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Phage display identifies two Caprine Arthritis Encephalitis Virus env epitopes

Karlen Gazarian1*, Alvaro Aguilar Setién2, Tatiana Gazarian3 and Sebastian Aguilar Pierle4

Author Affiliations

1 Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México (UNAM), México D.F., Ciudad Universitaria, 04510, México

2 Unidad de Investigación Médica en Inmunología, Coordinación de Investigación IMSS Hospital de Pediatría 3er piso, Centro Medico Nacional Siglo XXI, Av. Cuauhtemoc 330, Doctores, México D. F., 06720 México

3 Facultad de Medicina, Universidad Nacional Autónoma de México, (UNAM), México D.F., Ciudad Universitaria, 04510, México

4 Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, PO Box 647040 Pullman, WA, USA

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Veterinary Research 2011, 42:87  doi:10.1186/1297-9716-42-87

The electronic version of this article is the complete one and can be found online at: http://www.veterinaryresearch.org/content/42/1/87


Received:30 December 2010
Accepted:22 July 2011
Published:22 July 2011

© 2011 Gazarian et al; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Using phage display and IgG of a goat infected with Caprine Arthritis Encephalitis Virus (CAEV) we obtained families of 7 mer constrained peptides with consensus motifs LxSDPF/Y and SWN/KHWSY and mapped the epitopes mimicked by them at the Env 6-LISDPY-11 and 67-WNTYHW-72 sites of the mature gp135 amino acid sequence. The first epitope fell into the N-terminal immunogenic aa1-EDYTLISDPYGFS- aa14 site identified previously with a synthetic peptide approach; the second epitope has not been described previously. The first epitope is mostly conserved across CAEV isolates whereas the second newly described epitope is extremely conserved in Small Ruminant Lentiviruses env sequences. As being immunodominant, the epitopes are candidate targets for mimotope-mediated diagnosis and/or neutralization.

Introduction, Methods and Results

Caprine arthritis-encephalitis virus, CAEV [1], and ovine maedi-visna virus, MVV [2] belong to the Lentivirus genus of the Retroviridae family. Since the first comparison in 1995 of pol gene sequences of French MVV isolates with CAEV [3] and subsequent extensive analyses (reviewed by [4]), studies have established that ovine MVV and caprine CAEV constitute a genetically heterogeneous group of pathogens that have evolved in small ruminants and are known as small ruminant lentiviruses (SRLV) [5-7]. Multiple observations support interspecies transmission of CAEV and MVV [5,8,9] in small domestic and wild ruminants, causing neurological, pulmonary, articular and mammary symptoms [1,4,10]. As transmission of CAEV repeatedly occurs in different regions [6,8,11,12], it affects herds worldwide and has a significant economic impact. The infection is clinically difficult to detect, hence sensitive diagnostic methods are of primary importance to prevent further distribution of the virus. Despite the availability of a large number of serological tests for CAEV [13], immunological detection is challenging due to the lack of low-cost and readily available recombinant antigens (see [14]). Testing sera with recombinant and synthetic peptides from different CAEV envelope (Env) regions permitted identification of several immunogenic regions [15-18]. Here we describe the first phage display mapping of epitopes of CAEV.

We used the VR905, CAEV 75-G63 strain, cloned lot 2D, 91-12, ATCC (American Type Culture Collection). Cell cultures of Mycoplasma-free goat synovial membrane (GSM) cells that had been propagated in Dulbecco-modified Eagle's medium (DMEM) containing 10% fetal bovine serum, penicillin 100 U/mL, streptomycin 100 mg/mL, and 2 µM L-glutamine were used. Goat synovial membrane cell monolayers were used for virus multiplication and were monitored twice a week for evidence of cytopathic effects (multinucleated giant cells, syncytia). The virus in cell supernatants was then titered through an endpoint dilution assay.

We infected a naïve 4-month old goat with the CAEV75-G63 strain. The goat was previously screened with a standard ELISA kit (CHEKIT CAEV/Maedi Visna Virus®, Behring, IL, USA) [19]). The goat was inoculated intravenously with 30 mL of GSM supernatant containing 1.5 × 105 TCID50 of CAEV. The titer was obtained indirectly by counting the number of syncytia. Goats infected with this inoculum consistently show signs of disease. Seroconversion of the infected goat can be detected by serological diagnosis (POURQUIER® ELISA Maedi-Visna/CAEV; One IDEXX Drive Westbrook, Maine, New England, USA).

