetic mechanisms responsible for generation of antibodies recognizing the dominant epitope within a synthetic peptide PS1CT3 were examined. PS1CT3 is a peptide model antigen containing residues 28-42 of the large protein of the surface antigen of hepatitis B virus as B epitope (designated PS1), and the known T-helper-cell epitope derived from the circumsporozoite protein of the malaria parasite Plasmodium falciparum (designated CT3). To characterize the repertoire generated, the immunoglobulin heavy chain variable regions from IgM and IgG monoclonal antibodies against PS1CT3 were sequenced. Although all IgG monoclonal antibodies were directed against the immunodominant epitope, the genetic elements used were diverse. Comparison of the sequence of germ line precursor IgM to a mature IgG revealed that during maturation of the primary IgM response only the heavy chain fragment of the antibody molecule underwent somatic mutation.     

Fixed, epitope-specific, cytophilic antibody response to the polymorphic block 2 domain of the Plasmodium falciparum merozoite surface antigen MSP-1 in humans living in a malaria-endemic area

The MSP-1 merozoite surface antigen of the human malaria parasite Plasmodium falciparum is a major target of immune response. The domain called block 2 shows extensive allelic diversity, with more than 50 alleles identified, grouped into three allelic families. Presence of anti-block 2 antibodies has been associated with reduced risk for clinical malaria, but whether or not allele-specific antibodies are implicated remains unclear. To study the fine specificity of the human antibody response, we have used a series of 82 overlapping, N-biotinylated, 15-mer peptides scanning reference alleles and including numerous sequence variants. Peptide antigenicity was validated using sera from mice immunized with recombinant proteins. A cross-sectional survey conducted in a Senegalese village with intense malaria transmission indicated an overall 56 % seroprevalence. The response was specific for individuals and unrelated to the HLA type. Each responder reacted to a few peptides, unrelated to the infecting parasite genotype(s). Seroprevalence of each individual peptide was low, with no identifiable immunodominant epitope. Anti-block 2 antibodies were mostly of the IgG3 isotype, consistent with an involvement in cytophilic antibody-mediated merozoite clearance. The number of responders increased with age, but there was no accumulation of novel specificities with age and hence with exposure to an increasingly large number of alleles. A 15-month longitudinal follow up outlined a remarkably fixed response, with identical reactivity profiles, independent of the past or current parasite types, a pattern reminiscent of clonal imprinting. The implications of the characteristics of the anti-block 2 antibody response in parasite clearance are discussed.     

Protection against malaria in Aotus monkeys immunized with a recombinant blood-stage antigen fused to a universal T-cell epitope: correlation of serum gamma interferon levels with protection.

The major surface antigen p190 of the human malaria parasite Plasmodium falciparum contains nonpolymorphic, immunogenic stretches of amino acids which are attractive components for a subunit vaccine against malaria. One such polypeptide, termed 190L, is contained in the 80-kDa processing product of p190, which constitutes the major coat component of mature merozoites. We report here that immunization of Aotus monkeys with 190L gives only poor protection against P. falciparum challenge. However, addition by genetic engineering of a universal T-cell epitope (CS.T3) to 190L improved immunity, and as a result three of four monkeys were protected following challenge infection with blood-stage parasites. Neither antibody against the immunizing antigens or against blood-stage parasites nor the capacity of the monkeys' sera to inhibit in vitro parasite invasion correlated with protection. However, in contrast to sera from nonprotected monkeys, sera from protected animals contained elevated levels of gamma interferon. These results suggest that gamma interferon is directly or indirectly involved in the process of asexual parasite control in vivo.     

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.     

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.     

