Solution structure of RicC3, a 2S albumin storage protein from Ricinus communis

The three-dimensional structure in aqueous solution of recombinant (15)N labeled RicC3, a 2S albumin protein from the seeds of castor bean (Ricinus communis), has been determined by NMR methods. The computed structures were based on 1564 upper limit distance constraints derived from NOE cross-correlation intensities measured in the 2D-NOESY and 3D-HSQC-NOESY experiments, 70 phi torsion angle constraints obtained from (3)J(HNH)(alpha) couplings measured in the HNHA experiment, and 30 psi torsion angle constraints derived from (3)J(H)(alpha)(Ni+1) couplings measured in the HNHB experiment. The computed structures showed a RMSD radius of 0.64 A for the structural core. The resulting structure consists of five amphipatic helices arranged in a right-handed super helix, a folding motif first observed in nonspecific lipid transfer proteins. Different than the latter, RicC3 does have not an internal cavity, a fact that can be related to the exchange in the pairing of disulfide bridges in the segment.CXC. Previous attempts to determine high resolution structures of a 2S albumin protein by either X-ray crystallography or NMR methods failed because of the heterogeneity of the protein prepared from natural sources. Both 2S albumins and nonspecific lipid transfer proteins belong to the prolamine superfamily, some of whose members are food allergens. The solution structure for recombinant RicC3 determined here is a suitable representative structure for the broad family of seed 2S albumin proteins, which may help to establish meaningful relationships between structure and allergenicity. RicC3 is also the peptidic component of the immunomodulator Inmunoferon, a widely used pharmaceutical product, and its structure is expected to help understand its pharmaceutical activity.     

A simple model of backbone flexibility improves modeling of side-chain conformational variability

The considerable flexibility of side-chains in folded proteins is important for protein stability and function, and may have a role in mediating allosteric interactions. While sampling side-chain degrees of freedom has been an integral part of several successful computational protein design methods, the predictions of these approaches have not been directly compared to experimental measurements of side-chain motional amplitudes. In addition, protein design methods frequently keep the backbone fixed, an approximation that may substantially limit the ability to accurately model side-chain flexibility. Here, we describe a Monte Carlo approach to modeling side-chain conformational variability and validate our method against a large dataset of methyl relaxation order parameters derived from nuclear magnetic resonance (NMR) experiments (17 proteins and a total of 530 data points). We also evaluate a model of backbone flexibility based on Backrub motions, a type of conformational change frequently observed in ultra-high-resolution X-ray structures that accounts for correlated side-chain backbone movements. The fixed-backbone model performs reasonably well with an overall rmsd between computed and predicted side-chain order parameters of 0.26. Notably, including backbone flexibility leads to significant improvements in modeling side-chain order parameters for ten of the 17 proteins in the set. Greater accuracy of the flexible backbone model results from both increases and decreases in side-chain flexibility relative to the fixed-backbone model. This simple flexible-backbone model should be useful for a variety of protein design applications, including improved modeling of protein-protein interactions, design of proteins with desired flexibility or rigidity, and prediction of correlated motions within proteins.     

Solution structure of a methionine-rich 2S albumin from sunflower seeds: relationship to its allergenic and emulsifying properties

The three-dimensional structure in aqueous solution of SFA-8, a 2S albumin 103-residue protein from seeds of sunflower (Helianthus anuus L.), has been determined by NMR methods. An almost complete (1)H resonance assignment was accomplished from analysis of two-dimensional (2D) COSY and 2D TOCSY spectra, and the structure was computed by using restrained molecular dynamics on the basis of 1393 upper limit distance constraints derived from NOE cross-correlation intensities measured in 2D NOESY spectra. In contrast with most other 2S albumins, SFA-8 consists of a single polypeptide chain without any cleavage in the segment of residues 30-46. The computed structures exhibited an rmsd radius of 0.52 A for the backbone structural core (residues 11-30 and 46-101) and 1.01 A for the side chain heavy atoms. The resulting structure consists of five amphipathic helices arranged in a right-handed superhelix, a folding motif first observed in nonspecific lipid transfer (nsLTP) proteins, and common to other 2S albumins. In contrast to nsLTP proteins, neither SFA-8 nor RicC3 (a 2S albumin from castor bean) has an internal cavity that is able to host a lipid molecule, which results from an exchange in the pairing of disulfide bridges in the CXC segment. Both 2S albumins and nonspecific lipid transfer proteins belong to the prolamin superfamily, which includes a number of important food allergens. Differences in the extension and solvent exposition of the so-called "hypervariable loop" (which connects helices III and IV) in SFA-8 and RicC3 may be responsible for the different allergenic properties of the two proteins. SFA-8 has been shown to form highly stable emulsions with oil/water mixtures. We propose that these properties may be determined partly by a hydrophobic patch at the surface of the protein which consists of five methionines that partially hide the Trp76 residue. The flexibility of the loop which contains Trp76 and the hydrophobicity of the whole environment may favor a conformational change, by which the Trp76 side chain may become inserted into the oil phase.     

