isolation and immunological characterization of fatty acid ... - BioOne

J. Parasitol., 87(5), 2001, pp. 1028–1033 q American Society of Parasitologists 2001

ISOLATION AND IMMUNOLOGICAL CHARACTERIZATION OF FATTY ACID BINDING PROTEIN ISOFORMS FROM FASCIOLA HEPATICA Ana M. Espino, Jose´ R. Rodrı´guez Medina*, and George V. Hillyer†‡ Department of Pathology and Laboratory Medicine, University of Puerto Rico, School of Medicine, Suite 617-A, P.O. Box 365067, San Juan, PR 00936 ABSTRACT: A combination of molecular sieving chromatography and 2-step preparative isoelectric focusing showed that native Fh12, a fatty acid–binding protein isolated from Fasciola hepatica adult worms, is a protein complex of at least 8 isoforms with identical molecular mass but different isoelectric points. Using enzyme-linked immunosorbent assay (ELISA) and inhibition ELISA assays, immunological differences were observed between native (nFh12) and a recombinant molecule denoted rFh15 that was obtained after screening a cDNA library from F. hepatica adult worms with an anti-Fh12 monospecific polyclonal antibody. It was confirmed that in infected rabbits, antibodies to nFh12 appear by the second week postinfection, whereas antibodies to rFh15 appear much later, by 6 wk postinfection. Four acidic forms (Fh121–4) showed more immunological identity with rFh15 than with nFh12, based on the observation that they inhibited ELISA activity by nearly 50% when they were added to the anti-rFh15 polyclonal antibody at 20 mg/ml of protein concentration. Moreover, the Fh121–4 isoforms were poorly reactive with sera from rabbits 2–4 wk postinfection. However, the 2 acidic forms, denoted Fh125 and Fh126, and the neutral/basic forms, denoted Fh127 and Fh128, showed more immunological identity with the native nFh12 molecule than with the recombinant rFh15 because they were highly reactive with sera of rabbits with early 2-wk F. hepatica infection and inhibited ELISA activity nearly 50% when they were quantitatively added to the anti-nFh12 polyclonal antibody. These results suggest that rFh15 could be one of the acidic forms of nFh12, and that it, in fact, may be one of the less immunogenic or immunoprotective members, or both, of the nFh12 protein complex.

Studies have shown that several homologous purified native and recombinant molecules have the potential of immunoprophylaxis to challenge infection with Fasciola hepatica (Hillyer, 1995; Spithill and Dalton, 1998). One of the native molecules is a fatty acid–binding protein, denoted nFh12, purified from F. hepatica adult worm extract (Hillyer, Soler de Galanes et al., 1988). This molecule appears to be expressed early after transformation of the metacercariae to the juvenile stage, because different animal species, including mice, calves, and rabbits, with fascioliasis develop antibodies to nFh12 within 2 wk of infection (Hillyer, Garcıá Rosa et al., 1988). This molecule has also been shown to be a cross-reactive antigen against Schistoma mansoni because mice infected with S. mansoni develop antibodies to nFh12 by 5 wk postinfection (Hillyer, Soler de Galanes et al., 1988). Another such molecule is a recombinant protein denoted rFh15, which was obtained after screening a cDNA library from F. hepatica adult worms with an anti-Fh12 monospecific polyclonal antibody (Rodrı´guez Pe´rez et al., 1992). This molecule is also a cross-protective antigen against S. mansoni (Hillyer, 1987; Hillyer et al.; Hillyer, Garcıá Rosa et al., 1988). Several immunoprophylaxis studies have repeatedly shown that the purified native molecule (nFh12) always induces higher levels of protection against F. hepatica than does the recombinant (rFh15) one (Muro et al., 1997, Lo´pez Aban et al., 1999). This suggests that the 2 proteins may be slightly different. The current study concerns the purification of at least 8 isoforms of the nFh12 protein using a combination of molecular sieve chromatography and isoelectric focusing. Competitive enzyme-linked immunosorbent assay (ELISA) studies using monospecific polyclonal antisera against the naReceived 30 March 2000; revised 20 November 2000; accepted 12 March 2001. * Department of Biochemistry, University of Puerto Rico, School of Medicine, Suite 617-A, P.O. Box 365067, San Juan, Puerto Rico 00936. † Department of Pathology and Laboratory Medicine and Department of Biochemistry, University of Puerto Rico, School of Medicine, Suite 617-A, P.O. Box 365067, San Juan, Puerto Rico 00936. ‡ To whom correspondence should be addressed.

