ELSEVIER Molecular and Biochemical Parasitology 82 (1996) 113- 116

MOLECULAR

iii&EMIcAL PARAsIToLoGY

Short communication

Sequence and immunogenicity of Taenia saginata ferritin’

Laura Beniteza, Leslie J.S. Harrison b,* R. Michael E. Parkhouse”, Teresa Garate” ,

“Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III, CNMVIS, 28220 Majadahonda. Madrid, Spain blJnirlersity of Edinburgh, Centre for Tropical Veterinary Medicine, Easter Bush, Roslin, Midlothian, Scotland EH25 9RG. UK

“Institute for Animal Health, Pirbright Laboratories. Ash Road, Pirbright, Woking, Surrey, England, UK

Received 29 March 1996; revised 9 July 1996; accepted 18 July 1996

Keywords: Taenia saginata; Ferritin’

The zoonotic cestode, Taenia saginata is cosmo- politan in its distribution but the prevalence is greater in the tropics and sub-tropics. The para- site causes public health problems and production losses due to condemnation of infected carcasses or down-grading of carcass value [l]. The devel- opment of specific diagnostic procedures and the preparation of vaccines would greatly aid the control of the parasite. Diagnostic and protective antigens have already been described in the para- site oncosphere stage [2,3]. In order to study such relevant antigens, a T. saginata oncosphere cDNA library was prepared in hUni-Zap-II (Stratagene Ltd) from 1.2 ug of mRNA isolated from 3 x lo6 hatched and activated oncospheres [4]. The resul-

* Corresponding author. Tel.: + 44 131 6506217; fax: + 44 131 4455099; e-mail: leslie.harrison@ed.ac.uk

’ Note: Nucleotide sequence data reported in this paper are available in the EMBL, GenbankTM and DDJB databases under the accession number X95840.

tant amplified library (1.2 x 10” pfu ml- I) was screened using sera pooled from six cattle, immu- nised following primary (week 0) and secondary (week 6) intramuscular injection with a whole extract of T. saginata oncospheres along with saponin [5]. These cattle were resistant to an oral challenge with T. saginata oncospheres delivered 1 week after the secondary boosting injection. Se- lected positive recombinant phage were purified and the clones then transformed into phagemid (pBluescript) by helper phage rescue [6] and se- quenced using fluorescence base labelling and the automatic ALF System (Pharmacia). One clone, named C-Tso20 comprised 694 bp with an open reading frame of 173 amino acids corresponding to a deduced molecular mass of 19 943 Da and isoelectric point of 5.1. Comparison of the pre- dicted amino acid sequence with EMBL and SWISS-PROT databanks using software packages from Genetics Computer Group [7], revealed a high degree of similarity with heavy and light

0166-6851/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved

PIZ S166-6851(96)02713-7

114 L. Benitez et al. / Molecular and Biochemical Parasitology 82 (1996) I13- I16

” :: :: 9 z P TS

Eg _““‘SLV---s-E__

.%I-1

Sm-2

--T_-NM------L_-

:: s 0 P E 0

TS GLGEYLFDKETLNGGE’D 173

Eg -SQ-CE- _,______Q___‘K 1?3 Sm-1 _T__C__{N___ N_____T______‘H__SQ 173 Sm-2 j72

Fig. 1. (a) Alignment of ferritins from different organisms. Ts, Taenia saginata: Eg, Echinococcus granulosus [8]; Sm-l/2, Schistosoma mansorzi ferritin clones 1 and 2 [ll]. Hyphens indicate identity with the T. saginata ferritin sequence and stars represent gaps introduced to maximise alignment. Residues boxed (GluZ8. Glu6’, His66. G~IJ’~* and Gin“‘*) designate amino acids involved in binding metal or hydrogen, conserved in mammalian heavy chains [12]. The optimal alignment between T. saginata ferritin and the heavy chain of human ferritin required the introduction of nine gaps, at the same places as for Echinococcus granulosus [9] and Sclaisrosoma mansoni Sm-1 [1 I] ferritins. The sequence comparison revealed that 7’. saginata ferritin shares 80.9% identity with E. granulosus ferritin, while a lower degree of similarity was obtained with Sm-1 and Sm-2 ferritins from S. mansoni (61.8% and 49.4%, respectively). The similarity with heavy chains from human [13], rat [14], chicken [15], frog [16] and toad [17] ferritins was 50 to 60% (data not shown). Less homology was observed when I”. saginata ferritin was compared to the ferritin light chains. (b) The antigenic index according to Jameson-Wolf [lo].