Capsid intermediates assembled in a foot-and-mouth disease virus genome RNA-programmed cell-free translation system and in infected cells.

Structural protein complexes sedimenting at 140S, 70S (empty capsids), and 14S were isolated from foot-and-mouth disease virus-infected cells. The empty capsids were stable, while 14S complexes were relatively short-lived. Radioimmune binding assays involving the use of neutralizing monoclonal antibodies to six distinct epitopes on type A12 virus and polyclonal antisera to A12 structural proteins demonstrated that native empty capsids were indistinguishable from virus. Infected cell 14S particles possessed all the neutralizing epitopes and reacted with VP2 antiserum. Cell-free structural protein complexes sedimenting at 110S, 60S, and 14S containing capsid proteins VP0, VP3, and VP1 are assembled in a rabbit reticulocyte lysate programmed with foot-and-mouth viral RNA. These structures also contain the six epitopes, and cell-free 14S structures like their in vivo counterparts reacted with VP2 antiserum. Capsid structures from infected cells and the cell-free complexes adsorbed to susceptible cells, and this binding was inhibited, to various degrees, by saturating levels of unlabeled virus. These assays and other biochemical evidence indicate that capsid assembly in the cell-free system resembles viral morphogenesis in infected cells. In addition, epitopes on the virus surface possibly involved in interaction with cellular receptor sites are found early in virion morphogenesis.     

Evidence for a precursor molecule of Brazil nut 2 S seed proteins from biosynthesis and cDNA analysis

Summary Seed storage proteins were extracted from Brazil nut (Bertholletia excelsa H.B.K.) seed embryos at various maturation stages. Salt-soluble and water-soluble proteins (globulins and albumins) were separated by gel chromatography and exhaustive dialysis against water. Both fractions were analysed by one- and two-dimensional polyacrylamide gel electrophoresis. Amino acid analysis revealed that both fractions are unusually high in methionine. The albumins consist of a family of low molecular weight polypeptides that are heterogeneous with respect to pI and are identical to the high methionine 2 S proteins described by Youle and Huang (1981). The biosynthesis of this class of proteins in maturing embryos was followed by in vivo labelling combined with immunological studies. Western blotting with monospecific antibodies against purified 2 S albumins and sequencing of a nearly complete cDNA clone revealed that they are synthesized via a precursor polypeptide.     

Foot and mouth disease virus polyepitope protein produced in bacteria and plants induces protective immunity in guinea pigs

The goal of this project was to develop an alternative foot and mouth disease (FMD) vaccine candidate based on a recombinant protein consisting of efficient viral epitopes. A recombinant gene was designed that encodes B-cell epitopes of proteins VP1 and VP4 and T-cell epitopes of proteins 2C and 3D. The polyepitope protein (H-PE) was produced in E. coli bacteria or in N. benthamiana plants using a phytovirus expression system. The methods of extraction and purification of H-PE proteins from bacteria and plants were developed. Immunization of guinea pigs with the purified H-PE proteins induced an efficient immune response against foot and mouth disease virus (FMDV) serotype O/Taiwan/99 and protection against the disease. The polyepitope protein H-PE can be used as a basis for developing a new recombinant vaccine against FMD.     

