Epitope Mapping of the Antibody Response Against the Envelope Proteins of the Feline Foamy Virus.

Foamy viruses (FV) are retroviruses that infect several species without pathological signs, but induce substantial antibody responses in the infected host. In the case of feline FV (FFV), antibodies against Gag, Bet, and Env have been used to indicate infection; however, it is unclear whether the response to specific epitopes correlates with immunity. Here, we investigated the epitope specificity of antibodies targeting the Env protein using peptide microarrays. Sera from naturally and experimentally FFV-infected cats and pumas and from rats immunized with FFV Env expression plasmids were analyzed. An immunodominant epitope was identified in the Env leader protein (Elp), and a strong reactivity to two epitope clusters in the transmembrane (TM) subunit of Env was observed. Moreover, a short stretch of residues in the C-terminal part of the surface (SU) protein was found to be significantly associated with FFV serotype FUV-mediated neutralization. Taken together, our results add a new level of detail on the B cell epitope repertoire induced during FFV infection. Furthermore, our results provide a basis for current attempts to modify FV vectors to express and present vaccine epitopes for the directed induction of humoral immunity.

Antigenic and immunosuppressive properties of a trimeric recombinant transmembrane envelope protein gp41 of HIV-1.

The transmembrane envelope (TM) protein gp41 of the human immunodeficiency virus-1 (HIV-1) plays an important role during virus infection inducing the fusion of the viral and cellular membranes. In addition, there are indications that the TM protein plays a role in the immunopathogenesis leading to the acquired immunodeficiency syndrome (AIDS). Inactivated virus particles and recombinant gp41 have been reported to inhibit lymphocyte proliferation, as well as to alter cytokine release and gene expression. The same was shown for a peptide corresponding to a highly conserved domain of all retroviral TM proteins, the immunosuppressive domain. Due to its propensity to aggregate and to be expressed at low levels, studies comprising authentic gp41 produced in eukaryotic cells are extremely rare. Here we describe the production of a secreted, soluble recombinant gp41 in 293 cells. The antigen was purified to homogeneity and characterised thoroughly by various biochemical and immunological methods. It was shown that the protein was glycosylated and assembled into trimers. Binding studies by ELISA and surface plasmon resonance using conformation-specific monoclonal antibodies implied a six-helix bundle conformation. The low binding of broadly neutralising antibodies (bnAb) directed against the membrane proximal external region (MPER) suggested that this gp41 is probably not suited as vaccine to induce such bnAb. Purified gp41 bound to monocytes and to a lesser extent to lymphocytes and triggered the production of specific cytokines when added to normal peripheral blood mononuclear cells. In addition, gp41 expressed on target cells inhibited the antigen-specific response of murine CD8+ T cells by drastically impairing their IFNγ production. To our knowledge, this is the first comprehensive analysis of a gp41 produced in eukaryotic cells including its immunosuppressive properties. Our data provide another line of evidence that gp41 might be directly involved in HIV-1 immunopathogenesis through modulation of the cytokine release and active inhibition of immune responses.

The immunosuppressive domain of the transmembrane envelope protein gp41 of HIV-1 binds to human monocytes and B cells.

The induction of the acquired immunodeficiency syndrome by the human immunodeficiency virus-1 (HIV-1) is a complex process which is not yet understood in full detail. Still open is the question whether the highly conserved so-called immunosuppressive (Isu) domain in the transmembrane envelope (TM) protein gp41 of HIV-1 is actively participating in immunopathogenesis. Inactivated virus particles, recombinant gp41 and peptides corresponding to the Isu domain have been reported to inhibit lymphocyte proliferation, as well as to alter cytokine release and gene expression. Here we demonstrate, using fluorescence-activated cell sorting and competition experiments, that homopolymers of the Isu peptide of HIV-1 are binding specifically to human peripheral blood mononuclear cells, mainly to monocytes and B cells. These data suggest that a putative receptor might be involved in the immunomodulatory effects observed previously.

Construction and characterisation of replicating foamy viral vectors expressing HIV-1 epitopes recognised by broadly neutralising antibodies.

