Analysis of the Antibody Response to an Immunodominant Epitope of the Envelope Glycoprotein of a Lentivirus and Its Diagnostic Potential

Abstract

The envelope glycoprotein of small ruminant lentiviruses (SRLV) is a major target of the humoral immune response and contains several linear B-cell epitopes. We amplified and sequenced the genomic segment encoding the SU5 antigenic site of the envelope glycoprotein of several SRLV field isolates. With synthetic peptides based on the deduced amino acid sequences of SU5 in an enzyme-linked immunosorbent assay (ELISA), we have (i) proved the immunodominance of this region regardless of its high variability, (ii) defined the epitopes encompassed by SU5, (iii) illustrated the rapid and peculiar kinetics of seroconversion to this antigenic site, and (iv) shown the rapid and strong maturation of the avidity of the anti-SU5 antibody. Finally, we demonstrated the modular diagnostic potential of SU5 peptides. Under Swiss field conditions, the SU5 ELISA was shown to detect the majority of infected animals and, when applied in a molecular epidemiological context, to permit rapid phylogenetic classification of the infecting virus.Caprine arthritis-encephalitis virus (CAEV) of goats and maedi-visna virus (MVV) of sheep belong to the genus Lentivirus of the family Retroviridae (18,22,39). Strong evidence indicates that cross-species transmission from sheep to goats and vice versa occurs under field conditions (16, 24, 33). Therefore, these viruses are no longer considered to be species specific and are referred to as small-ruminant lentiviruses (SRLV).The majority of infected animals mount a strong immune response to these viruses but remain persistently infected. Only one-third of infected goats develop overt clinical disease, and in sheep the percentage of animals with clinical symptoms differs greatly between breeds, strongly suggesting that, in both species, genetic factors play a key role in determining the clinical outcome of infection (11,28,29).In several countries, eradication programs have been initiated to control SRLV-induced diseases with the aim of eliminating these viruses (23). The Swiss CAEV eradication program, initiated in 1984, has reduced the seroprevalence from 60 to 80% to less than 1% and eliminated clinical cases in the goat population (14, 15). However, in the last phase of the eradication program, detection and elimination of the remaining virus carriers appear to be very difficult. The serological tools currently used (37, 38) are of limited use when applied to screen a population with a low seroprevalence. In the absence of reliable tools to directly detect SRLV, consistent serological diagnoses depend on the costly use of a combination of tests run in parallel (4). Major problems in SRLV serology are slow seroconversions and low titers of antibody to Gag in some animals. In this respect, the strong immunogenicity of the envelope glycoprotein (Env) and especially the rapid seroconversion induced by this antigen in infected animals make it an ideal candidate for diagnostic applications (2, 19). Goats and sheep infected with SRLV develop high titers of antibodies to several conformational and linear ...

Seroconversion against SU5 derived synthetic peptides in sheep experimentally infected with different SRLV genotypes

“…Five epitopes have been identified in the SU subunit of Env (Bertoni et al, 2000 andValas et al, 2000). SU5 in particular, a 25 aminoacid sequence located at the C terminus of SU, is immunodominant and partly type--specific: it carries at least three antibody--binding sites, one in the N--terminal conserved region, one within the C--terminal most variable domain, and the third encompassing the junction between the variable and conserved regions (Mordasini et al, 2006). In this study, two env gene fragments encoding respectively It--561 and It--Pi1 SU5, were sequenced.…”

“…The avidity index values of SU5--specific antibodies were measured by testing the stability of the antigenantibody complexes following a wash in 8 M urea (Mordasini et al, 2006). Antibodies with avidity indexes <30% were considered to be of low affinity; those with values between 30% and 50% of intermediate avidity and those with values >50% of high avidity.…”

“…Cloning, sequencing and sequence analysis of env region coding for SU5 An env gene fragment encompassing the 75 bp "SU5 total" domain ( Mordasini et al, 2006) coding sequence (nt 7800-7874) was amplified from DNA of foetal ovine lung fibroblasts infected respectively with It--561 and It--Pi1 viral stocks. Primer sequences ( Bertoni et al, 2000) were as follows: 563F: GAYATGRYRGARCAYATGAC (nt.…”

Presence of specific antibodies and proviral DNA of small ruminant lentiviruses in lambs in their first weeks of life

“…Two fragments, V4V5 (608 bp) and V1V2 (394 bp), and one selected from the variable part of env gene as well as the fragment of gag gene encoding capsid protein CA (624 bp), were amplified by nested PCR. The V4V5 and V1V2 fragments were amplified with Ptat and Penv primers (21), in the first round of PCR and primers A51/B31 and 567/564 in the second run (21,10). The primer GAGf1/P15 was used for the first amplification and CAGAG5/CAGAG3 for the second when PCR amplified the Gag fragment (22,27).…”

Impact of Corynebacterium Pseudotuberculosis Infection on Serologic Surveillance for Johne's Disease in Goats

“…a,2 Blood samples were collected from the jugular vein into tubes without anticoagulant and centrifuged within 24 hours for serum collection. Each serum sample was tested with 2 commercially available JD ELISAs: one licensed for use in small ruminants (Parachek) b and the other, in 2004, containing a protein G conjugate (HerdChek) c known to be capable of binding antibodies from multiple species, 9,12,13,[16][17][18] including goats. Both assays are used globally for JD surveillance in small ruminants (unpublished survey, 8th Int Colloq Paratuberculosis).…”