tive and recombinant proteins were performed to investigate the immunological relatedness between the rFh15 and nFh12 molecules. The results demonstrate that nFh12 is a protein complex with at least 8 isoforms and that rFh15 could be one of the acidic forms of this protein complex. MATERIALS AND METHODS Animal infection and serum sample collection Ten New Zealand White rabbits were infected orally with 60 F. hepatica metacercariae each. Blood was obtained for the collection of serum from each rabbit before infection and then at weekly intervals through 12 wk postinfection. Sera were stored at 220 C until used. Rabbit anti-rFh12 and anti-rFh15 antisera A New Zealand White rabbit was immunized with the nFh12 antigen purified by a combination of molecular sieving chromatography on sephadex G-50 and ion exchange chromatography using DEAE-sephadex A-120, as previously described (Hillyer, Soler de Galanes et al., 1988). The nFh12 antigen (100 mg) was emulsified in Freund’s complete adjuvant and injected subcutaneously on the dorsal surface of a rabbit. After 2 wk, 100 mg of nFh12 antigen emulsified in Freund’s incomplete adjuvant was injected subcutaneously, followed by 2 similar injections at weekly intervals. Blood was collected 1 wk after the last injection, and the serum was stored at 220 C until used. The recombinant molecule (rFh15) was obtained as previously reported (Rodrı´guez-Pe´rez et al., 1992). A rabbit antiserum against rFh15 was obtained following a similar protocol described above.

Fasciola hepatica whole worm extract preparation Fasciola hepatica adult worms were obtained from the bile ducts of cattle at a local abattoir. The flukes were repeatedly washed with 0.01 M phosphate-buffered saline (PBS), pH 7.2, supplemented with 5 mM ethylenediaminetetraacetic acid (EDTA), 8 mM phenylmethylsulfonyl fluoride (PMSF), 2 mM iodoacetamide, and 2 mM leupeptin to inhibit proteases. Fasciola hepatica whole-worm extract (FhWWE) was obtained after homogenization of adult worms on ice in PBS using a Ten Broeck tissue grinder followed by centrifugation at 30,000 g for 30 min. The protein concentration was determined using the bicinchoninic acid method (Smith et al., 1985). Between 30 and 50 mg FhWWE was analyzed with the use of 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli (1970) using the Mini Protean II System (Bio Rad, Hercules, California). The samples were electrophoresed at a constant current of 25 mM/ 77 mm gel. A mixture of a broad range of proteins (Bio Rad) was used

