Naturally occurring antibodies to an epitope on Plasmodium falciparum gametes detected by monoclonal antibody-based competitive enzyme-linked immunosorbent assay.

The antibody response to an epitope on gamete antigens of Plasmodium falciparum in persons naturally exposed to malaria has been investigated by competitive enzyme-linked immunosorbent assay. The assay detects antibodies to an epitope on the 48/45-kilodalton (kDa) gamete surface antigen by competition with horseradish peroxidase-labeled monoclonal antibody IIC5-B10. Five sera previously shown to immunoprecipitate the 230- and 48/45-kDa antigens significantly inhibited IIC5-B10 binding to an average of 24.2% of control. The one serum which precipitated only the 48/45-kDa antigen did not inhibit IIC5-B10 binding. For 26 sera which were negative by immunoprecipitation, mean binding in the assay was 112.7% of control (pooled London nonimmune sera). Recognition of both 230-kDa and 48/45-kDa antigens was associated with a titer of 1:9 or greater (reciprocal geometric mean titer, 27.6) for inhibition to more than 2 standard deviations from the mean of the negative sera. The results show that the IIC5-B10 binding site is a naturally immunogenic epitope recognized by the majority of persons who had antibodies to the 48/45-kDa protein. An additional finding was enhancement of binding of IIC5-B10 to an average of 154.4% of control by five sera which recognized only the 230-kDa antigen, presumably due to conformational alteration of the gamete antigen complex.     

Biosynthesis of the target antigens of antibodies blocking transmission of Plasmodium falciparum

We have studied the biosynthesis of three proteins of Mr 260 000, 59 000 and 53 000 previously identified on the surface of extracellular gametes of Plasmodium falciparum as the targets of monoclonal antibodies which block infectivity of P. falciparum to mosquitoes. In cultures of P. falciparum pulse labeled with [35S]methionine we have found that these proteins are synthesized by gametocytes from an early stage in their maturation but are not synthesized by asexual blood stage parasites. The target proteins synthesized by the gametocytes become expressed on the surface of the extracellular gametes but the gametes themselves no longer synthesize these proteins. The 59 000 and 53 000 Mr proteins do not result from processing from the 260 000 Mr protein. The 59 000 and 53 000 Mr protein, but not the 260 000 Mr proteins, were glycosylated by either glucosamine or mannose.     

Complement-mediated lysis of Plasmodium falciparum gametes by malaria-immune human sera is associated with antibodies to the gamete surface antigen Pfs230.

Antibodies to the sexual-stage surface antigens of Plasmodium falciparum, Pfs230 and Pfs48/45, can abolish the infectivity of gametes to mosquitoes; these antigens have been proposed as candidates for inclusion in a malaria transmission-blocking vaccine. One possible mechanism of antibody-mediated transmission blocking is complement-mediated gamete lysis. We have used a panel of human sera from geographically distinct regions where malaria is endemic to investigate whether this may be a mechanism of naturally acquired transmission-blocking immunity to P. falciparum. By immunoprecipitation, we have shown that antibody recognition of Pfs230 and Pfs48/45 is limited, despite universal exposure to P. falciparum gametocytes. In vitro complement-mediated lysis of P. falciparum gametes was positively associated with the presence of antibodies to Pfs230 but not with antibodies to the N-terminal region of the precursor molecule (Pfs260), which is shed from the gametocyte surface at the time of gametogenesis. Similarly, antibodies to two other gametocyte-specific proteins, Pfs48/45 and Pfg27/25, were not associated with gamete lysis. All sera which mediate gamete lysis contain immunoglobulin G1 (IgG1) and/or IgG3 antibodies to gamete surface proteins as determined by an enzyme-linked immunosorbent assay. These data suggest that Pfs230 is a major target of complement-fixing antibodies which may be important for antibody-mediated transmission-blocking immunity.     

Saccharomyces cerevisiae recombinant Pfs25 adsorbed to alum elicits antibodies that block transmission of Plasmodium falciparum.