Passive immunization with a multicomponent vaccine against conserved domains of apical membrane antigen 1 and 235-kilodalton rhoptry proteins protects mice against Plasmodium yoelii blood-stage challenge infection

During malaria parasite invasion of red blood cells, merozoite proteins bind receptors on the surface of the erythrocyte. Two candidate Plasmodium yoelii adhesion proteins are apical membrane antigen 1 (AMA1) and the 235-kDa rhoptry proteins (P235). Previously, we have demonstrated that passive immunization with monoclonal antibodies (MAbs) 45B1 and 25.77 against AMA1 and P235, respectively, protects against a lethal challenge infection with P. yoelii YM. We show that MAb 45B1 recognizes an epitope located on a conserved surface of PyAMA1, as determined by phage display and analysis of the three-dimensional structure of AMA1, in a region similar to that bound by the P. falciparum AMA1-specific inhibitory antibody 4G2. The epitope recognized by 25.77 could not be assigned. We report here that MAbs 45B1 and 25.77 also protect against challenge with the nonlethal parasite line 17X, in which PyAMA1 has a significantly different amino acid sequence from that in YM. When administered together, the two MAbs acted at least additively in providing protection against challenge with the virulent YM parasite. These results support the concept of developing a multicomponent blood-stage vaccine and the inclusion of polymorphic targets such as AMA1, which these results suggest contain conserved domains recognized by inhibitory antibodies.     

Development of immunity in natural Plasmodium falciparum malaria: antibodies to the falciparum sporozoite vaccine 1 antigen (R32tet32).

Antibodies that reacted with a candidate sporozoite vaccine antigen (R32tet32) were found in 20 of 21 patients treated for acute infection with Plasmodium falciparum and monitored longitudinally over 67 days. R32tet32 contains 32 tandem copies of a tetrapeptide sequence that constitutes the immunodominant epitope of the circumsporozoite surface protein. The magnitude of the antibody response varied considerably among individuals and appeared to be independent of the number of previous clinical infections. Recrudescence of infection or infection with Plasmodium vivax had no demonstrable effect on antibody levels, although reinfection with P. falciparum produced a rapid rise in antibody titer. Antibody levels were observed to decline rapidly after treatment of clinical infection with mefloquine. The apparent antibody half-life was 27 days, which is comparable to the half-life of circulating immunoglobulin G in humans. The data suggest that antisporozoite antibody production ceased on about day 4 after treatment of acute infection. A similar pattern of response was observed for antibodies against the erythrocytic forms of the parasite. The cessation of antibody synthesis was interpreted as being due to immunosuppression induced by the presence of intraerythrocytic parasites and may explain in part why protective immunity is poorly developed in natural malaria infections.     

Antibodies to malaria peptide mimics inhibit Plasmodium falciparum invasion of erythrocytes

Apical membrane antigen 1 (AMA1) is expressed on the surfaces of Plasmodium falciparum merozoites and is thought to play an important role in the invasion of erythrocytes by malaria parasites. To select for peptides that mimic conformational B-cell epitopes on AMA1, we screened a phage display library of >10(8) individual peptides for peptides bound by a monoclonal anti-AMA1 antibody, 4G2dc1, known to inhibit P. falciparum invasion of erythrocytes. The most reactive peptides, J1, J3, and J7, elicited antibody responses in rabbits that recognized the peptide immunogen and both recombinant and parasite AMA1. Human antibodies in plasma samples from individuals exposed to chronic malaria reacted with J1 and J7 peptides and were isolated using immobilized peptide immunoadsorbents. Both rabbit and human antibodies specific for J1 and J7 peptides were able to inhibit the invasion of erythrocytes by P. falciparum merozoites. This is the first example of phage-derived peptides that mimic an important epitope of a blood-stage malaria vaccine candidate, inducing and isolating functional protective antibodies. Our data support the use of J1 and J7 peptide mimics as in vitro correlates of protective immunity in future AMA1 vaccine trials.     

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.     

Plasmodium chabaudi antigen Pch105, Plasmodium falciparum antigen Pf155, and erythrocyte band 3 share cross-reactive epitopes.

By immunoblotting with a number of monoclonal antibodies raised in human and murine malaria systems, we have been able to establish the presence of cross-reactive epitopes on the Plasmodium falciparum vaccine candidate antigen Pf155/RESA and its proposed Plasmodium chabaudi analog Pch105. These findings were confirmed when the same antibodies were tested in an immunofluorescence assay. By using short synthetic peptides corresponding to repeated sequences in the C terminus of the Pf155 and enzyme-linked immunosorbent assays, the cross-reacting epitope was found to be localized to this repeat segment. Furthermore, a monoclonal antibody to murine erythrocyte band 3 which also cross-reacted with human band 3 bound to both Pch105 and Pf155 as well as to the synthetic peptides, suggesting that these proteins share a related epitope. The cross-reactions reflect the existence of sequence homologies of band 3 with these plasmodial proteins. This molecular similarity may be used by the parasite to disturb the rigidity of the erythrocyte membrane, thereby facilitating its entrance into the cell.     