IgG fraction purified from serum (see [20]) at 12-week post-infection (pi) was used to perform three-round panning of a 12 mer linear and a 7 mer constrained phage library (New England BioLabs Inc.,240 County Road, Ipswich, MA,USA) as previously described [20,21]. Twenty phage clones were isolated from each library; the sequences of DNA inserts in the phage pIII gene N-terminal region were determined using Sequenase kit and the amino acid sequences of peptides they encoded were deduced; reactivity of the IgG with phage clones was determined by standard phage ELISA [20,21]. Figures 1 and 2 depict the deduced sequences and reactivity levels shown by the selecting IgG with these clones in an ELISA test (clone no. 20 from the 7 mer library was tested for its reactivity but not sequenced). The shaded bars in Figure 2 show optical densities for wells containing PBS instead of IgG (blank). Clones with low signal (clone 14 from the 12 mer, and clones 12, 4, 6 from the 7 mer collections) serve as internal controls to indicate that the high optical density levels shown by the majority of the clones were due to reactivity with peptides and not phage antigens.

thumbnailFigure 1. Sequences of peptides retrieved by infected goat IgG from the 12 mer linear (A) and 7 mer constrained (B) random peptide libraries. B1 - non-homologous peptides, B2 - homologous motif group 1 peptides, B3 - homologous motif group 2 peptides. Consensus sequences are shown in bottom of both groups. EP 1 and EP2 - CAEV Env sequences matching the motif sequences and considered the identified epitope sites (the SU mature protein amino acids numbering). Note: the clone 20 peptide shown in Figure 2 was not sequenced.

thumbnailFigure 2. Reactivity of the goat IgG with clones selected from the 12 mer linear (A) and the 7 mer constrained (B) libraries. The shaded bars: PBS was added instead of the phage. Motif groups are shown.

The deduced sequences of peptides from the 12-mer linear library did not show a detectable consensus motif (Figure 1a). Conversely, two motif groups designated as motif groups 1 and 2 (Figure 1 panels b2 and b3, respectively) comprising altogether 50% of the deduced sequences (the rest of sequences showed no similarity with other ones, Figure 1, b1) were derived from the 7 mer constrained library.

A homology search revealed two sites in the Env N-terminal region (Figure 3) matching the above mimotope motifs. One of these (EP1), with CAEV gp135 aa6-LISDPY-aa11 mature protein sequence, falls into the immunogenic amino acids 1 to 14 segment described by Valas et al. [18]. The second epitope (EP2), located in the mature protein aa67-WNTYHW-aa72 site and matching the mimotopes (SWNHWSY, Figure 1, clones 11,17,20) has not been described previously. Alignments and other bioinformatic analyses were performed using the CLC Protein Workbench version 3.0 (CLC bio), which aligns nucleotides and proteins using a progressive alignment algorithm [22], and performs secondary structure [23], antigenicity [24] and hydropathic [25] predictions. The first epitope is located at the very end of a beta strand and the second in a region that showed no particular structures but is adjacent to an alpha helix. The first epitope site was in a region whose antigenicity varied in different isolates from -0.04 to -0.02. For the second epitope site the values ranged from -0.03 to -0.04. The predicted hydrophobicity for the first epitope site was significantly higher (from 1.5 to 1.9 in the isolates) than for the second epitope (-2.0 to -2.1). Sixteen different origin SRLV Env aligned sequences (Additional file 1) show that the second newly described epitope is extremely conserved across SRLV sequences. In contrast, the first previously described epitope is mostly conserved across CAEV isolates.

thumbnailFigure 3. Localization of epitopes on the CAEV Env N-terminal part of the mature protein.

Additional file 1. Alignment of 16 env sequences showing the Ep1 (red) and Ep2 (yellow) sequence conservation.