Antibodies to Pfs25, a cysteine-rich 25-kDa protein present on the surface of Plasmodium falciparum zygotes, can completely block the transmission of malaria parasites when mixed with infectious blood and fed to mosquitoes through a membrane feeding apparatus. Recently, a polypeptide analog, Pfs25-B, secreted from recombinant Saccharomyces cerevisiae was found to react with conformation-dependent, transmission-blocking monoclonal antibodies and to elicit transmission-blocking antibodies in experimental animals when emulsified in either Freund's or muramyl tripeptide adjuvant. In this study, Pfs25-B adsorbed to alum induced transmission-blocking antibodies in both rodents and primates. Bacterially produced Pfs25, however, did not elicit complete transmission-blocking antibodies in rodents. Furthermore, unlike monoclonal antibodies to Pfs25, which block transmission only after ookinete development, antisera to Pfs25-B adsorbed to alum appeared to block the in vivo development of zygotes to ookinetes as well.     

Use of a specific immunogenic region on the Cowdria ruminantium MAP1 protein in a serological assay.

Currently available serological tests for cowdriosis (Cowdria ruminantium infection) in domestic ruminants are hampered by their low specificities because of cross-reactivity with Ehrlichia spp. The use of recombinant major antigenic protein (MAP1) of C. ruminantium for serodiagnosis was investigated. Overlapping fragments of the MAP1 protein were expressed in Escherichia coli and were reacted with sera from sheep infected with either C. ruminantium or Ehrlichia ovina. Two immunogenic regions on the MAP1 protein, designated MAP1-A and MAP1-B, were identified. MAP1-A was reactive with C. ruminantium antisera, E. ovina antisera, and three MAP1-specific monoclonal antibodies, whereas MAP1-B reacted only with C. ruminantium antisera. An indirect enzyme-linked immunosorbent assay (ELISA) based on MAP1-B was further developed and validated with sera from animals experimentally infected with C. ruminantium or several Ehrlichia spp. Antibodies raised in sheep, cattle, and goats against nine isolates of C. ruminantium reacted with MAP1-B. Cross-reactivity with MAP1-B was limited to Ehrlichia canis and Ehrlichia chaffeensis, two rickettsias which do not infect ruminants. Antibodies to Ehrlichia spp. which do infect ruminants (E. bovis, E. ovina, and E. phagocytophila) did not react with MAP1-B. Antibody titers to C. ruminantium in sera from experimentally infected cattle, goats, and sheep were detectable for 50 to 200 days postinfection. Further validation of the recombinant MAP1-B-based ELISA was done with sera obtained from sheep raised in heartwater-free areas in Zimbabwe and from several Caribbean islands. A total of 159 of 169 samples which were considered to be false positive by immunoblotting or indirect ELISA did not react with MAP1-B.(ABSTRACT TRUNCATED AT 250 WORDS)     

A surface protein expressed during the transformation of zygotes of Plasmodium gallinaceum is a target of transmission-blocking antibodies

Antibodies against gametes of malarial parasites (Plasmodium spp.) have previously been shown to block infectivity of the parasites to mosquitoes by preventing fertilization of the parasites in the insect midgut. These antibodies did not have any effect on the development of fertilized parasites. We now report that a surface protein of Mr 26,000 synthesized by zygotes of P. gallinaceum is the target of antibodies which block infectivity of the fertilized parasites to mosquitoes. Identification of this target antigen offers a new stage of the parasite against which a malaria transmission-blocking vaccine could be developed.     

N-terminal prodomain of Pfs230 synthesized using a cell-free system is sufficient to induce complement-dependent malaria transmission-blocking activity

The aim of a malaria transmission-blocking vaccine is to block the development of malaria parasites in the mosquito and thus prevent subsequent infection of the human host. Previous studies have demonstrated that the gametocyte/gamete surface protein Pfs230 can induce transmission-blocking immunity and have evaluated Escherichia coli-produced Pfs230 as a transmission-blocking vaccine candidate. In this study, we used the wheat germ cell-free expression system to produce N-terminal fragments of Pfs230 and evaluated the transmission-blocking activity of antisera raised against the recombinant Pfs230 protein. The rabbit antisera reacted to the surface of cultured gametocytes and gametes of the Plasmodium falciparum NF54 line, recognized the 360-kDa form of parasite-produced Pfs230 by Western blot assay, and reduced the infectivity of NF54 parasites to Anopheles stefensi mosquitoes in the presence of complement in a standard membrane feeding assay. Thus, our data demonstrate that the N-terminal pro domain of Pfs230 is sufficient to induce complement-dependent transmission-blocking activity against P. falciparum.     