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as molecular weight markers. Proteins were visualized with silver stain using a kit from Bio-Rad. Molecular sieving chromatography Approximately 100 mg FhWWE in 10 ml PBS was chromatographed through a 2.5- by 90-cm Sephadex G-50 column equilibrated with PBS supplemented with 8 mM PMSF, 5 mM EDTA, 2 mM iodoacetamide, and 2 mM leupeptin. Fractions were eluted at a flow rate of 3 cm/hr and then analyzed by SDS-PAGE. Samples containing the 12-kDa polypeptide were pooled, desalted against 1% glycine using prepacked sephadex G-25 columns (PD-10 columns, Amersham-Pharmacia, Newark, New Jersey) and concentrated down to 50 ml using a YM-3 ultrafiltration membrane (Millipore, Bedford, Massachusetts). Preparative isoelectric focusing The concentrated fraction was mixed with 2.5 ml of Bio-Rad biolyte, an ampholyte mixture with a pH range between 3 and 10. The sample was then injected into the focusing chamber of a Rotofor Cell (BioRad). The anion and cation exchange membranes of the apparatus were previously equilibrated in 0.1 M NaOH and 0.1 M H3PO4 solutions, respectively. Isoelectric focusing (IEF) was performed over 4 hr at 4 C with the power supply set on constant (12W), a starting voltage of 500 V, and a current of 25 mA. During the run, the diminution of current was monitored. Once 2 consecutive, equal milliamp periods were obtained, the run was stopped and samples were aspirated from the cell by applying a vacuum to the collection box. The pH and absorbance at 280 nm from each sample collected was measured, and the presence of the 12-kDa antigen was determined by SDS-PAGE. Samples containing the 12-kDa antigen were pooled and dialyzed, first against 10 mM TrisHCL, pH 7.5, and afterwards against 1% glycine buffer. The antigen was then refractionated by IEF as above, but using an ampholyte mixture with pH 4–7. When the second isoelectric focusing run was finished, samples were collected as above, and the pH and absorbance at 280 nm of each sample was measured. Samples were analyzed by SDSPAGE. Those samples in which the 12-kDa polypeptide appears to be free of contaminants were selected and analyzed by western blot. Western blot analysis Samples selected from the second run of IEF were analyzed on 15% SDS-PAGE as above, but using a prestained molecular weight marker (Bio Rad) of broad range pH (3–10). Afterwards, they were transferred to a 0.45-mm nitrocellulose membrane (Bio Rad) by the method of Towbin et al. (1979). The nitrocellulose (NC) membrane was first blocked with 5% nonfat dry milk in PBS containing 0.05% Tween 20 (PBST) then incubated 2 hr at room temperature with the anti-Fh12 antiserum diluted 1:400 in PBST. After 3 washes with PBST, NC membrane was incubated 2 hr at room temperature with a goat anti-rabbit IgG peroxidase conjugate (Bio Rad) diluted 1:5,000 in PBST milk. After another wash step, the NC membrane was incubated at room temperature with the substrate solution (50 mg diaminobenzidine 1 100 ml H2O2, 30% [wt/v], 1 100 ml PBS) until bands were visible. The reaction was stopped by soaking the NC membrane in distilled water. Indirect enzyme immunoassay (ELISA) The ELISA was performed in disposable polystyrene plates (Costar, Corning, New York), which were coated overnight at 4 C with rFh15 or nFh12 molecules diluted in 0.05 M carbonate buffer (pH 9.6). After coating, the plates were washed 3 times with PBST, and unbound sites in the wells were blocked with 2.5% nonfat dry milk diluted in PBST. After incubation for 1 hr at 37 C, the plates were emptied by suction, and various rabbit serum dilutions of 100 ml in PBST were added and incubated for 2 hr at room temperature, after which they were washed 6 times. Conjugate (100 ml/well peroxidase-labeled goat anti-rabbit IgG) diluted in PBST-milk was added. The plates were incubated 2 hr at room temperature and rinsed 6 times. Substrate (100 ml/well of 20 ml H2O2, 30% [wt/v], 1 50 ml 0.1 M citrate buffer, pH 5.0, 1 20 mg ophenylenediamine hydrochloride) was added. The plates were incubated in the dark at room temperature for 20 min, and the reaction was stopped with 50 ml/well of 12.5% sulfuric acid. Absorbance was read at 492 nm using a Microplate ELISA reader (Bio Rad). The optimal dilution (OD) of antigens, serum, and conjugate were

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previously established by checkerboard titration using a pool of 5 normal rabbit sera (NRS) as the negative control and a pool of 5 sera from rabbits 12 wk postinfection as the positive control. Different concentrations of rFh15 and Fh12 antigens (from 2.5 to 60 mg/ml) were simultaneously tested with various dilutions of negative and positive control sera (from 1:100 to 1:6,400) and various conjugate dilutions (from 1: 2,000 to 1:10,000) in such a way that only 1 combination of antigen and positive or negative serum dilution for each conjugate dilution existed. The results were analyzed using a signal to noise (S/N) ratio against conjugate dilution. The S/N ratio was the observed OD reading of the positive serum to the observed OD reading of the negative serum at the same serum dilution. Once the optimal concentration of antigen, serum, and conjugate was established, individual NRS and sera collected from rabbits 1–12 wk postinfection were tested. A ELISA result was considered positive when individual OD492 exceeded the mean 1 2 SD of the NRS pool. Inhibition ELISA assay The inhibition ELISA assay was performed at the optimal antigen, serum, and conjugate dilutions previously established for the indirect ELISA, but using anti-rFh15 and anti-nFh12 antisera. The plates were coated with rFh15 or nFh12 diluted in carbonate buffer, pH 9.6, and the unbound sites in the wells were blocked as described above. After blocking, the anti-nFh12 or anti-rFh15 antiserum diluted in PBST was added. Before addition to the plate, each antiserum was mixed with increasing amounts of each 12-kDa fraction purified by IEF at concentrations ranging between 2.5 and 40 mg/ml and incubated 1 hr at 37 C to favor antigen–antibody complex formation. Afterwards, the antiserum was added to the plate and incubated 2 hr at room temperature. After incubation, the plates were washed and then the conjugate solution was added and incubated 2 hr at room temperature. After another washing step, the substrate solution was added, and after 30 min of incubation at room temperature in the dark the reaction was stopped and absorbance was read as above. Two controls were used in this assay. One was a negative control in which PBST was added to immune serum at a similar volume to the antigen added. The second was a positive control made by adding nFh12 or rFh15 at 20 mg/ml to their respective antiserum. Statistical analysis Each ELISA assay was done in triplicate, and the results were expressed as the mean absorbance value for each determination. The inhibition ELISA assay was repeated 10 times. The OD492 obtained for each determination was used to calculate the inhibition percentage at each antigen concentration added to the antiserum. Inhibition percentage (I%) was calculated as I 5 1 2 (OD490 serum 1 antigen/OD490 serum 1 PBS) 3 100. Statistical analysis of the data obtained was made using a Student’s t-test and 1-way ANOVA test. Differences were considered significant at P , 0.05.