Immunofluorescent reaction of antibodies directed against antigens from the intestinal epithelium of Schistosoma mansoni. III. Study of the reactivity of monoclonal antibodies

A monoclonal mouse antibody of IgM class was raised against an epitope of the gut epithelium of the adult worm and was applied to the detection of antigen in parasite infection. The antigen was found in urine from mice and hamsters infected with Schistosoma mansoni; a good correlation between the concentration of antigen and worm burden was observed. The antigen was thermostable, soluble in trichloracetic acid; it was not hydrolysed by proteinase K but it was destroyed by metaperiodate. The antigen was shown to be Schistosoma genus specific. It was found in different developmental stages of the parasite. High levels were detected in egg extracts.     

High levels of antibodies to multiple domains and strains of VAR2CSA correlate with the absence of placental malaria in Cameroonian women living in an area of high Plasmodium falciparum transmission

Placental malaria, caused by sequestration of Plasmodium falciparum-infected erythrocytes in the placenta, is associated with increased risk of maternal morbidity and poor birth outcomes. The parasite antigen VAR2CSA (variant surface antigen 2-chondroitin sulfate A) is expressed on infected erythrocytes and mediates binding to chondroitin sulfate A, initiating inflammation and disrupting homeostasis at the maternal-fetal interface. Although antibodies can prevent sequestration, it is unclear whether parasite clearance is due to antibodies to a single Duffy binding-like (DBL) domain or to an extensive repertoire of antibodies to multiple DBL domains and allelic variants. Accordingly, plasma samples collected longitudinally from pregnant women were screened for naturally acquired antibodies against an extensive panel of VAR2CSA proteins, including 2 to 3 allelic variants for each of 5 different DBL domains. Analyses were performed on plasma samples collected from 3 to 9 months of pregnancy from women living in areas in Cameroon with high and low malaria transmission. The results demonstrate that high antibody levels to multiple VAR2CSA domains, rather than a single domain, were associated with the absence of placental malaria when antibodies were present from early in the second trimester until term. Absence of placental malaria was associated with increasing antibody breadth to different DBL domains and allelic variants in multigravid women. Furthermore, the antibody responses of women in the lower-transmission site had both lower magnitude and lesser breadth than those in the high-transmission site. These data suggest that immunity to placental malaria results from high antibody levels to multiple VAR2CSA domains and allelic variants and that antibody breadth is influenced by malaria transmission intensity.     

Antibodies to a Single,Conserved Epitope in Anopheles APN1 Inhibit Universal Transmission of Plasmodium falciparum and Plasmodium vivax Malaria

Malaria transmission-blocking vaccines (TBVs) represent a promising approach for the elimination and eradication of this disease. AnAPN1 is a lead TBV candidate that targets a surface antigen on the midgut of the obligate vector of the Plasmodium parasite, the Anopheles mosquito. In this study, we demonstrated that antibodies targeting AnAPN1 block transmission of Plasmodium falciparum and Plasmodium vivax across distantly related anopheline species in countries to which malaria is endemic. Using a biochemical and immunological approach, we determined that the mechanism of action for this phenomenon stems from antibody recognition of a single protective epitope on AnAPN1, which we found to be immunogenic in murine and nonhuman primate models and highly conserved among anophelines. These data indicate that AnAPN1 meets the established target product profile for TBVs and suggest a potential key role for an AnAPN1-based panmalaria TBV in the effort to eradicate malaria.     