A plant-derived human monoclonal antibody induces an anti-carbohydrate immune response in rabbits

A common argument against using plants as a production system for therapeutic proteins is their inability to perform authentic N-glycosylation. A major concern is the presence of beta 1,2-xylose and core alpha 1,3-fucose residues on complex N-glycans as these nonmammalian N-glycan residues may provoke unwanted side effects in humans. In this study we have investigated the potential antigenicity of plant-type N-glycans attached to a human monoclonal antibody (2G12). Using glyco-engineered plant lines as expression hosts, four 2G12 glycoforms differing in the presence/absence of beta 1,2-xylose and core alpha 1,3-fucose were generated. Systemic immunization of rabbits with a xylose and fucose carrying 2G12 glycoform resulted in a humoral immune response to both N-glycan epitopes. Furthermore, IgE immunoblotting with sera derived from allergic patients revealed binding to plant-produced 2G12 carrying core alpha 1,3 fucosylated N-glycan structures. Our results provide evidence for the adverse potential of nonmammalian N-glycan modifications present on monoclonal antibodies produced in plants. This emphasizes the need for the use of glyco-engineered plants lacking any potentially antigenic N-glycan structures for the production of plant-derived recombinant proteins intended for parenteral human application.     

An electrophile-nucleophile interaction in metalloprotein structures.

A new attractive interaction in metalloprotein structures, between the thiolate anion of a metal-bound cysteine (acting as a nucleophile) and a carbonyl carbon of a peptide group (an electrophile), has been identified. From 82 cases extracted from 23 metalloprotein structures, the interacting S and C atoms are found to be at a distance of 3.2 (+/-2) A, such that the angle S ... C-O is 109 degrees (+/-15 degrees). Usually, the interacting atoms are from the same Cys residue, and to allow the S to interact with the carbonyl group the side-chain and the main-chain torsion angles deviate from those found in cysteines not bound by metals. There is a good correlation between the S...C distance and the angular deviation of the S...C vector from the normal to the peptide plane. Various data points may be envisaged to represent "snapshots" along the reaction coordinate for the intra-residue attack of Cys S on the CO group.     

Fusion proteins encoded by orf 129L of ectromelia and orf A30L of smallpox viruses cross-react with neutralizing monoclonal antibodies

Open reading frame (orf) 129L of ectromelia (EV) and orf A30L of smallpox viruses (SPV) encoding fusion proteins were cloned and expressed in E. coli cells. The recombinant polypeptides (prA30L H pr129L) were purified from cell lysates by Ni-NTA chromatography. Recombinant polypeptides were able to form trimers in buffered saline and they destroyed under treatment with SDS and 2-mercaptoethanol. Reactivity of prA30L, pr129L and orthopoxvirus proteins was analyzed by ELISA and Western blotting with panel of 22 monoclonal antibodies (MAbs) against orthopoxviruses (19 against EV, 2 MAbs against vaccinia virus and 1 Mabs against cowpox virus). This data allowed us to conclude that there are 12 EV-specific epitopes of pr129L and EV fusion proteins, ten orthopox-specific epitopes of EV, VV, CPV fusion proteins, from them 9 orthopox-specific epitopes of prA30L and SPV fusion proteins. Five Mabs, which cross-reacted with orthopox-specific epitopes, were able to neutralize the VV on Vero cells and from them two MAbs has neutralizing activity against smallpox virus. Our findings demonstrate that 129L fusion protein have EV-specific epitopes, that EV 129L and SPV A30L fusion proteins have a several orthopox-specific epitopes to induce a neutralizing antibodies against human pathogenic orthopoxviruses.     

Neutralizing Monoclonal Antibodies Cross-React with Fusion Proteins Encoded by <Emphasis Type="Italic">129L</Emphasis> of the Ectromelia Virus and <Emphasis Type="Italic">A30L</Emphasis> of the Variola Virus

PCR fragments containing the fusion protein genes 129L of the ectromelia virus (EV) and A30L of the variola virus (VARV) were cloned in pQE32. The expression products, recombinant prA30L and pr129L, were isolated from Escherichia coli cell lysates by metal-chelate affinity chromatography. The recombinant proteins retained the capability of oligomerization, characteristic of their natural analogs. ELISA and immunoblotting were used to test 22 monoclonal antibodies (mAbs) to orthopoxviruses (19 mAbs to EV, 2 mAbs to the vaccinia virus (VACV), and 1 mAb to the cowpox virus (CPXV)) for interaction with prA30L, pr129L, and orthopoxviruses. Twelve species-specific epitopes were found in the EV fusion protein 129L and its recombinant analog. Ten cross-reacting epitopes were found in the EV, CPXV, and VACV fusion proteins. Of these, nine epitopes were present both in prA30L and in the VARV fusion protein. Five mAbs interacting with cross-reacting epitopes were capable of efficient neutralization of VACV; two of these mAbs neutralized VARV. It was demonstrated that there are species-specific epitopes in EV 129L and cross-reacting epitopes in the EV, VARV, CPXV, and VACV fusion proteins, including epitopes that induced synthesis of virus-neutralizing antibodies against VACV and VARV.     