RESULTS Purification of nFh12 isoforms FhWWE consists of a complex mixture of proteins ranging in molecular mass (MW) from ,14 kDa to .200 kDa. When FhWWE was fractionated by gel filtration with Sephadex G50, 2 major peaks were observed. The first peak was basically composed of polypeptides above the 40-kDa range. The second peak contained both intermediate- and low-MW polypeptides (from 3 3 104 to 1.5 3 103 MW). Fractions from the second peak were pooled and fractionated by preparative IEF. Absorbance and pH profiles, after focusing with biolytes in the pH range 3–10 followed by pH 4–7, are shown in Figures 1 and 2, respectively. From the first IEF run, fractions 3–15 with pH 3–7.52 were collected and refocused using biolytes with pH 4– 7. After the second fractionation, the IEF samples were analyzed by SDS-PAGE and western blot. Eight fractions with isoelectric points (pI) of 5.11, 5.24, 5.33, 5.44, 5.72, 6.3, 7.25, and

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FIGURE 1. Isoelectric focusing run in Rotofor Cell using biolytes with pH 3–10. Fractions 3–15 with pH 3–7.52 were collected because they contained the 12-kDa polypeptide as demonstrated by SDS-PAGE.

7.82 (arbitrarily designated as Fh121, Fh122, Fh123, Fh124, Fh125, Fh126, Fh127, and Fh128, respectively) were selected because they contained the 12-kDa polypeptide (Fig. 3). Because all of these samples contained the 12-kDa polypeptide and because each differed in pI, they were likely 8 different isoforms of nFh12. ELISA results The rFh15, nFh12, and Fh121–8 antigens were titrated using a checkerboard titration in which each row of wells from A to H was coated with a different concentration of antigens from 2.5 to 60 mg/ml protein. In all cases, the concentrations of 2.5, 5, and 10 mg/ml were inadequate to saturate the wells. The best results were obtained when the plate was coated with 20 mg/m antigen. The antibody dose–response effect was studied using the previously determined optimal dilution of antigen and various dilutions of conjugate in the presence of increasing positive and negative serum dilutions. The rFh15 and the Fh12 proteins were all highly reactive with sera from 12 wk F. hepatica– infected rabbits at all serum dilutions. However, the highest S/ N ratio value was obtained at 1:800 serum dilution and 1:6,000 conjugate dilution. Using the optimal concentration of all reagents, NRS and sera from rabbits infected with F. hepatica were studied. Neither rFh15 nor nFh12 antigens were reactive with NRS. The mean OD of the NRS group obtained with both antigens was similar. It ranged from 0.08 to 0.14, with a mean value of 0.09 6 0.07. Thus, sera with OD . 0.23 (mean OD 6 2 SD) was considered positive. Following this criterion, the rFh15 antigen was found to be reactive only with sera from rabbits 6–12 wk postinfection, but not earlier (Fig. 4). The mean OD at this infection period ranged from 0.6 to 2.2 (0.04 , SD , 0.09), which was significantly different from controls (P , 0.01). The nFh12 antigen was not reactive with sera from rabbits 1 wk F. hepatica postinfection. However, notable reactivity was observed with sera from rabbits 2 to 12 wk postinfection. At this period of infection, the mean absorbance ranged from 0.8 to 2.7 (0.09 , SD , 0.12), which was significantly different