Boosting of transmission-blocking immunity during natural Plasmodium vivax infections in humans depends upon frequent reinfection.

The infectivity to mosquitoes of 31 acute Plasmodium vivax patients was measured by permitting mosquitoes to feed directly on the patients. The infectivity of these patients correlated closely with titers of antibodies in their serum as measured by indirect immunofluorescence against air-dried female gametes of P. vivax. Infectivity by direct feeding was also closely parallel to the transmission-blocking activity of the sera of patients as measured by the suppression of infectivity of parasitized blood by autologous serum relative to normal (nonmalarial) human serum when fed to mosquitoes through a membrane. These results are consistent with serum antibodies in human P. vivax infections as major factors determining the infectivity of an infected individual to mosquitoes. It was further noted that individuals having a second attack of P. vivax within less than 4 months were considerably less infectious to mosquitoes than first-attack patients were. This "boosting" of transmission-blocking immunity was much less if longer intervals intervened between attacks. We discuss the immunological implications and possible epidemiological significance of this short-term boosting of transmission-blocking immunity by successive P. vivax infections.     

Structural diversity in the 45-kilodalton merozoite surface antigen of Plasmodium falciparum

An integral membrane protein associated with the merozoite surface of Plasmodium falciparum termed merozoite surface antigen 2 (the 45-kDa merozoite surface antigen), occurs in antigenically diverse forms. Here we report the sequences of the MSA 2 gene from two other isolates of P. falciparum. The 43 N-terminal residues and the 74 C-terminal residues of all three MSA 2 sequences are highly conserved, but between these conserved regions there are dramatic differences among the alleles. Instead of the two copies of a 32-amino-acid repeat present in the MSA 2 of isolate FC27, MSA 2 from clone 3D7 and isolate Indochina 1 contain 5 and 12 copies respectively of the four amino acid sequence Gly Gly Ser Ala. The sequences flanking the repeats also differ among the three antigens. The repeats in MSA 2 appear to be immunodominant during natural infection, and antibodies to the repeat regions of different alleles react with a restricted number of parasite isolates.     

Fungal-strain-dependent alterations in the time course and mortality of chronic murine pulmonary blastomycosis.

Intratracheal injection of 10(4) conidia of Blastomyces dermatitidis strain M1-A into mice was shown in previous work to induce chronic pulmonary and disseminated infection with histopathologic features of chronic human blastomycosis. Furthermore, 10-fold variations in inoculum density produced marked changes in mean survival times (MSTs), i.e., 32 days at 10(6), 36 days at 10(5), 97 days at 10(4), and 172 days at 10(3). A second strain (M1-B) failed to induce death in this model. To assess fungal-strain-dependent virulence, we extended these previous studies to 11 additional human isolates. Groups of male BALB/cByJ mice (6 to 8 weeks old) were injected intratracheally with 10(4) conidia from each of the 13 strains; the mice were weighed weekly and monitored for mortality, and their lungs were examined histopathologically. Infection rate and mortality were 100% in all groups except for the M1-B inoculated mice. For strains M1-B and M1-A, MST and mortality curves were not significantly different from those observed in our previously reported experiments. Four different survival patterns were noted in infected mice. The shortest MSTs were produced by strains M2-E, M2-B, M2-K, M2-H, and M2-A (24, 26, 26, 27, and 31 days, respectively), and the longest MST was seen in animal groups inoculated with strains M2-J and M2-G (130 and 134 days, respectively). Strains M2-I, M2-F, and M2-D produced intermediate MSTs of 65, 79, and 80 days, respectively. The 107-day MST induced by M1-A did not differ from strain M2-C-induced MST but differed significantly from the MST produced by the other strains. Pulmonary histopathology was similar in all animals dying with blastomycosis. The wide spectrum in survival times was not related to differences in clinical status of the patient from whom the isolate had been obtained, to fungal inoculum viability, or to individual mouse weight at inoculation. Strain-dependent virulence factors present in these fungal isolates alter the disease course in inbred mice in a fashion similar to that induced by 10-fold inoculum variation of strain M1-A conidia. These 13 isolates of B. dermatitidis, 1 avirulent and 12 with differing levels of virulence, provide tools for future studies into the nature of fungal virulence determinants in chronic blastomycosis.     

