Immunological properties of the transmembrane envelope protein of the feline foamy virus and its use for serological screening

Abstract

The transmembrane envelope (TM) proteins of retroviruses are used as antigen in diagnostic immunoassays and they represent a conserved target for neutralizing antibodies. To analyze the situation in infections with the feline foamy virus (FFV), its recombinant TM protein was produced and used for ELISA and Western blot analyses. Screening sera from 404 German cats showed that 39% reacted against the TM protein, the same infection rate was determined using the Gag protein. Epitope mapping showed antibodies against the membrane proximal external region (MPER) of the TM protein in the sera from infected cats, but attempts to induce neutralizing antibodies by immunization with the recombinant TM protein failed. This is the first report demonstrating that the TM protein of the FFV is highly immunogenic and valuable for serological screening. Similar to HIV-1, but in contrast to different gammaretroviruses, immunization with the TM protein of FFV did not induce neutralizing antibodies.

Optimisation of expression and purification of the feline and primate foamy virus transmembrane envelope proteins using a 96 deep well screen

“…Both GST-fusion proteins were extracted from the insoluble fraction by incubation with 1% sarkosyl followed by a subsequent refolding step by dilution of the lysate before affinity purification (see Material and methods). Using this purification protocol the protein yields of the TM protein of FFV was drastically improved from about 0.5-0.8 mg/l originally obtained with the previously described on-column refolding protocol [17] up to 14-16 mg/l ( Table 1). The protein yields obtained for the TM protein of PFV were in the same range (15-18 mg/l, Table 1).…”

“…Detection was performed using OPD (o-phenylenediamine dihydrochloride) substrate dissolved in PBS containing hydrogen peroxide and the absorbance was measured at 492 nm and 620 nm after 10 minutes of incubation. As controls a TM protein specific antiserum (goat serum 348, [17]) and the secondary antibody were used. Cut offs were calculated as the mean absorbance of 14 included negative sera plus 2.743 times the mean standard deviation corresponding to a statistical confidence level of 99% [26].…”

“…To compare the ability to detect antibodies in sera of infected cats using the TM protein of FFV purified by the sarkosyl extraction method with that obtained by on-column refolding as used recently, [17], a comparative ELISA was performed. 30 sera with known FFV serological status (16 positive, 14 negative as determined previously by ELISA using Gag, Bet and TM proteins and immunoblots using infected CRFK cells [17,18]) were selected for this screening.…”

“…The TM proteins are larger, their central part is characterised by the presence of seven to eight cysteines and numerous potential glycosylation sites [14,15] and electron microscopic pictures demonstrate an unusual trimeric oligomerisation [16]. Recently the TM protein of the feline foamy virus (FFV) was produced and used for serological screening of infections in cats and for immunisation studies [17]. When compared with diagnostic methods based on the detection of antibodies against Gag, detection of antibodies against the TM protein was found to be a reliable diagnostic tool [17,18].…”

“…Recently the TM protein of the feline foamy virus (FFV) was produced and used for serological screening of infections in cats and for immunisation studies [17]. When compared with diagnostic methods based on the detection of antibodies against Gag, detection of antibodies against the TM protein was found to be a reliable diagnostic tool [17,18]. In this report, an improved protocol for the expression and purification of this protein as well as for the TM protein of the primate foamy virus (PFV)is described.…”