Vaccination-induced variation in the 140 kD merozoite surface antigen of Plasmodium knowlesi malaria.

Immunity to 143/140 kD schizont antigens of a monkey malaria, Plasmodium knowlesi, provides partial protection to lethal malaria infection in rhesus monkeys challenged with uncloned parasites. To determine the capacity of a cloned parasite to generate variants of the 143/140 kD antigens, immunized monkeys were challenged with a clone of P. knowlesi. Parasites recovered 8 d after inoculation with a cloned parasite retained the 143/140 kD antigens. Parasites recovered 30 d after challenge had undergone changes in the 143/140 kD antigens. Antibodies that block erythrocyte invasion in vitro of the inoculum parasites did not inhibit invasion of erythrocytes by two isolates recovered from the immunized monkeys. An isolate from one monkey recovered on day 30 contained clones expressing new 76/72 kD antigens reactive with rabbit antiserum against the 143/140 kD proteins, and other clones expressing no antigens crossreactive with antisera against the 143/140 kD proteins. An isolate from another monkey obtained 59 d after challenge expressed new antigens of 160/155, 115/113, and 87/85 kD. Using monoclonal antibodies, we found that epitopes were lost from the variant proteins, but we were unable to determine whether new epitopes had appeared. We conclude that clones of P. knowlesi can rapidly vary antigenic determinants on the 143/140 kD proteins in animals immunized with these antigens.

Multiple pathways of T-T interaction in the generation of cytotoxic T lymphocytes to alloantigens.

The interaction of T helper (Th) cells with syngeneic and allogeneic cytotoxic T lymphocyte precursors (CTL.P) has been investigated. Unprimed and mixed lymphocyte culture-primed peripheral T cells were used as a source of Th. Thymocytes, which depend upon exogenous Th cells for activation, were used as a source of cytotoxic precursors. Data is presented that demonstrates that at least two pathways of T-T interaction can lead to the activation of cytotoxic lymphocytes. The first is an allogeneic effect, in which Th cells recognize and respond to alloantigens expressed on CTL.P. The second is the interaction of Th cells with syngeneic CTL.P, in which both cell types are thought to respond to alloantigens on stimulator cells. The latter interaction can be shown to be restricted by H-2-linked determinants when primed Th cells are used and allogeneic effects against thymocytes are minimized. Restricted interactions between unprimed Th cells and thymocyte CTL.P have never been observed. Mechanisms that may explain the difference between the interaction of unprimed and primed Th cells with CTL.P are discussed.

Receptor-like specificity of a Plasmodium knowlesi malarial protein that binds to Duffy antigen ligands on erythrocytes.

A 135-kD parasite protein, a minor component of the Plasmodium knowlesi malaria radiolabeled proteins released into culture supernatant at the time of merozoite release and reinvasion, specifically bound to human erythrocytes that are invaded and carry a Duffy blood group determinant (Fya or Fyb), but did not bind to human erythrocytes that are not invaded and do not carry a Duffy determinant (FyFy). Specific anti-Duffy antibodies blocked the binding of the 135-kD protein to erythrocytes carrying that specific Duffy determinant. Purified 135-kD protein bound specifically to the 35-45-kD Duffy glycoprotein on a blot of electrophoretically separated membrane proteins from Fya and Fyb erythrocytes but not from FyFy erythrocytes. Binding of the 135-kD protein was consistently greater to Fyb than to Fya both on the blot and on intact erythrocytes. The 135-kD protein also bound to rhesus erythrocytes that are Fyb and are invaded, but not to rabbit or guinea pig erythrocytes that are Duffy-negative and are not invaded. Cleavage of the Duffy determinant by pretreating Fyb human erythrocytes with chymotrypsin greatly reduced both invasion and binding of the 135-kD protein, whereas pretreating Fyb erythrocytes with trypsin had little effect on the Duffy antigen, the 135-kD protein binding, or on invasion. However, instances of invasion of other enzyme-treated erythrocytes that are Duffy-negative and do not bind the 135-kD protein suggest that alternative pathways for invasion do exist.

Resistance of Melanesian elliptocytes (ovalocytes) to invasion by Plasmodium knowlesi and Plasmodium falciparum malaria parasites in vitro.

Erythrocytes from humans with Melanesian elliptocytosis are resistant to invasion by Plasmodium falciparum in vitro and epidemiological evidence suggests they may be resistant to P. vivax and P. malariae. We have examined the ability of P. knowlesi merozoites to invade Melanesian elliptocytes in vitro as a definitive means of examining these cells for resistance to invasion by malarial species with different receptor requirements. The Melanesian elliptocytes were highly resistant to invasion by P. knowlesi merozoites showing that the resistance associated with this erythrocyte variant lies at a level common to the invasion pathway(s) of P. falciparum and P. knowlesi. This makes Melanesian elliptocytosis unique as no other human erythrocyte variant has been shown to be resistant to invasion by both species.