chains of ferritin from different organisms (Fig.

la). Experimental evidence suggests the physiologi-

cal role for ferritin is to protect cells from oxidis-

ation and this could well be the function of T.

saginata ferritin [8]. E. granulosus ferritin has

been reported to be highly immunogenic in echi-

nococcosis patients as well as a useful diagnostic

antigen for the disease [9]. In the case of the

cloned ferritin expression product its antigenicity

was deduced from the Jameson-Wolf [lo] anti-

genie index (Fig. lb) and confirmed using sera from patients with relevant helminth infections,

sera from the immunised cattle (see above) and

sera similarly prepared in rabbits (Fig. 2).

Both the cattle and rabbit sera recognised the

20 kDa expression product, and no reaction was

obtained with normal bovine or rabbit sera.

Four of the five sera taken from cysticercosis patients and four of the six sera taken from

hydatidosis patients recognised the in vitro trans-

lated ferritin gene. T. saginata ferritin therefore

appears to share epitopes present? on E. granulo- sus ferritin, while the corollary is not true of E.

granulosus ferritin [9]. In addition, indications are that the molecule also shares epitopes found on

L. Benitez et al. / Molecular and Biochemical Parasitology 82 (1996) 113- 116 115

123456789 10 11 12 13 14 15 16 17 18 19

KDa t

32.5 _ 27 5 _

185, ; ll< _^

Fig. 2. lmmunoprecipitation of the in vitro translated ferritin clone by sera from animals immunised with T. saginata extracts and

humans with helminth infections. (I) Total proteins obtained from in vitro translation of the ferritin clone; (2) rabbit anti-human

ferritin serum; (3) bovine anti-i’ saginata oncospheres extract serum; (4) normal cow serum; (5) rabbit anti-T. saginata oncospheres

extract serum; (6) normal rabbit serum: (7-11) cysticercosis human sera; (12-17) hydatidosis human sera; (18, 19) normal human

sera. The in vitro transcription/translation (TnT Coupled Reticulocyte Lysate System, Promega) using T3 RNA polymerase was

carried out directly employing pBluescript cDNA insert from the clone. The [35S]methionine-labelled proteins (Amersham) were

immunoprecipitated with slight modification according to Ersfeld and Craig [9]. The polyclonal anti-human ferritin rabbit sera

(Sigma) was included as a positive control and normal rabbit, cattle and human sera served as negative controls.

trematodes, as sera from schistosomiasis (1:2) and fasciolosis (1:2) patients also reacted with the T. suginata ferritin (data not shown). Thus T. sugi- nata ferritin is not an appropriate antigen for specific diagnosis of T. saginata infection. The results differ from those obtained with E. granulo- sus ferritin, which was only recognised by sera from cystic and alveolar echinococcosis patients, and not by patients with other helminthic diseases [9]. Interestingly the anti-human ferritin poly- clonal rabbit sera, did not immunoprecipitate the T. saginata ferritin, even though these molecules share a high homology (57.2%).

Since sera from human patients with cestode and trematode infections recognised T. saginata ferritin, and there is a lack of cross reactivity with human ferritin, the molecule may have a role as a potential vaccine candidate against helminth in- fection.

Acknowledgements

The research presented in this paper was sup- ported by a grant from the European Union, by the FISS (0321/94) and by the ODA/NRRD Ani- mal Health Programme. We would like to thank Dr. J.A. Onyango-Abuje of NVRC, KARI Kenya

for supplying the T. suginata eggs, Mr. S.H. Wright CTVM for his skilful technical assistance, Dr. Paul Kirkham, IAH Pirbright for his help with the preparation of the T. suginata library, Dr. J. Nieto, CNMVIS for his assistance in the screening of the T. saginata library and Dr. M. Rodriguez, CNMVIS for providing the human sera.