Format: DOC Size: 45KB Download file

This file can be viewed with: Microsoft Word ViewerOpen Data

Discussion

Previous work by Bertoni's [16,17] and Valas' [18] groups with recombinant and synthetic peptides describe several multi-epitope immunogenic regions throughout the length of Env subunit SU. In this study, we took advantage of the following characteristics of high throughput phage display epitope mapping [26]: (1) apart from identification of epitopes, it highlights their crucial amino acids; (2) it provides families of antigenic mimics, mimotopes, that may be used as novel diagnostic and antiviral antigens, (3) the epitopes identified are very likely to be immunodominant (gauged by the appearance of its mimics in 50% of the 19 sequenced clones randomly picked from the screened 7 mer library of 108 phages). Two libraries with different peptide lengths and conformation were used. As shown by our previous screenings, one of the libraries can mimic an epitope more successfully than the other depending on its structure [20,21]. Here the 7 mer cyclic peptides mimicked the epitopes more adequately. To obtain serum-containing antibodies elicited by immunodominant epitopes, we infected a goat with CAEV. Although goats infected with CAEV75-G63 mount an early (3 to 4 weeks) response to Gag and Env, seroconversion to immunodominant epitopes such as TM3 [15], a homologue of the HIV-1 principal immunodominant epitope [20,26] was shown to delay significantly, appearing between week 12 and 28 pi. Considering this, we took serum at 12 weeks pi and obtained antibodies that successfully selected groups of five and four mimics of epitopes 1 and 2, respectively. Our data show that seroconversion took place when using immunodominant epitope Ep2 at 12-weeks pi.

The coincidence of one of our mapped epitopes, aa6-LISDPY-aa11, with the previously identified immunogenic aa1- aa14 site [18] validates our selection results. The second epitope, aa-67 PWNTYHW-aa72, has not been described previously. This epitope was not precisely mapped in Bertoni's experiment due to the large size of peptides they used as antigens [16].

In the experiments by Valas et al. [18], peptide No. 10 with aa64-aa77 sequence covering our epitope aa67-aa72 sequence was recognized by 4% of the tested sera but in their study the criterion for the immunodominance was the number of positive immune sera to an individual peptide and not the dominance of the antibody in the serum which is the criterion in phage display mapping. Hence, the authors focused on the peptides recognized by more than 10% of sera. Both of these epitopes fell within a relatively short (72 aa) N-terminal section of the mature protein corresponding to SU1 and SU2 [16]. The fact that phage display did not detect the earlier reported [16,17] C-terminal SU5 immunogenic domain (underlined in Figure 3) confirms the relevance of the conformational structure of epitopes in this region because the method experiences difficulties with this type of epitope [21] (reviewed by [27]).

In conclusion, this first phage display study describes two discrete epitopes on CAEV gp135 N-terminal segment preceding the first variable V1 domain of the protein. One of them coincides with the previously reported N-terminal immunogenic site; the other was discovered for the first time. Apart from precise mapping of these epitope cores, the study highlights their critical (contact) amino acids and some secondary structure features. In forthcoming experiments the epitopes will be subjected to functional analysis to determine the potentials of their mimotopes for diagnosis of the infection and for virus neutralization (as done for HIV-1 [26,28]).

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

KG conceived the study, and participated in its design, coordination, contributed to the analysis of the results and preparation of initial and revised manuscript versions. AGS participated in designing of the experimental strategy, carried out the goat immunization, obtaining and characterization of sera; TG carried out the biopanning experiments, immunization of mice with mimotopes and immuno-testing the anti-mimotope sera; SAP - carried out the computer analysis of selected peptides and Env sequences, contributed to the preparation of the manuscript. All authors read and approved the final manuscript.

Acknowledgements

This study was in part supported by the grant from Mexican National University D.G.A.P.A. Foundation to KG and CONACYT-SAGARPA 11734 grant to AAS.

References

  1. Crawford TB, Adams DS, Cheevers WP, Cork L: Chronic arthritis in goats caused by a retrovirus.

    Science 1980, 207:997-999. PubMed Abstract | Publisher Full Text OpenURL

  2. Pepin M, Vitu C, Russo P, Mornex JF, Peterhans E: Maedi-visna virus infection in sheep: a review.

    Vet Res 1998, 29:341-367. PubMed Abstract OpenURL

  3. Leroux C, Vuillermoz S, Mornex J-F, Greenland T: Genomic heterogeneity in the pol region of ovine lentiviruses obtained from bronchoalveolar cells of infected sheep from France.