With the aim to develop a replicating vector system for the delivery of HIV-1 antigens on the basis of an apathogenic foamy virus we recently showed that immunisation with purified recombinant hybrid antigens composed of the feline foamy virus Bet protein and parts of the transmembrane envelope protein of HIV-1 induced antibodies with an epitope specificity identical to that of the broadly neutralising antibody 2F5 (Mühle et al., Immunol Res., 2013, 56:61-72). Here we set out to further improve the HIV-1 inserts consisting of the membrane proximal external region (MPER) and the fusion peptide proximal region (FPPR) by stepwise shortening distinct linker residues between both domains. In a subset of these antigens, enhanced recognition by 2F5 and 4E10 was observed, indicating that a specific positioning of FPPR and MPER domains is critical for improved antibody binding. Introduction of these optimised inserts as well as of the MPER domain alone into the feline foamy virus backbone was compatible with virus replication, giving viral titres similar to wild-type virus after extended passaging. Most importantly, expression of the HIV-1 transgenes in infected feline CRFK cells remained stable in three out of four constructs and was detectable after serial passages for several weeks. These data encourage further testing of these vectors in vivo, which may allow insights into the necessity of affinity maturation for the induction of broadly reactive HIV-1 antibodies.

Identification of the feline foamy virus Bet domain essential for APOBEC3 counteraction.

BACKGROUND: APOBEC3 (A3) proteins restrict viral replication by cytidine deamination of viral DNA genomes and impairing reverse transcription and integration. To escape this restriction, lentiviruses have evolved the viral infectivity factor (Vif), which binds A3 proteins and targets them for proteolytic degradation. In contrast, foamy viruses (FVs) encode Bet proteins that allow replication in the presence of A3, apparently by A3 binding and/or sequestration, thus preventing A3 packaging into virions and subsequent restriction. Due to a long-lasting FV-host coevolution, Bet proteins mainly counteract restriction by A3s from their cognate or highly related host species. RESULTS: Through bioinformatics, we identified conserved motifs in Bet, all localized in the bel2 exon. In line with the localization of these conserved motifs within bel2, this part of feline FV (FFV) Bet has been shown to be essential for feline A3 (feA3) inactivation and feA3 protein binding. To study the function of the Bet motifs in detail, we analyzed the ability of targeted deletion, substitution, and chimeric FFV-PFV (prototype FV) Bet mutants to physically bind and/or inactivate feA3. Binding of Bet to feA3Z2b is sensitive to mutations in the first three conserved motifs and N- and C-terminal deletions and substitutions across almost the complete bel2 coding sequence. In contrast, the Bel1 (also designated Tas) domain of Bet is dispensable for basal feA3Z2b inactivation and binding but mainly increases the steady state level of Bet. Studies with PFV Bel1 and full-length FFV Bel2 chimeras confirmed the importance of Bel2 for A3 inactivation indicating that Bel1 is dispensable for basal feA3Z2b inactivation and binding but increases Bet stability. Moreover, the bel1/tas exon may be required for expression of a fully functional Bet protein from a spliced transcript. CONCLUSIONS: We show that the Bel2 domain of FV Bet is essential for the inactivation of APOBEC3 cytidine deaminase restriction factors. The Bel1/Tas domain increases protein stability and can be exchanged by related sequence. Since feA3 binding and inactivation by Bet are highly correlated, the data support the view that FV Bet prevents A3-mediated restriction of viral replication by creating strong complexes with these proteins.

Immunisation with foamy virus Bet fusion proteins as novel strategy for HIV-1 epitope delivery.

The induction of 2F5- and 4E10-like antibodies broadly neutralising HIV-1 and targeting the membrane external proximal region (MPER) of the transmembrane envelope protein gp41 would be a major advancement for the development of a preventive HIV-1 vaccine, but successful attempts remain rare. Recent studies demonstrated that broadly reactive antibodies develop relatively late during infection and after intensive affinity maturation. Therefore, a prolonged antigen delivery might be beneficial to induce them. Replicating foamy viruses which are characterised by apathogenic but persistent infection could represent suitable carrier viruses for this purpose. In order to develop such a system, we modified the accessory foamy virus Bet protein to contain the MPER of gp41, or the MPER linked to the stabilising fusion peptide proximal region of gp41 and analysed here the antigenic and immunogenic properties of such hybrid proteins. The antigens, expressed and purified to homogeneity, were recognised by the monoclonal antibodies 2F5 and 4E10 with nanomolar affinities and induced high levels of antibodies specific to gp41 after immunisation of rats. The antisera also bound to virus particles attached to infected cells, and peptide-based epitope mapping showed that they recognised the 2F5 epitope. Although no HIV-1 neutralising activity was observed, the presented data demonstrate that using the foamy virus Bet for HIV-1 epitope delivery is successfully applicable. Together with the attractive potential for sustained antigen expression after transfer to replicating virus, these results should therefore provide a first basis for the development of chimeric foamy viruses as novel HIV-1 vaccine vectors.