Alpha-tubulin II is a male-specific protein in Plasmodium falciparum.

The tubulin gene family in Plasmodium falciparum consists of one beta-tubulin and two alpha-tubulin genes (alpha-tubulin I and II). We present here data indicating that alpha-tubulin II is expressed only in male sexual stage parasites. An IgM mAb, 5E7, specifically reacted with stage III (day 4-5) through mature (day 10-11) male gametocytes and with emerging, exflagellating, or freely moving male gametes. No reactivity was detected in female gametocytes, female gametes, sporozoites, or asexual parasites. mAb 5E7 also specifically recognized male gametes of the avian parasite, Plasmodium gallinaceum, and immunoblotted a 50 kDa protein in extracts of male gametes from both species. This 50 kDa antigen was localized by immunoelectron microscopy to axonemes of male gametes in a pattern similar to that obtained with anti-alpha- and anti-beta-tubulin antibodies. Furthermore, mAb 5E7 specifically reacted with recombinant alpha-tubulin II protein obtained using the PCR-amplified alpha-tubulin II gene from a gametocyte-specific cDNA library. The sex-specific expression of alpha-tubulin II and its localization to axoneme of the male parasite suggest a role for this molecule in the morphologic changes that occur during exflagellation and in the motility of the parasite. alpha-Tubulin II and mAb 5E7 may prove useful tools in studies of the biology of sexual stage differentiation and development in P. falciparum in addition to the general understanding of post-translational modifications of tubulin isoforms.     

Monoclonal anti-gametocyte antibodies identify an antigen present in all blood stages of Plasmodium falciparum

Two polypeptides of 150 and 130 kDa present in all asexual and sexual blood stages of Plasmodium falciparum have been identified with anti-gametocyte monoclonal antibodies. The apparent molecular mass of these antigens is identical in different developmental stages of the parasite and in different isolates. These antigens are released in the culture supernatant during the process of schizogony and are also detected in the sera of patients undergoing a primary P. falciparum infection. Antibodies against these antigens occur in sera of a large percentage of children and most adults living in malaria-endemic areas, suggesting that they are highly immunogenic. The anti-gametocyte monoclonal antibodies react with a synthetic peptide (Glu-Glu-Asn-Val)4, present in antigen Pf155 [Perlmann, H. et al. (1984) J. Exp. Med. 159, 1686-1704] and in the ring-infected erythrocyte surface antigen [Coppel, R.L. et al. (1984) Nature 310, 789-792], indicating that these polypeptides are closely related. In contrast, two glycophorin-binding proteins of similar molecular mass [Perkins, M.E. (1984) J. Exp. Med. 160, 788-798] appear to be entirely distinct from the presently described antigens. We failed to observe any in vitro inhibitory activity of the monoclonal antibodies on merozoite invasion and on gametocyte infectivity.     

Antigenic differences among isolates of Plasmodium falciparum demonstrated by monoclonal antibodies.

Hybridomas raised against two Papua New Guinea (PNG) isolates of Plasmodium falciparum secreted monoclonal antibodies which bound to schizonts of all seven PNG isolates tested but not to schizonts of four non-PNG isolates from Thailand, Nigeria, Ghana, and The Netherlands. Some of the monoclonal antibodies were tested for their ability to inhibit the growth of one PNG isolate, one Thai isolate, and one Nigerian isolate in vitro. Only the growth of the PNG isolate was inhibited, thus demonstrating functional antigenic differences among isolates of P. falciparum.     

Planned immunization of human volunteers with HLA-DR incompatible lymphocytes

Aiming at the production of anti HLA-DR test sera, eight healthy human volunteers were immunized by repeated intradermal injections of lymphocytes which were selected to be incompatible for one HLA-DR antigen, and matched as well as possible for HLA-A,-B,-C antigens. One out of 3 recipients immunized exclusively against HLA-DR produced lyrnphocytotoxic HLA-DR antibodies. The remaining 5 recipients were immunized against 1 or more HLA-A,-B,-C antigens in addition to one HLA-DR antigen. After 3 immunizations, 3 of these reacted with strong HLA-A or -B antibody production; however, only one showed a parallel anti HLA-DR antibody response detectable by complement dependent lymphocytotoxicity.
Testing of the recipient sera in the antibody dependent cell-mediated cytotoxicity (ADCC) assay revealed that 6 of the 8 recipients did react early to the immunizations with HLA specific antibody production. However, in spite of repeated booster injections it was not possible to obtain more than the above-mentioned 2 sera with HLA-DR antibodies strong enough to react in the lymphocytotoxicity microtechique.     