    J Gen Virol 1995, 76:1533-1537. PubMed Abstract | Publisher Full Text OpenURL

  4. Leroux C, Cruz JC, Mornex JF: SRLVs: a genetic continuum of lentiviral species in sheep and goats with cumulative evidence of cross species transmission.

    Curr HIV Res 2010, 8(1):94-100. PubMed Abstract | Publisher Full Text OpenURL

  5. Leroux C, Chastang J, Greenland T, Mornex JF: Genomic heterogeneity of small ruminant lentiviruses: existence of heterogeneous populations in sheep and of the same lentiviral genotypes in sheep and goats.

    Arch Virol 1997, 142:1125-1137. PubMed Abstract | Publisher Full Text OpenURL

  6. Peterhans E, Greenland T, Badiola J, Harkiss G, Bertoni G, Amorena B, Eliaszewicz M, Juste RA, Krassnig R, Lafont JP, Lenihan P, Petursson G, Pritchard G, Thorley J, Vitu C, Mornex JF, Pepin M: Routes of transmission and consequences of small ruminant lentiviruses (SRLVs) infection and eradication schemes.

    Vet Res 2004, 35:257-274. PubMed Abstract | Publisher Full Text OpenURL

  7. Valas S, Benoit C, Guionaud C, Perrin G, Mamoun RZ: North American and French arthritis-encephalitis viruses emerge from ovine maedi-visna viruses.

    Virology 1997, 237:307-318. PubMed Abstract | Publisher Full Text OpenURL

  8. Pisoni G, Bertoni G, Puricelli M, Maccalli M, Moroni P: Demonstration of coinfection with and recombination by caprine arthritis-encephalitis virus and maedi-visna virus in naturally infected goats.

    J Virol 2007, 81(10):4948-4955. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  9. Shah C, Boni J, Huder JB, Vogt HR, Muhlherr J, Zanoni R, Miserez R, Lutz H, Schupbach J: Phylogenetic analysis and reclassification of caprine and ovine lentiviruses based on 104 new isolates: evidence for regular sheep-to-goat transmission and worldwide propagation through livestock trade.

    Virology 2004, 319:12-26. PubMed Abstract | Publisher Full Text OpenURL

  10. Crawford TB, Adams DS: Caprine arthritis encephalitis: clinical features and presence of antibody in selected goat populations.

    J Am Vet Med Assoc 1981, 178:713-719. PubMed Abstract OpenURL

  11. Daltabuit Test M, de la Concha Bermejillo A, Espinosa LEL, Loza Rubio E, Aguilar Setién A: Isolation of caprine arthritis encephalitis virus from goats in Mexico.

    Can J Vet Res 1999, 63:212-215. PubMed Abstract | PubMed Central Full Text OpenURL

  12. Torres-Acosta JFJ, Gutierrez EJ, Butler V, Schmidt A, Evans J, Babington J, Bearman K, Fordham T, Brownlie T, Schroer S, Camara-G E, Lightsev J: Serological survey of caprine arthritis-encephalitis virus in 83 goat herds of Yucatan, Mexico.

    Small Rumin Res 1999, 49:207-211. OpenURL

  13. De Andrés D, Klein D, Watt NJ, Berriatua E, Torsteinsdottir S, Blacklaws BA, Harkiss GD: Diagnostic tests for small ruminant lentiviruses.

    Vet Microbiol 2005, 107(1-2):49-62. PubMed Abstract | Publisher Full Text OpenURL

  14. Konishi M, Yamamoto T, Shimada T, Shirafuji H, Kameyama K, Sentsui H, Murakami K: Development of enzyme-linked immunosorbent assay for detection of antibody against Caprine arthritis encephalitis virus using recombinant protein of the precursor of the major core protein, p55gag.