IgM antibody responses to the circumsporozoite protein in naturally acquired falciparum malaria

The circumsporozoite (CS) protein on the surface of sporozoites is the major target for antibody response(s) to the infective stage of the malaria parasite. Sera from malaria endemic areas contain both IgM and IgG antibodies that react with a dominant epitope in the tetrapeptide repeat region of the CS protein. In order to characterize the IgM CS antibody response inPlasmodium falciparum (PF) malaria, a prospective study was conducted in Thai Rangers. Variable IgM responses against the CS protein were detected in 81% of 47 PF-infected subjects. Similar to IgG response kinetics, IgM levels rose to a peak 6–10 days after detection of parasitemia and showed an apparent serum half-life of less than 25 days. The classic difference in isotype ratio (IgG/IgM) between primary and secondary antibody responses was observed to bloodstage, but not CS, antigens.     

Plasmodium vivax promiscuous T-helper epitopes defined and evaluated as linear peptide chimera immunogens

Clinical trials of malaria vaccines have confirmed that parasite-derived T-cell epitopes are required to elicit consistent and long-lasting immune responses. We report here the identification and functional characterization of six T-cell epitopes that are present in the merozoite surface protein-1 of Plasmodium vivax (PvMSP-1) and bind promiscuously to four different HLA-DRB1* alleles. Each of these peptides induced lymphoproliferative responses in cells from individuals with previous P. vivax infections. Furthermore, linear-peptide chimeras containing the promiscuous PvMSP-1 T-cell epitopes, synthesized in tandem with the Plasmodium falciparum immunodominant circumsporozoite protein (CSP) B-cell epitope, induced high specific antibody titers, cytokine production, long-lasting immune responses, and immunoglobulin G isotype class switching in BALB/c mice. A linear-peptide chimera containing an allele-restricted P. falciparum T-cell epitope with the CSP B-cell epitope was not effective. Two out of the six promiscuous T-cell epitopes exhibiting the highest anti-peptide response also contain B-cell epitopes. Antisera generated against these B-cell epitopes recognize P. vivax merozoites in immunofluorescence assays. Importantly, the anti-peptide antibodies generated to the CSP B-cell epitope inhibited the invasion of P. falciparum sporozoites into human hepatocytes. These data and the simplicity of design of the chimeric constructs highlight the potential of multimeric, multistage, and multispecies linear-peptide chimeras containing parasite promiscuous T-cell epitopes for malaria vaccine development.     

Analysis of human B‐cell responses following ChAd63‐MVA MSP1 and AMA1 immunization and controlled malaria infection

Acquisition of non‐sterilizing natural immunity to Plasmodium falciparum malaria has been shown in low transmission areas following multiple exposures. However, conflicting data from endemic areas suggest that the parasite may interfere with the induction of effective B‐cell responses. To date, the impact of blood‐stage parasite exposure on antigen‐specific B cells has not been reported following controlled human malaria infection (CHMI). Here we analysed human B‐cell responses in a series of Phase I/IIa clinical trials, which include CHMI, using candidate virus‐vectored vaccines encoding two blood‐stage antigens: merozoite surface protein 1 (MSP1) and apical membrane antigen 1 (AMA1). Previously vaccinated volunteers show boosting of pre‐existing antigen‐specific memory B‐cell (mBC) responses following CHMI. In contrast, unvaccinated malaria‐naive control volunteers developed an mBC response against MSP1 but not AMA1. Serum IgG correlated with the mBC response after booster vaccination but this relationship was less well maintained following CHMI. A significant reduction in peripheral MSP1‐specific mBC was observed at the point of diagnosis of blood‐stage infection. This was coincident with a reduction in peripheral blood B‐cell subsets expressing CXCR3 and elevated serum levels of interferon‐γ and CXCL9, suggesting migration away from the periphery. These CHMI data confirm that mBC and antibody responses can be induced and boosted by blood‐stage parasite exposure, in support of epidemiological studies on low‐level parasite exposure.     