Transmission blockade of Plasmodium falciparum malaria by anti-Pfs230-specific antibodies is isotype dependent.

By use of the parental hybridoma cell line 63F2A2 that produces specific antibodies of immunoglobulin isotype G1 (IgG1; 63F2A2.1) against Pfs230, we attempted to enrich for the synthesis of the downstream switch variant IgG2b and IgG2a monoclonal antibodies (MAbs) of the hybridoma cell line (63F2A2.2b and 63F2A2.2a, respectively). The parental IgG1 did not reduce the Plasmodium falciparum transmission in a bioassay irrespective of the presence of complement. MAbs 63F2A2.2b and 63F2A2.2a were effective in reducing the infectivity of P. falciparum parasites to Anopheles gambiae mosquitoes in membrane-feeding experiments. A transmission reduction of 91% was accomplished by the 63F2A2.2b switch variant, and a reduction of greater than 99% was accomplished by the 63F2A2.2a switch variant, but only in the presence of active human complement. Subsequently, the transmission-reducing effect of MAb 63F2A2.2b or 63F2A2.2a was confirmed in vitro by the rapid lysis of newly formed macrogametes or zygotes in the presence of active complement. MAb 63F2A2.1 did not lyse the newly formed macrogametes or zygotes irrespective of the presence of complement.     

Antigenic and genetic variations of the 15 kD nucleocapsid protein of porcine reproductive and respiratory syndrome virus isolates

Summary.  The antigenic variability of the 15 kD nucleocapsid protein of porcine reproductive and respiratory syndrome (PRRS) virus was characterized with a panel of 24 monoclonal antibodies (MAbs) raised against the American PRRS virus isolate ISU-P. Five continuous epitopes designated EpORF7-A through E were revealed by the reactivity pattern of these MAbs with 67 American field isolates, two modified-live vaccine viruses, and the European Lelystad virus as determined by the indirect immnofluorescence assay and Western immunoblotting and confirmed by additivity and blocking enzyme-linked immunosorbent assays. The reactivity pattern of isolates in the IFA permitted their subdivision into 4 American antigenic groups which represented 84.1, 11.6, 2.9 and 1.4% of viruses tested. The antigenic variation among isolates was correlated to single, group specific nucleotide substitutions and mediated by a combination of at least 4 of the 5 epitopes. EpORF7-A was conserved in all American isolates and the Lelystad virus which constituted a separate antigenic group. Consequently, monoclonal antibodies specific for EpORF7-A may prove useful as the antigenic basis for a universal diagnostic test for the PRRS virus. EpORF7-C, D and E were only present in the American isolates tested. Received April 6, 1998 Accepted September 22, 1998     

Identification of p53 peptides recognized by CD8(+) T lymphocytes from patients with bladder cancer

In many types of cancer, p53 frequently accumulates in tumor cells and anti-p53 antibodies can be detected. However, only four CD8(+) T-cell epitopes from p53 have been identified in humans so far. To further analyze the development of a T-cell response against p53, peptides having binding motifs specific for HLA-A1, -A2, -A3, -A24, -B7, -B35, -B44, and -B51 molecules have been defined. The HLA-binding capacity of those peptides was tested, and the stability of formed complexes was defined. Thirteen peptides that bound to HLA-A24 and -B44 molecules are presented. The positive peptides were then used to detect the anti-p53 response of CD8(+) T lymphocytes from patients with bladder cancer. Six peptides, presented by HLA-A2, -B51, or -A24, were able to stimulate T cells from two patients (among 16) with tumor cells that strongly accumulated p53. On the contrary, p53 peptides systematically failed to stimulate T cells from healthy donors or patients with low or undetectable levels of p53 in their tumor cells. These results have led to the identification of four new potential T CD8(+) epitopes from p53: 194-203 associating with HLA-B51 and 204-212, 211-218, and 235-243 associating with HLA-A24.     