FIGURE 2. Isoelectric focusing run in Rotofor Cell using biolytes with pH 4–7. Fractions with IPs 5.11, 5.24, 5.33, 5.44, 5.72, 6.3, 7.25, and 7.82 were selected because they contained the 12-kDa polypeptide, as demonstrated by SDS-PAGE and western blot analysis.

from controls (P , 0.001). Similarly, Fh121–8 molecules were not reactive with sera from rabbit 1 wk postinfection, but when they were tested against sera from rabbits 2 to 12 wk postinfection, a significant reactivity was observed. Although the reactivity of all Fh12 isoforms was significantly different from controls during 2–12 wk postinfection, differences in reactivity were observed between some of them. The more acidic isoforms (Fh121–4) were less reactive with sera from rabbits 2 to 4 wk postinfection than the less acidic isoforms (Fh125–8). The OD492 range obtained with each of the Fh121–4 molecules was similar and ranged between 0.29 and 0.45 (0.12 , SD , 0.18), but it was statistically different (P , 0.01) from the OD492 range

FIGURE 3. Western blot analysis of the samples isolated after the second IEF run using ampholytes with pH 4–7 against a rabbit antinFh12 antiserum. Samples denoted as Fh121, Fh122, Fh123, Fh124, Fh125, Fh126, Fh127, and Fh128 with IPs 5.11, 5.24, 5.33, 5.44, 5.72, 6.3, 7.25, and 7.82, respectively, were selected because they contained the 12-kDa polypeptide, mostly free of contaminants. The arrow shows the 12-kDa polypeptide.


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FIGURE 4. Detection of antibodies to Fasciola hepatica infection by ELISA in the sera of infected rabbits. The solid line indicates mean OD obtained with rFh15 or nFh12 antigens or with the Fh12 isoforms. Vertical line indicates the mean OD 6 SD at each week postinfection. The horizontal dashed line represents the mean OD 1 2 SD of normal rabbit sera (0.23).

obtained with the Fh125–8 molecules (0.66 – 1.42; 0.09 , SD , 0.11). No statistical differences were observed between Fh121–4 and Fh125–8 molecules after 4 wk postinfection (Figure 4). Inhibition ELISA All inhibition assays were performed at the previously determined optimal dilution of antigen (20 mg/ml), serum (1:800), and conjugate (1:6,000) established for the indirect ELISA. When the plates were coated with rFh15 antigen and incubated with the anti-rFh15 antiserum previously mixed with concentrations of each Fh12 isoform between 2.5 and 40 mg/ml, an increasing inhibition of ELISA activity was observed. The inhibition curves obtained with each isoform are shown in Figure 5A. In all cases, statistical differences (P , 0.05) were found between mean inhibition percentages obtained at each protein antigen concentration. When inhibition curves obtained with the Fh12 isoforms were compared, 3 statistically different groups were identified. These groups were formed with the Fh121–4, Fh125–6, and Fh127–8 molecules. When the more acidic forms of Fh121–4 were added to serum at concentrations between 2.5 and 40 mg/ml, mean inhibition of ELISA activity ranged from 26.5 to 63.2% (0.73 , SD , 2.67), respectively, and reached values nearly 50% of inhibition at the antigen concentration of 20 mg/ml. In fact, the inhibition curve obtained with the acidic isoforms Fh121–4 was similar to that obtained when rFh15 was added to immune serum at the same range of antigen concentration (Fig. 5A), but it was statistically different (P , 0.05) from the inhibition curves obtained with the other acidic forms (Fh125–6) and the neutral/ basic isoforms (Fh27–8). These last two ranged between 17.1 and 43.5% (0.5 , SD , 1.5) and between 8.5 and 31% (0.25 , SD , 1.0), respectively. It was observed that at any antigen concentration added to serum, the inhibition percentage was inversely proportional to the observed pI of the molecules. Thus, molecules with more acidic pI (i.e., 5.11) showed the higher inhibition percentage, whereas the molecules with neu-