Western Blot Detection of Human Anti-Chikungunya Virus Antibody with Recombinant Envelope 2 Protein

Chikungunya virus (CHIKV), a tropical pathogen, has re-emerged and has massive outbreaks abruptly all over the world. Containing many dominant epitopes, the envelope E2 protein of CHIKV has been explored for the vaccination or diagnosis. In the present study, the antigenicity of a recombinant expressed intrinsically disorder domain (IUD) of E2 was tested for the detection of the antibody against CHIKV through western blot method. The gene of the IUD of E2 was inserted into 2 different vectors and expressed as recombinant GST-E2 and recombinant MBP-E2 fusion protein, respectively. Two kinds of fusion proteins were tested with 30 CHIKV patient sera and 30 normal sera, respectively. Both proteins were detected by 25 patients sera (83.3%) and 1 normal serum (3.3%). This test showed a relatively high sensitivity and very high specificity of the recombinant E2 proteins to be used as diagnostic antigens against CHIKV infection.     

Characterization of a metalloprotease inhibitor protein (SmaPI) of Serratia marcescens.

As suggested by Y. Suh and M.J. Benedik (J. Bacteriol. 174: 2361-2366, 1992), Serratia marcescens ATCC 27117 produced very small amounts (0.8 U ml-1) of an inhibitor protein (SmaPI) that shows an inhibitory activity against extracellular 50-kDa metalloprotease (SMP) of S. marcescens and that is localized in the periplasm of cells at the optimal growth temperature of 25 degrees C. A recombinant S. marcescens harboring plasmid pSP2 encoding SMP and SmaPI genes produced 20 U of SmaPI ml-1 that is also localized in the periplasm of cells at 25 degrees C. However, a large amount of SmaPI (86 Uml-1) was extracellularly produced at the supraoptimal growth temperature 37 degrees C from the recombinant S. marcescens (pSP2). We purified SmaPI from the culture supernatant of S. marcescens (pSP2) grown at 37 degrees C, and some biochemical properties were characterized. SmaPI had a pI value of about 10.0 and was a monomeric protein with a molecular mass of 10,000. SmaPI was produced from a precursor SmaPI by cleavage of a signal peptide (26 amino acid residues). The inhibitor was stable in boiling water for up to 30 min. The thermostability of SmaPI can be attributed to its reversible denaturation. SmaPI inhibited SMP by formation of a noncovalent complex with a molar ratio of 1:1 and showed a high protease specificity, which inhibited only SMP among the various proteases we examined.     

Cross-reactivity of pollen and food allergens: soybean Gly m 4 is a member of the Bet v 1 superfamily and closely resembles yellow lupine proteins

In many cases, patients allergic to birch pollen also show allergic reactions after ingestion of certain fruits or vegetables. This observation is explained at the molecular level by cross-reactivity of IgE antibodies induced by sensitization to the major birch pollen allergen Bet v 1 with homologous food allergens. As IgE antibodies recognize conformational epitopes, a precise structural characterization of the allergens involved is necessary to understand cross-reactivity and thus to develop new methods of allergen-specific immunotherapy for allergic patients. Here, we report the three-dimensional solution structure of the soybean allergen Gly m 4, a member of the superfamily of Bet v 1 homologous proteins and a cross-reactant with IgE antibodies originally raised against Bet v 1 as shown by immunoblot inhibition and histamine release assays. Although the overall fold of Gly m 4 is very similar to that of Bet v 1, the three-dimensional structures of these proteins differ in detail. The Gly m 4 local structures that display those differences are also found in proteins from yellow lupine with known physiological function. The three-dimensional structure of Gly m 4 may thus shed some light on the physiological function of this subgroup of PR10 proteins (class 10 of pathogenesis-related proteins) and, in combination with immunological data, allow us to propose surface patches that might represent cross-reactive epitopes.     

Molecular characterization and allergenic activity of Lyc e 2 (beta-fructofuranosidase), a glycosylated allergen of tomato.