Enhancement or inhibition of Plasmodium falciparum erythrocyte reinvasion in vitro by antibodies to an asparagine rich protein

A clone encoding a recombinant protein which reacted strongly with human antibodies from a donor clinically immune to malaria, was isolated from a genomic Plasmodium falciparum library. Mice injected with this protein, designated 10b, produced antibodies which reacted with all developmental stages of erythrocytic asexual parasites in indirect immunofluorescence. In immunoblotting, the same antibodies recognized two P. falciparum polypeptides of 36 kDa and 33 kDa. Of three monoclonal antibodies raised against the 10b recombinant protein, two inhibited parasite reinvasion of erythrocytes in an isolate specific manner. Surprisingly, however, the third was found to significantly enhance reinvasion of erythrocytes and also to induce a more rapid maturation of intraerythrocytic parasites in all isolates tested. Nucleotide sequence analysis of the 1124 bp insert revealed that it encodes a protein which consists of 30% asparagine and contains three asparagine rich, imperfect tandem repeats: Lys-Lys-Asn-Asn (3x), Met-Asn-His/Gln-Pro-Asn-Asn (14x), and Lys-Asn-Asn-Asn-Asn (7x).     

采用静态培养装置培养恶性疟原虫配子体的研究

应用自制恒温恒气装置静态培养恶性疟原虫配子体。培养第１４ｄ，４个恶性疟原虫分离株Ｂ－１，ＦＣＣ－９０１／ＹＮ，ＦＣＣ－９０２／ＹＮ，ＦＣＣ－９０３／ＹＮ的配子体率为０。４６～１。５４％，对照组为０。４４～１．３８％；Ｖ期配子体率为５－２０％，对照组为０－４％；Ｖ期配子体雄雌比例为１：４．００～１：９．００。结果表明，实验组与对照组的配子体绝对数没有明显差别，但前者的成熟配子体数明显多于后者，说明该培养方法更有利于配子体的成熟。４株恶性疟原虫配子体经人工膜饲喂按蚊，均未见卵囊形成。对ＦＣＣ－９０３／ＹＮ株配子生殖过程的观察表明，其Ｖ期配子体可以发育成雌雄配子，在蚊胃血涂片中观察到极少量合子和动合子，说明上述配子体功能上已经成熟，但没有发现卵囊形成。本文对其原因进行了讨论。     

Monoclonal antibodies directed against conserved epitopes on the nucleocapsid protein and the major envelope glycoprotein of equine arteritis virus

We recently developed a highly effective immunization procedure for the generation of monoclonal antibodies (MAbs) directed against the porcine reproductive and respiratory syndrome virus (E. Weiland, M. Wieczorek-Krohmer, D. Kohl, K. K. Conzelmann, and F. Weiland, Vet. Microbiol. 66:171-186, 1999). The same method was used to produce a panel of 16 MAbs specific for the equine arteritis virus (EAV). Ten MAbs were directed against the EAV nucleocapsid (N) protein, and five MAbs recognized the major viral envelope glycoprotein (G(L)). Two of the EAV G(L)-specific MAbs and one antibody of unknown specificity neutralized virus infectivity. A comparison of the reactivities of the MAbs with 1 U.S. and 22 newly obtained European field isolates of EAV demonstrated that all N-specific MAbs, the three nonneutralizing anti-G(L) MAbs, and the weakest neutralizing MAb (MAb E7/d15-c9) recognized conserved epitopes. In contrast, the two MAbs with the highest neutralization titers bound to 17 of 23 (MAb E6/A3) and 10 of 23 (MAb E7/d15-c1) of the field isolates. Ten of the virus isolates reacted with only one of these two MAbs, indicating that they recognized different epitopes. The G(L)-specific MAbs and the strongly neutralizing MAb of unknown specificity (MAb E6/A3) were used for the selection of neutralization-resistant (NR) virus variants. The observation that the E6/A3-specific NR virus variants were neutralized by MAb E7/d15-c1 and that MAb E6/A3 blocked the infectivity of the E7/d15-c1-specific NR escape mutant confirmed that these antibodies reacted with distinct antigenic sites. Immunoelectron microscopy revealed for the first time that the antigenic determinants recognized by the anti-G(L) MAbs were localized on the virion surface. Surprisingly, although the immunofluorescence signal obtained with the neutralizing antibodies was relatively weak, they mediated binding of about three times as much gold granules to the viral envelope than the nonneutralizing anti-G(L) MAbs.