Molecular basis for mutation in a surface protein expressed by malaria parasites.

Plasmodium knowlesi parasites isolated from a rhesus monkey vaccinated with a 143,000/140,000 Mr merozoite surface protein no longer expressed this protein. To study the molecular basis for the mutations, a lambda gt11 cDNA expression library constructed from the original parasite clone was screened with rabbit antiserum specific for the 143,000/140,000 Mr protein. Two cDNA clones that mapped to the 5' and 3' ends of the gene hybridized to two chromosomes of 3.6 x 10(6) kilobases and 1.8 x 10(6) kilobases. The gene on the 3.6 x 10(6) base chromosome was identified as the gene expressing the 143,000/140,000 Mr protein. Since the two cDNA clones also hybridized at high stringency with the 1.8 x 10(6) base chromosome, it appears that the 143,000/140,000 Mr gene was involved in an ancestral duplication and interchromosomal transposition. We have analyzed mutant parasites, using the cDNA clones and a 7000 base fragment of genomic DNA that contains the 143,000/140,000 Mr gene. In one type of mutation, the 143,000/140,000 Mr protein was replaced by a 76,000/72,000 Mr protein. The identical restriction sites and the identical size of the mRNA indicated that a point mutation resulted in premature interruption of translation. Sequence analysis revealed an AT substitution for a C in the middle of the coding region of the gene that created a frameshift and a stop codon. In a second type of mutation, no protein was expressed; a 4000 base deletion encompassed the transcriptional unit of the gene. The rapid mutation under vaccine pressure of an otherwise stable parasite protein emphasizes the need to identify vaccine candidates in which mutations would be lethal.

The role of H2O2 and the lactoperoxidase-SCN(-)-H2O2 system on the interaction between two bacteria originating from human dental plaque, Streptococcus rattus (mutans) BHT and Streptococcus mitior LPA-1, grown on human teeth in an artificial mouth.

Teeth were inoculated with either the organisms separately or with a freshly-prepared mixture of both. The apparatus was swept with 5 per cent (v/v) CO2 in either air or N2, and incubated for 90 h. A nutrient supplement containing 1 per cent (w/v) glucose was supplied for 1 h in every 6 h. Both organisms achieved similar numbers when grown aerobically in pure culture, yet in mixed culture there was pronounced inhibition of BHT (p less than 0.001). When the synthetic saliva was supplemented with catalase the strain BHT count in mixed culture was much higher (p less than 0.001). It was concluded, therefore, that the strain LPA-1 produced inhibitory levels of hydrogen peroxide (H2O2) on the tooth surface under aerobic conditions. This was supported by finding that a lower viable count of LPA-1 in pure culture was attained when lactoperoxidase (LPO) was included in the saliva (p less than 0.005), as all components of the LPO-SCN-H2O2 system were presumably present. With the N2-CO2 mixture, conditions were not strictly anaerobic and both catalase and LPO increased all viable counts. Under these conditions, therefore, when H2O2 was limiting, LPO protected bacteria against its bactericidal effect.

A laboratory microcosm (artificial mouth) for the culture and continuous pH measurement of oral bacteria on surfaces.

A laboratory microcosm has been designed for the cultivation of bacteria on surfaces subjected to an adjustable supply of fluids. Bacteria are grown as a microbial film on halved premolar teeth, mounted back to back. Synthetic saliva is dropped slowly over the teeth throughout experiments. A nutrient supplement is provided at regular intervals. The drops of fluid retained by the teeth can be sampled for metabolic end-products. Alternatively, a miniature glass electrode may be set into one half of a tooth assembly to monitor the pH continuously at the stagnation site between tooth segments. Up to six replicate culture flasks and six electrodes can be accommodated in a single experiment. Satisfactory electrode performance was maintained during 66 h experiments. In initial 48 h experiments, teeth were inoculated with Streptococcus rattus BHT or 'Streptococcus mitior' LPA-1 in pure culture and provided with 1% (w/v) glucose for 1 h every 6 h. Bacteria produced typical responses to glucose feeds leading to the formation of 'Stephan'-like curves of pH-fall. Under these conditions, 'Strep. mitior' was more acidogenic than Strep. rattus and the pattern of acid production was distinct for each organism.

The -55 C/T polymorphism within the UCP3 gene and performance during the South African Ironman Triathlon.