FIGURE 5. Inhibition curves, obtained at different protein antigen concentrations, of each Fh12 isoform added to serum at a concentrations of 2.5–40 mg/ml. (A) Plate was coated with the rFh15 antigen at 20 mg/ml then incubated with anti-rFh15 antiserum diluted 1:800 and previously mixed with protein antigen concentration from 5 to 40 mg/ml of rFh15, Fh121–4, Fh125–6, or Fh127–8 molecules. Statistical differences (P , 0.05) were found between the three groups of isoforms. (B) Plate was coated with the nFh12 antigen then incubated with anti-nFh12 antiserum diluted 1:800 and previously mixed with protein antigen concentrations from 5 to 40 mg/ml of nFh12, Fh121–4, Fh125–6, or Fh127–8 molecules. Statistical differences (P , 0.05) were found between the 3 groups of isoforms.

tral or slightly basic pI (i.e., 7.82) exhibited the lowest inhibition percentage (Fig. 6). When plates were coated with nFh12 and the experiment was performed using anti-nFh12 immune serum incubated with increasing amounts of Fh121–8 isoforms, the results obtained were inverse to the above. In this case, the neutral/basic Fh127–8 molecules showed a higher inhibition percentage (18.75–61.1%;

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FIGURE 6. Inhibition ELISA curve obtained when each Fh12 isoform was added to both anti-rFh15 and anti-nFh12 antisera at 20 mg/ ml. In both cases, the inhibition percentage was inversely proportional to the isoelectric point of the Fh12 molecules.

0.15 , SD , 0.5) than the acidic Fh121–4 and Fh125–6 molecules, which showed an inhibition percentage range between 6.6 and 32.7% (0.19 , SD , 3.3) and 12.9 and 53.5% (0.15 , SD , 0.5), equivalent to the protein concentration range of 2.5–40 mg/ml, respectively. Significant differences (P , 0.05) were found between the 3 groups of molecules. Neutral/basic isoforms (Fh127–8) inhibited ELISA activity nearly 50% when they were added to anti-nFh12 immune serum at 20 mg/ml. The inhibition curve obtained with Fh127–8 was similar to that obtained when nFh12 antigen was added to the immune sera at identical protein antigen concentrations (Fig. 5B) but differed slightly from that obtained with Fh125–6. Moreover, it was statistically different (P , 0.05) from Fh121–4 molecules. Similar to the above, the inhibition percentage obtained at each antigen concentration was inversely proportional to the observed isoelectric point of the molecules. In this case, molecules with neutral/basic pI (i.e., 7.82) showed the highest inhibition percentage, whereas the molecules with more acidic pI (i.e., 5.11) exhibited the lowest inhibition percentage. DISCUSSION The purified nFh12 protein was found to induce significant levels of protection to challenge infection with F. hepatica in mice (Hillyer, 1985), cattle (Hillyer et al., 1987), and rabbits (Muro et al., 1997). In addition to numerous studies demonstrating cross-protection against Schistosoma mansoni (Hillyer, Garcıá Rosa et al., 1988; Hillyer, Soler de Galanes et al., 1988), purified nFh12 also cross-protects against infection with S. bovis in mice (Lo´pez Aban et al., 1999). An antiserum to nFh12 has been used to identify a gene encoding a recombinant protein denoted rFh15, a polypeptide of 132 amino acids with a predicted MW of 14.7 kDa (Rodrı´guez Pe´rez et al., 1992). This polypeptide also has significant homology with a 14.8-kDa S. mansoni fatty acid–binding protein (Moser et al., 1991). The F. hepatica rFh15 has been shown to induce resistance to challenge infection with F. hepatica in rabbits (Muro et al., 1997).