    J Vet Diagn Invest 2010, 22(3):415-419. PubMed Abstract | Publisher Full Text OpenURL

  15. Bertoni G, Zahno M-L, Zanoni R, Vogt HR, Peterhans E, Ruff G, Cheevers WP, Sonigo P, Pancino G: Antibody reactivity to the immunodominant epitopes of the caprine arthritis-encephalitis virus gp38 transmembrane protein associates with the development of arthritis.

    J Virol 1994, 68:7139-7147. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  16. Bertoni GC, Hertig ML, Zahno HR, Vogt S, Dufour P, Cordano E, Peterhans WP, Cheevers PW, Sonigo G, Pancino G: B-cell epitopes of the envelope glycoprotein of caprine arthritis-encephalitis virus and antibody response in infected goats.

    J Gen Virol 2000, 81:2929-2940. PubMed Abstract | Publisher Full Text OpenURL

  17. Mordasini F, Vogt HR, Zahno M-L, Maeschli A, Nenci C, Zanoni R, Peterhans E, Bertoni G: Analysis of the antibody response to an immunodominant epitope of the envelope glycoprotein of a lentivirus and its diagnostic potential.

    J Clin Microbiol 2006, 44(3):981-991. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  18. Valas S, Benoit C, Baudry C, Perrin G, Mamoun RZ: Variability and immunogenicity of caprine arthritis-encephalitis virus surface glycoprotein.

    J Virol 2000, 74(13):6178-6185. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  19. Juste RA, Kwang J, de la Concha-Bermejillo A: Dynamics of cell-associated viremia and antibody response during the early phase of lentivirus infection in sheep.

    Am J Vet Res 1998, 59:563-568. PubMed Abstract OpenURL

  20. Palacios-Rodriguez Y, Gazarian T, Rowley M, Majluf-Cruz A, Gazarian K: Collection of phage-peptide probes for HIV-1 immunodominant loop-epitope.

    J Microbiol Methods 2007, 68:225-235. PubMed Abstract | Publisher Full Text OpenURL

  21. Gazarian T, Selisko B, Gurrola GB, Hernández R, Possani LD, Gazarian K: Potential of peptides selected from random phage-displayed libraries to mimic conformational epitopes: a study on scorpion toxin Cn2 and the neutralizing monoclonal antibody BCF2.

    Comb Chem High Throughput Screen 2003, 6:119-132. PubMed Abstract | Publisher Full Text OpenURL

  22. Eddy SR: Where did the BLOSUM62 alignment score matrix come from?

    Nat Biotechnol 2004, 22(8):1035-1036. PubMed Abstract | Publisher Full Text OpenURL

  23. Rost B: Review: protein secondary structure prediction continues to rise.

    J Struct Biol 2001, 134(2-3):204-218. PubMed Abstract | Publisher Full Text OpenURL

  24. Welling GW, Weijer WJ, van der Zee R, Welling-Wester S: Prediction of sequential antigenic regions in proteins.

    FEBS Lett 1985, 188(2):215-218. PubMed Abstract | Publisher Full Text OpenURL

  25. Kyte J, Doolittle RF: A simple method for displaying the hydropathic character of a protein.

    J Mol Biol 1982, 157(1):105-132. PubMed Abstract | Publisher Full Text OpenURL

  26. Gnann JW Jr, Nelson JA, Oldstone MBA: Fine mapping of an immunodominant domain in the transmembrane glycoprotein of human immunodeficiency virus.

    J Virol 1987, 61:2639-2641. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  27. Gazarian K: Drug Discovery and Design Via High Throughput Screening of Combinatorial Phage-Display Protein-Peptide Libraries. In Frontiers in Drug Design & Discovery. Volume 1. Edited by Caldwell G, Springer B, Atta-u-Rahman. Bentham Science Publishers, New York; 2005::29-67. PubMed Abstract | Publisher Full Text OpenURL

  28. Keller PM, Arnold BA, Shaw AR, Tolman RL, Van Middlesworth F, Bondy S, Rusiecki VK, Koenig S, Zolla-Pazner S, Conard P, Emini FA, Conley AJ: Identification of HIV vaccine candidate peptides by screening random phage epitope libraries.

    Virology 1993, 193:709-716. PubMed Abstract | Publisher Full Text OpenURL