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

The production of recombinant transmembrane proteins is due to their biochemical properties often troublesome and time consuming. Here the prokaryotic expression and purification of the transmembrane envelope proteins of the feline and primate foamy viruses using a screening assay for optimisation of expression in 96 deep well plates is described. Testing simultaneously various bacterial strains, media, temperatures, inducer concentrations and different transformants, conditions for an about twentyfold increased production were quickly determined. These small scale test conditions could be easily scaled up, allowing purification of milligram amounts of recombinant protein. Proteins with a purity of about 95% were produced using a new purification protocol, they were characterised by gel filtration and circular dichroism and successfully applied in immunological assays screening for foamy virus infection and in immunisation studies. Compared to the previously described protocol (M. Mühle, A. Bleiholder, S. Kolb, J. Hübner, M. Löchelt, J. Denner, Immunological properties of the transmembrane envelope protein of the feline foamy virus and its use for serological screening, Virology 412 (2011) 333-340), proteins with similar characteristics but about thirtyfold increased yields were obtained. The screening and production method presented here can also be applied for the production of transmembrane envelope proteins of other retroviruses, including HIV-1.

Pattern of seroreactivity against feline foamy virus proteins in domestic cats from Germany.

The prevalence of feline foamy virus (FFV, spumaretrovirinae) in naturally infected domestic cats ranges between 30 and 80% FFV positive animals depending on age, sex and geographical region analyzed. Two serotypes have been reported for FFV designated FUV7-like and F17/951-like. Serotype-specific neutralization has been shown to correlate with sequence divergence in the surface (SU) domain of the envelope protein (Env). We analyzed a serum collection of 262 domestic cat sera from Germany using a GST-capture ELISA setup screening for Gag and Bet specific antibodies and identified 39% FFV positive animals. Due to the heterogeneity of the serological samples, cut-offs for Gag and Bet reactivity had to be experimentally determined since application of calculated cut-off values yielded some false-positive results; the new cut-off values turned out to be also fully applicable to a previous study. Using the already established FUV7 ElpSU antigen and the newly cloned and produced F17/951 ElpSU antigen, both consisting of the corresponding ectodomains of the envelope leader protein (Elp) and SU protein, we aimed at the detection of Env-specific antibodies and discrimination between the two known FFV serotypes within the diagnostic FFV ELISA. We validated the ElpSU antigens using cat reference sera of known serotype and screened with this assay domestic cat sera from Germany. Use of the FUV7- and F17/951 ElpSU antigens in ELISA resulted in the detection of Env-specific antibodies in both cat reference sera and sera from domestic cats in Germany, but failed to allow serotyping at the same time.

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

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.

A var gene promoter implicated in severe malaria nucleates silencing and is regulated by 3' untranslated region and intronic cis-elements.

Questions surround the mechanism of mutually exclusive expression by which Plasmodium falciparum mediates activation and silencing of var genes. These encode PfEMP1 proteins, which function as cytoadherent and immunomodulatory molecules at the surface of parasitised erythrocytes. Current evidence suggests that promoter silencing by var introns might play a key role in var gene regulation. To evaluate the impact of cis-acting regulatory regions on var silencing, we generated P. falciparum lines in which luciferase was placed under the control of an UpsA var promoter. By utilising the Bxb1 integrase system, these reporter cassettes were targeted to a genomic region that was not in apposition to var subtelomeric domains. This eliminated possible effects from surrounding telomeric elements and removed the variability inherent in episomal systems. Studies with highly synchronised parasites revealed that the UpsA element possessed minimal activity in comparison with a heterologous (hrp3) promoter. This may result from the integrated UpsA promoter being largely silenced by the neighbouring cg6 promoter. Our analyses also revealed that the DownsA 3' untranslated region further decreased the luciferase activity from both cassettes, whereas the var A intron repressed the UpsA promoter specifically. By applying multivariate analysis over the entire cell cycle, we confirmed the significance of these cis-elements and found the parasite stage to be the major factor regulating UpsA-promoter activity. Additionally, we observed that the UpsA promoter was capable of nucleating reversible silencing that spread to a downstream promoter. We believe these studies are the first to analyse promoter activity of Group A var genes, which have been implicated in severe malaria, and support the model that var introns can further suppress var expression. These data also suggest an important suppressive role for the DownsA terminator. Our findings imply the existence of multiple levels of var gene regulation in addition to intrinsic promoter-dependent silencing.