However, in every instance in which the immunoprophylactic potential of nFh12 and rFh15 has been tested simultaneously, the native molecule always induced higher levels of protection compared to the recombinant form. The observed immunogenic differences between rFh15 and nFh12 can be explained with the results obtained in the present study. It was demonstrated that nFh12 is actually a complex of proteins with similar molecular mass and different pIs. These proteins not only differ in pI, but also in immunological behavior. For example, the 2 acidic molecules (Fh125–6) and 2 neutral/basic molecules (Fh127–8) were more reactive with serum from rabbits 2 to 4 wk postinfection than the other acidic molecules (Fh121–4). Furthermore, the Fh121–4 forms showed higher inhibition percentages during ELISA when added to anti-rFh15 antiserum, and no statistical differences were observed between the inhibition curves obtained with these isoforms and those obtained with rFh15. This suggests that Fh121–4 possess more immunological relatedness with the rFh15 recombinant molecule than with the nFh12 native protein. On the other hand, the observation that the Fh125–8 forms show a higher inhibition ELISA percentage when they were added to anti-nFh12 antiserum and that their inhibition curves were statistically different from that obtained with rFh15 suggests that the Fh125–8 isoforms possess more immunological relatedness with the native than with the recombinant molecule. If one considers these findings and that it has been repeatedly demonstrated that rFh15 (whose pI is 5.44) only reacts with sera from mice and rabbits that have been infected for 6 wk or longer (Hillyer, 1995), it is likely that rFh15 is one of the acidic isoforms of nFh12 reported in this study as well as one of the less immunogenic proteins of the complex. It also suggests that other recombinant proteins with higher immunoprophylactic potentials exist. The previously published procedure for nFh12 purification involved a combination of molecular sieving chromatography and anion exchange chromatography using DEAE sephadex A120 equilibrated with 10–15 mM NaCL buffer, pH 7.2 (Hillyer, Soler de Galanes et al., 1988). Using this procedure, one obtains nFh12 free of contaminants. However, this technique does not allow the identification or the presence of the different isoforms because at pH 7.2, all acidic molecules have an overall negative charge, whereas the neutral or basic molecules remain positive or acquire a slight overall negative charge. As a result, the nFh12 antigen purified as described by Hillyer, Soler de Galanes et al. (1988) only contains the acidic forms (Fh121–6) because the neutral/basic forms would not have bound to the matrix. Consequently, the immune serum obtained against the nFh12 purified in this manner, and which was then used later for the immunoscreening of a cDNA library to identify the gene encoding for this protein (Rodrı´guez Pe´rez et al., 1992), was obtained against the protein complex in which the acidic forms were in significantly higher proportion with respect to the neutral/basic molecules and which also overlapped with one another. Thus, screening of the cDNA library using this polyclonal serum significantly increased the chance of detecting a form of the molecule with an acidic pI and, thus, rFh15. Because nFh12 induced higher protective antibody levels than rFh15 (Muro et al., 1997), it is now clear that further studies are necessary to obtain antiserum against the more neutral/basic isoforms of Fh12 to identify recombinant molecules with even higher im-


munoprophylaxis potential. Two fatty acid–binding protein isoforms have been reported by Bozas and Spithill (1996). Their relationship to the protein identified in the present study needs to be determined. Now that Fh12 has been shown to be a complex of different forms with multiple isoelectric points, it is necessary to investigate whether such differences in charge are attributable to discrete differences in amino acid composition or to various ligands bound to the protein. Further studies on these aspects are in progress. ACKNOWLEDGMENTS These studies were supported by grants from the University of The United Nations in Tokyo, Japan, to Ana M. Espino, NSF-EPSCOR SPACS, and The University of Puerto Rico Central Administration. Ana M. Espino is a visiting scientist from the Institute of Tropical Medicine ‘‘Pedro Kourı´, Havana City, Cuba. Jose´ R. Rodrı´guez Medina received partial support from NIH-RCMI grant G12RR 03051.

LITERATURE CITED BOZAS, E., AND T. W. SPITHILL. 1996. Identification of 3-hydroxyproline residues in several proteins of Fasciola heatica. Experimental Parasitology 82: 69–72. HILLYER, G. V. 1985. Induction of immunity in mice to Fasciola hepatica with a Fasciola/Schistosoma cross-reactive defined immunity antigen. American Journal of Tropical Medicine and Hygiene 34: 1127–1131. ———. 1987. Heterologous resistance in schistosomiasis. Memorias Do Instituto Oswaldo Cruz 82: 171–174. ———. 1995. Comparison of purified 12 KDa and recombinant 15 KDa Fasciola hepatica antigens related to a Schistosoma mansoni fatty acid binding protein. Memorias Do Instituto Oswaldo Cruz 90: 249–253. ———, M. I. GARCIÁ ROSA, H. ALICEA, AND A. HERNAŃDEZ. 1988. Successful vaccination against murine Schistosoma mansoni infection with a purified 12 KDa Fasciola hepatica cross-reactive anti-

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