Until now, only a small amount of information is available about tomato allergens. In the present study, a glycosylated allergen of tomato (Lycopersicon esculentum), Lyc e 2, was purified from tomato extract by a two-step FPLC method. The cDNA of two different isoforms of the protein, Lyc e 2.01 and Lyc e 2.02, was cloned into the bacterial expression vector pET100D. The recombinant proteins were purified by electroelution and refolded. The IgE reactivity of both the recombinant and the natural proteins was investigated with sera of patients with adverse reactions to tomato. IgE-binding to natural Lyc e 2 was completely inhibited by the pineapple stem bromelain glycopeptide MUXF (Man alpha 1-6(Xyl beta 1-2)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-3)GlcNAc). Accordingly, the nonglycosylated recombinant protein isoforms did not bind IgE of tomato allergic patients. Hence, we concluded that the IgE reactivity of the natural protein mainly depends on the glycan structure. The amino acid sequences of both isoforms of the allergen contain four possible N-glycosylation sites. By application of MALDI-TOF mass spectrometry the predominant glycan structure of the natural allergen was identified as MMXF (Man alpha 1-6(Man alpha 1-3)(Xyl beta 1-2)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-3) GlcNAc). Natural Lyc e 2, but not the recombinant protein was able to trigger histamine release from passively sensitized basophils of patients with IgE to carbohydrate determinants, demonstrating that glycan structures can be important for the biological activity of allergens.     

Expression and epitope analysis of the major allergenic protein Fag e 1 from buckwheat

Seeds of common buckwheat (Fagopyrum esculentum) contain valuable nutritive substances but also allergenic proteins that cause hypersensitive reactions. Thus, the development of hypoallergenic buckwheat would make this important pseudo-cereal available to allergic people. A major allergenic protein of buckwheat is Fag e 1. We isolated the respective cDNA, coding for a 22 kDa protein, from a recently developed autogamous strain of common buckwheat and confirmed its immunoglobulin E (IgE)-binding activity using recombinant Fag e 1 and sera of allergic patients. The derived amino acid sequence from Fag e 1 cDNA was used to synthesize an overlapping peptide library on nitrocellulose membranes for the determination of the Fag e 1 epitopes. We identified eight epitopes and the critical amino acids for IgE-binding within the epitopes. This epitope analysis of a major allergenic protein of buckwheat should help therapeutic efforts and aid in the development of hypoallergenic buckwheat.     

Identification and analysis of a conserved immunoglobulin E-binding epitope in soybean G1a and G2a and peanut Ara h 3 glycinins

To identify conserved immunoglobulin E (IgE)-binding epitopes among legume glycinins, we utilized recombinant soybean G2a and G2a-derived polypeptide fragments. All of these fusion polypeptides bound IgE, and the C-terminal 94-residue fragment appeared to bind more IgE. Using synthetic peptides we identified S219-N233 (S(219)GFAPEFLKEAFGVN(233)) as the dominant IgE-binding epitope. Alanine scanning of this epitope indicated that six amino acids (E224, F225, L226, F230, G231, and V232) contributed most to IgE binding. Among these amino acids, only G231 of soybean G2a is not conserved in soybean G1a (S234) and peanut Ara h 3 (Q256). Synthetic peptides corresponding to the equivalent regions in G1a and Ara h 3 bound IgE in the order Ara h 3>/=soybean G2a>soybean G1a. This sequence represents a new IgE-binding epitope that occurs in a highly conserved region present in legume glycinins. Such IgE-binding sites could provide a molecular explanation for the IgE cross-reactivity observed between soybean and peanut proteins.     

Expression and characterization of chimeric hepatitis B surface antigen particles carrying preS epitopes.

Many studies have provided evidence that hepatitis B surface antigen (HBsAg) including preS1 and preS2 sequences could be an ideal candidate for a new hepatitis B virus (HBV) vaccine with higher efficacy. However, the large (L) protein containing the entire preS region expressed in mammalian cells is not efficiently assembled into particles and secreted. Here we report an alternative approach to include the dominant epitopes of preS1 and preS2 to the small (S) protein as fusion proteins by the recombinant DNA technology. Three fusion proteins containing preS2(120-146) and preS1(21-47) at the N-terminus and/or truncated C-terminus of S protein were expressed using the recombinant vaccinia virus system. All these fusion proteins were efficiently secreted in the particulate form, and displayed S, preS1 and/or preS2 antigenicity. Further analysis showed that these chimeric HBsAg particles elicited strong antibody responses against S, preS1 and preS2 antigens in BALB/c mice, suggesting that they could be promising candidates for a new recombinant vaccine to induce broader antibody response required for protection against hepatitis B viral infection.     