Uncoupling protein 3 is believed to be involved in total body energy expenditure, including the regulation of fat and glucose metabolism. These biochemical processes may distinguish top ultra-endurance triathletes from slower competitors. The aim of this study was to determine whether the uncoupling protein 3 gene is associated with the performance capacity of ultra-endurance Ironman triathletes. Two triathlete groups consisting of the 89 fastest and 89 slowest Caucasian, male triathletes who completed either the 2000 or 2001 South African Ironman triathlon events were genotyped for the -55 C/T polymorphism within the uncoupling protein 3 gene. A control group consisting of 92 Caucasian males who had not trained for or participated in an ultra-endurance athletic event was also genotyped. There was no significant difference in the genotype (CC, CT and TT) frequency distribution of the -55 C/T polymorphism within the uncoupling protein 3 gene between the fast triathlete, slow triathlete and control groups. In addition, no significant differences were observed between the frequencies of the C and T alleles between the three groups. The two triathlete groups were combined and grouped according to their genotype. No particular genotype or allele was associated with the time taken by the triathletes to complete the entire triathlon, or either the swim, cycle or run legs of the event. Thus no association was found between the -55 C/T polymorphism within the uncoupling protein 3 gene and the ultra-endurance performance of triathletes who completed either the 2000 or 2001 South African Ironman triathlons.

The malaria merozoite surface: a 140,000 m.w. protein antigenically unrelated to other surface components on Plasmodium knowlesi merozoites.

We previously identified three proteins on the surface of merozoites (140,000, 105,000 and 75,000 m.w.). To determine if 140,000 m.w. protein was related to other surface proteins, we immunized mice with liposomes containing merozoite proteins from the 140,000 m.w. region of the polyacrylamide gel. The immune sera reacted with the surface of viable merozoites and acetone-fixed schizonts by immunofluorescence. The sera immunoprecipitated only the 140,000 m.w. protein from surface-labeled merozoites. We demonstrated that monoclonal antibody 13C11 immunoprecipitated a 250,000 m.w. protein from metabolically labeled schizonts and bound to the merozoite surface. This monoclonal antibody immunoprecipitated the 75,000 and lower m.w. proteins from surface-labeled merozoites but did not bring down the 140,000 m.w. protein. Because the mouse immune sera did not immunoprecipitate the 250,000 m.w. protein from metabolically labeled schizonts or proteins other than the 140,000 m.w. protein from surface-labeled merozoites, we conclude that the 140,000 m.w. protein is unrelated to other merozoite surface antigens identified to date. The mouse immune sera against the 140,000 m.w. protein on the merozoite surface did not immunoprecipitate a 140,000 m.w. protein from metabolically labeled schizonts. Instead, the major protein immunoprecipitated had a m.w. of 144,000. By analogy to the 250,000 m.w. protein and its cleavage products, we propose that the 140,000 m.w. protein on the merozoite surface is a cleavage product of the higher m.w. protein.

Monoclonal antibodies to a 140,000-m.w. protein on Plasmodium knowlesi merozoites inhibit their invasion of rhesus erythrocytes.

Merozoites are the invasive stage of the malaria parasite, which are released from infected erythrocytes to invade other erythrocytes. Antibody to surface antigens on merozoites may prevent invasion by agglutinating merozoites as they are released from infected erythrocytes or by blocking receptors before contact of merozoites with the host erythrocyte. Monoclonal antibodies were produced to a 140,000-m.w. protein on the merozoite surface. The protein was synthesized by the mature intraerythrocytic parasite, the schizont, as a 143,000-m.w. protein and had a m.w. of 140,000 on the surface of free merozoites. The monoclonal antibodies were shown to bind to the surface of merozoites by immune electron microscopy. Ascitic fluid containing four of 11 anti-140,000 monoclonal antibodies partially blocked invasion of erythrocytes by merozoites released from schizont-infected cells. The low invasion rate was always associated with a high frequency of multiply infected erythrocytes (two or more rings per erythrocyte). Monoclonal antibodies purified by (NH4)2SO4 precipitation and diethylaminoethyl column fractionation also blocked invasion and caused multiple invasion of individual erythrocytes. The monoclonal antibodies, incubated with free merozoites, did not block invasion, indicating that the antibodies did not bind to merozoite receptors for erythrocytes. We propose that the reduced rate of invasion and the multiple invasion of erythrocytes, the characteristic of these monoclonal antibodies, was caused by weak agglutination of merozoites as they were released from infected erythrocytes.

Immunization of monkeys with a 140 kilodalton merozoite surface protein of Plasmodium knowlesi malaria: appearance of alternate forms of this protein.