Duffy antigen is important for the lethal effect of the lethal strain of<Emphasis Type="Italic"> Plasmodium yoelii</Emphasis> 17XL

We studied the potential role of the Duffy antigen and glycophorin A as receptors for rodent malaria parasite invasion of erythrocytes. Parasitemia increased exponentially after infection with Plasmodium berghei NK65, P. chabaudi, and P. vinckei in Duffy antigen knockout, glycophorin A knockout, and wild-type mice, indicating that the Duffy antigen and glycophorin A are not essential for these malaria parasites. However, parasitemia of the Duffy antigen knockout mice infected with P. yoelii 17XL remained constant from day 5 to 14 after infection, and then decreased, resulting in autotherapy. The treatment of P. yoelii 17XL-infected Duffy antigen knockout mice with anti-CD4 antibody increased the parasitemia 15 days after infection and the mice eventually died, indicating that CD-4-positive cells play an important role in the clearance of P. yoelii 17XL at the late stage of the infection.     

Discovery of GAMA, a Plasmodium falciparum merozoite micronemal protein, as a novel blood-stage vaccine candidate antigen

One of the solutions for reducing the global mortality and morbidity due to malaria is multivalent vaccines comprising antigens of several life cycle stages of the malarial parasite. Hence, there is a need for supplementing the current set of malaria vaccine candidate antigens. Here, we aimed to characterize glycosylphosphatidylinositol (GPI)-anchored micronemal antigen (GAMA) encoded by the PF08_0008 gene in Plasmodium falciparum. Antibodies were raised against recombinant GAMA synthesized by using a wheat germ cell-free system. Immunoelectron microscopy demonstrated for the first time that GAMA is a microneme protein of the merozoite. Erythrocyte binding assays revealed that GAMA possesses an erythrocyte binding epitope in the C-terminal region and it binds a nonsialylated protein receptor on human erythrocytes. Growth inhibition assays revealed that anti-GAMA antibodies can inhibit P. falciparum invasion in a dose-dependent manner and GAMA plays a role in the sialic acid (SA)-independent invasion pathway. Anti-GAMA antibodies in combination with anti-erythrocyte binding antigen 175 exhibited a significantly higher level of invasion inhibition, supporting the rationale that targeting of both SA-dependent and SA-independent ligands/pathways is better than targeting either of them alone. Human sera collected from areas of malaria endemicity in Mali and Thailand recognized GAMA. Since GAMA in P. falciparum is refractory to gene knockout attempts, it is essential to parasite invasion. Overall, our study indicates that GAMA is a novel blood-stage vaccine candidate antigen.     

Low CD4(+) T cell responses to the C-terminal region of the malaria merozoite surface protein-1 may be attributed to processing within distinct MHC class II pathways

The C-terminal fragment of merozoite surface protein-1 (MSP-1) of the mouse malaria parasite Plasmodium chabaudi chabaudi (AS) stimulates a weak CD4 T cell response when compared to the response to a more structurally simple region of the molecule. The tertiary structure of the C-terminal region of MSP-1 is maintained by five disulfide bonds. A peptide from this region could only be processed and loaded onto newly synthesized MHC class II molecules, whereas a peptide from the structurally simple region was available for loading onto recycling MHC class II. CD4(+) T cell hybridomas took longer to recognize an epitope derived from the disulfide-bonded region whether native parasite or recombinant MSP-1 antigen was used. Reduction of disulfide bonds in the C-terminal region subsequently allowed peptides to be loaded onto recycling MHC class II and greatly enhanced the rapidity of the T cell response. These data demonstrate that differential processing occurs intramolecularly in MSP-1, which may be responsible for the observed weak CD4 T cell responses against this region. The consequences of this in vivo may be that limited T cell help is available for protective antibody production which has important implications for designing vaccines based on MSP-1.     

Laser light scattering immunoassay for malaria.

Laser light scattering immunoassay (LIA) was proposed as a prospective diagnostic method for the detection of antibody (or antigen) by monitoring the agglutination of antigen (or antibody) coated carrier particles using dynamic light scattering (DLS) as probe. LIA is a very sensitive assay as it can detect microscopic immune complexes even when antibody (or antigen) level is low. A sizeable number of human sera collected from malaria endemic areas and hospitals have been analysed by ELISA using Pf parasite lysate or a RESA derived synthetic peptide as antigen parallel to LIA using Pf antigen coated polystyrene latex beads. Comparative analysis of data suggests LIA to be as good as ELISA and possibly better in terms of sensitivity and simplicity. LIA can be a simple and inexpensive immunoassay suitable for field use and mass application.