Antigenic and immunogenic characterization of recombinant baculovirus-expressed severe acute respiratory syndrome coronavirus spike protein: implication for vaccine design

The spike (S) glycoprotein of severe acute respiratory syndrome coronavirus (SARS-CoV) mediates the receptor interaction and immune recognition and is considered a major target for vaccine design. However, its antigenic and immunogenic properties remain to be elucidated. In this study, we immunized mice with full-length S protein (FL-S) or its extracellular domain (EC-S) expressed by recombinant baculoviruses in insect cells. We found that the immunized mice developed high titers of anti-S antibodies with potent neutralizing activities against SARS pseudoviruses constructed with the S proteins of Tor2, GD03T13, and SZ3, the representative strains of 2002 to 2003 and 2003 to 2004 human SARS-CoV and palm civet SARS-CoV, respectively. These data suggest that the recombinant baculovirus-expressed S protein vaccines possess excellent immunogenicity, thereby inducing highly potent neutralizing responses against human and animal SARS-CoV variants. The antigenic structure of the S protein was characterized by a panel of 38 monoclonal antibodies (MAbs) isolated from the immunized mice. The epitopes of most anti-S MAbs (32 of 38) were localized within the S1 domain, and those of the remaining 6 MAbs were mapped to the S2 domain. Among the anti-S1 MAbs, 17 MAbs targeted the N-terminal region (amino acids [aa] 12 to 327), 9 MAbs recognized the receptor-binding domain (RBD; aa 318 to 510), and 6 MAbs reacted with the C-terminal region of S1 domain that contains the major immunodominant site (aa 528 to 635). Strikingly, all of the RBD-specific MAbs had potent neutralizing activity, 6 of which efficiently blocked the receptor binding, confirming that the RBD contains the main neutralizing epitopes and that blockage of the receptor association is the major mechanism of SARS-CoV neutralization. Five MAbs specific for the S1 N-terminal region exhibited moderate neutralizing activity, but none of the MAbs reacting with the S2 domain and the major immunodominant site in S1 showed neutralizing activity. All of the neutralizing MAbs recognize conformational epitopes. These data provide important information for understanding the antigenicity and immunogenicity of S protein and for designing SARS vaccines. This panel of anti-S MAbs can be used as tools for studying the structure and function of the SARS-CoV S protein.     

Shared epitopes between the soluble proteins of Hypoderma lineatum and Hypoderma bovis first instars.

Cattle are known to acquire immunological resistance to hypodermyiasis by repeated exposure to both species of cattle grubs, Hypoderma lineatum (Villers) and Hypoderma bovis (L.). Vaccination of cattle with purified proteins of H. lineatum, particularly hypodermin A, is known to protect cattle against hypodermyiasis by this species. The development of a protective recombinant vaccine against both species using hypodermin A isolated from H. lineatum would require that immunological epitopes be shared by complementary proteins in H. bovis. The purpose of this study was to investigate the soluble proteins of H. bovis first-instars for shared epitopes with H. lineatum. Soluble H. lineatum and H. bovis first-instar larval proteins were resolved by nondenaturing polyacrylamide electrophoresis, blotted onto nitrocellulose paper, and probed with selected polyclonal cow, polyclonal rabbit, and mouse monoclonal antisera. Considerable cross-reactivity was demonstrated by antibodies in the serum of an H. lineatum-infested cow as 6 of 10 resolved H. bovis proteins were bound by the antibodies. The most common shared epitope(s) was associated with hypodermin C, a collagenolytic protease. Hypodermin A shared epitope(s) were noted on 1 prominent H. bovis band (HB1-2). Hypodermin B, a prominent protein in H. lineatum, did not appear to share epitopes with H. bovis proteins. Shared epitopes between H. bovis proteins and hypodermins A and C of H. lineatum would suggest that cross-protection of cattle against H. bovis can be expected by vaccination with recombinant proteins of H. lineatum.