The merozoite is the invasive stage of the malaria parasite which is released by rupture of the schizont-infected erythrocyte. A monoclonal antibody against a 140 kilodalton (kDa) merozoite surface antigen of Plasmodium knowlesi was used to characterize and to purify this antigen. It was shown by pulse-chase metabolic labeling of mature schizonts that the 140 kDa merozoite antigen was the processed product of a 143 kDa schizont component, and that processing occurred at the time of erythrocyte rupture. Antiserum, prepared by immunizing a rabbit with the 143/140 kDa antigen purified by immunoaffinity chromatography with the monoclonal antibody, strongly inhibited invasion of erythrocytes in vitro; Fab fragments prepared from purified rabbit IgG were inactive at blocking invasion, suggesting that agglutination of merozoites was the mechanism of invasion inhibition. The purified 143/140 kDa antigen was used in Freund's adjuvant to immunize four rhesus monkeys. Two of the immunized animals developed fulminating infections on challenge with 10(4) schizonts, as did the three control animals. The remaining two immunized animals controlled their infections and developed chronic low-grade parasitemias. The animals which were partially protected were those that had developed anti-143/140 kDa antibodies capable of blocking invasion in vitro. Parasites were isolated from the chronic stage of infection (V5 population) and were compared with the original parasite population used for challenge (P population). Inhibition of invasion, immunofluorescence, and immunoprecipitation with anti-143/140 kDa monoclonal antibody, with immune rabbit, and with monkey sera showed that the 143/140 kDa surface antigen had been replaced by multiple cross-reacting alternate antigenic forms of the molecule in the V population. Thus, specific immune response directed against a purified merozoite surface antigen resulted in the replacement of this antigen by variant or mutant forms.

Effect of inoculation sequence and nutrients upon Streptococcus mutans BHT and Streptococcus mitior LPA-1 growing on human teeth in an artificial mouth.

Human teeth in an artificial mouth were inoculated with Streptococcus mutans BHT, Streptococcus mitior LPA-1, or sequentially with both organisms. Incubation was continued for 90 h. Mixed populations were largest when a nutrient supplement containing 5.0% (w/v) sucrose was supplied. Fewer organisms were recovered from experiments with synthetic saliva only, or when a supplement containing 0.05% (w/v) glucose was available. The inoculation sequence determined the total viable count and a larger population resulted when Strep. mutans was the initial colonizer (P less than 0.01). Strep. mutans was always able to become established even when super-infected on to a 24 h plaque of Strep. mitior. The final proportion of Strep mutans was lower when it was the superinfecting organism and the sucrose (P less than 0.01) or glucose (P less than 0.05) nutrient supplement was provided. This work confirms the importance of inoculation sequence and presence of sugars in plaque accumulation and demonstrates the fundamental role of microbial interactions in this process.

The Duffy receptor family of Plasmodium knowlesi is located within the micronemes of invasive malaria merozoites.

Plasmodium vivax and Plasmodium knowlesi merozoites invade human erythrocytes that express Duffy blood group surface determinants. A soluble parasite protein of 135 kd binds specifically to a human Duffy antigen. Using antisera affinity purified on the 135 kd protein, we cloned a gene that encodes a member of a P. knowlesi family of erythrocyte binding proteins. The gene is a member of a family that includes three homologous genes located on separate chromosomes. Two genes are expressed as major membrane-bound products that give rise to soluble erythrocyte binding proteins: the 135 kd Duffy binding protein and a 138 kd protein that binds only rhesus erythrocytes. These different erythrocyte binding specificities may result from sequence divergence of the homologous genes. The Duffy receptor family is localized in micronemes, an organelle found in all organisms of the phylum Apicomplexa.

A merozoite receptor protein from Plasmodium knowlesi is highly conserved and distributed throughout Plasmodium.

The 66-kDa merozoite surface antigen (PK66) of Plasmodium knowlesi, a simian malaria, possesses vaccine-related properties that are thought to originate from a receptor-like role in parasite invasion of erythrocytes. We report the complete sequence of PK66 which allowed the demonstration that highly conserved analogues exist throughout Plasmodium including a recently reported gene from P. falciparum (Peterson, M. G., Marshall, V. M., Smythe, J. A., Crewther, P. E., Lew, A., Silva, A., Anders, R. F., and Kemp, D. J. (1989) Mol. Cell. Biol. 9, 3151-3155). These analogues are highly promising vaccination candidates. The distribution of PK66 changes after schizont rupture in a coordinate manner associated with merozoite invasion. The protein is concentrated at the apical end prior to rupture, following which it can distribute itself entirely across the surface of the free merozoite. During invasion, immunofluorescence studies suggest that, PK66 is excluded from the erythrocyte at, and behind, the invasion interface.