References

111

121

]31

]41

151

Harrison, L.J.S., Hammond, J.A. and Sewell, M.M.H.

(1996) Studies in helminthosis at the Centre for Tropical

Veterinary Medicine. Trop. Anim. Health Prod. 28, 23-

39.

Harrison, L.J.S. and Parkhouse, R.M.E. (1985) Antigens

of taeniid cestodes in protection, diagnosis and escape.

Curr. Top. Microbial. Immunol. 120, 159-172.

Harrison, L.J.S. and Parkhouse, R.M.E. (1986) Passive

protection against Taenia saginata infection in cattle by a

mouse monoclonal antibody reactive with the surface of

the invasive oncospheres. Parasite Immunol. 8, 319-332.

Benitez, L., Garate, T., Harrison, L.J.S., Kirkham, P.,

Brookes, S.M. and Parkhouse, R.M.E. (1996) Cloning

and sequencing of the gene encoding the principal ld-kDa

secreted antigen of activated oncospheres of Taenia sagi- nata. Mol. Biochem. Parasitol. 78, 2655268.

Harrison, L.J.S. and Parkhouse, R.M.E. (1989) Taenia saginata and Taenia solium - reciprocal models. Pro-

ceedings of the Symposium on Neurocysticercosis. Acta

Leidensia 57. 143- 152.

116 L. Benitez et al. / Molecular and Biochemical Parasitology 82 (1996) 1 i3-1 I6

[6] Short, J.M., Fernandez, J.M., Sorge, J.A. and Huse, W.D. (1988) h-ZAP: a bacteriophage expression vector with in-vivo excision properties. Nucleic Acids Res. 16, 7583-7600.

[7] Devereux, J.R., Haeberli, P.L. and Smithies, 0. (1984) A comprehensive set of sequence analysis programmes for the VAX. Nucleic Acids Res. 12, 387-395.

[8] Juckett, M.B., Balla, J., Jessurun, J. and Vercellotti, GM. (1995) Ferritin protects endothelial cells from oxidized low density lipoprotein in vitro. Am. J. Pathol. 147, 782-789.

[9] Ersfeld, K. and Craig, P.S. (1995) Cloning and immuno- logical characterisation of Echinococcus granulosus fer- ritin. Parasitol. Res. 81, 382-387.

[IO] Jameson, B.A. and Wolf, H. (1988) The antigenic index: a novel algorithm for predicting antigenic determinants. Comput. Appl. Biosci. 4, 181-186.

[11] Dietzel, J., Hirzmann, J., Preis, D., Symmons, P. and Kunz, W. (1992) Ferritins of Schistosoma mansoni: se- quence comparison and expression in female and male worms. Biochem. Parasitol. 50, 245-254.

[12] Treffy, A., Hirzmann, J., Yewdall, S.J. and Harrison, P.M. (1992) Mechanism of catalysis of Fe(H) oxidation by ferritin H chains. FEBS Lett. 302, 108-I 12.

[13] Boyd, D., Vecoli, C., Belcher, D.M., Jain, S.K. and Drysdale, J.W. (1985) Structural and functional relation- ships of human ferritin H and L chains deduced from cDNA clones. J. Biol. Chem. 260, 11755-11761.

[14] Murray, M.T., White, K. and Munro, H.N. (1987) Con- servation of ferritin heavy subunit gene structure: implica- tions for the regulation of ferritin gene expression. Proc. Natl. Acad. Sci. USA 84, 74387442.

[15] Stevens, P.W., Dodgson, J.B. and Engel, J.D. (1987) Structure and expression of the chicken ferritin H-subunit gene. Mol. Cell. Biol. 7, 1751-1758.

[16] Dickey, L.F., Sreedharan, S., Theil, EC., Didsbury, J.R., Wang, Y.-H. and Kaufman, R.E. (1987) Differences in the regulation of messenger RNA for housekeeping and specialized-cell ferritin. J. Biol. Chem. 262, 7901-7907.

[17] Moskaitis, J.E., Pastori, R.L. and Schoenberg, D.R. (1990) Sequence of Xenopus laeuis ferritin mRNA. Nu- cleic Acids Res. 18, 2184.