Magdalena Eschricht

Generation of neutralising antibodies against porcine endogenous retroviruses (PERVs)

Antibodies neutralising porcine endogenous retroviruses (PERVs) were induced in different animal species by immunisation with the transmembrane envelope protein p15E. These antibodies recognised epitopes, designated E1, in the fusion peptide proximal region (FPPR) of p15E, and E2 in the membrane proximal external region (MPER). E2 is localised in a position similar to that of an epitope in the transmembrane envelope protein gp41 of the human immunodeficiency virus-1 (HIV-1), recognised by the monoclonal antibody 4E10 that is broadly neutralising. To detect neutralising antibodies specific for PERV, a novel assay was developed, which is based on quantification of provirus integration by real-time PCR. In addition, for the first time, highly effective neutralising antibodies were obtained by immunisation with the surface envelope protein of PERV. These data indicate that neutralising antibodies can be induced by immunisation with both envelope proteins.

Modulation of cytokine release and gene expression by the immunosuppressive domain of gp41 of HIV-1.

The transmembrane envelope protein gp41 of the human immunodeficiency virus HIV-1 plays an important role during infection allowing fusion of the viral and cellular membrane. In addition, there is increasing evidence that gp41 may contribute to the immunodeficiency induced by HIV-1. Recombinant gp41 and a synthetic peptide corresponding to a highly conserved domain in gp41, the immunosuppressive (isu) domain, have been shown to inhibit mitogen-induced activation of human peripheral blood mononuclear cells (PBMCs) and to increase release of IL-6 and IL-10 from these cells. We recently reported that a single mutation in the isu domain of gp41 abrogated the immunosuppressive properties and that HIV-1 sequences containing such abrogating mutations had never been isolated from infected individuals. Here, we studied the influence of the isu peptide on the release of 66 cytokines and the expression of 27,000 genes in PBMCs. Incubation of PBMCs with isu peptide homopolymers increased the expression of 16 cytokines among them IL-6 and IL-10, and decreased that of IL-2 and CXCL9. Interestingly, the extend of cytokine modulation was donor-dependent. Among the genes up-regulated were IL-6, IL-8, IL-10 but also MMP-1, TREM-1 and IL-1beta. Most importantly, genes involved in innate immunity such as FCN1 and SEPP1 were found down-regulated. Many changes in cytokine expression demonstrated in our experiments were also found in HIV-1 infected individuals. These data indicate that the isu domain of gp41 has a broad impact on gene expression and cytokine release and therefore may be involved in HIV-1 induced immunopathogenesis.

Mode of interaction between the HIV-1-neutralizing monoclonal antibody 2F5 and its epitope.

Objective:To determine the mechanism of interaction between the HIV-1 gp41-specific broadly neutralizing monoclonal antibody (mAb) 2F5, its epitope in the membrane proximal external region and a domain located in the fusion peptide proximal region in the N-terminal region of gp41. Knowledge of these interactions would be useful for the design of antigens used to induce 2F5-like antibodies. Methods:The binding and avidity of the mAb 2F5 were analyzed using enzyme-linked immunosorbent assays, epitope mapping and surface plasmon resonance analysis. Inhibition of virus neutralization by 2F5 was analyzed using peptides corresponding to the gp41 sequence. Results:Using transmembrane envelope proteins of gammaretroviruses, we had previously induced neutralizing antibodies that recognize two epitopes, one located in the N-terminal part of the transmembrane protein (designated E1) and the other in the C-terminal membrane proximal external region (E2). The E2 epitope corresponds to the mAb 2F5/4E10 epitope in the gp41 of HIV and we have now identified a corresponding E1 domain in gp41. Although 2F5 did not bind directly to E1, the presence of E1 peptides increased the binding of 2F5 to peptides carrying its epitope. Neutralization of HIV-1 by 2F5 was inhibited more effectively by both gp41-derived peptides E1 and E2 together than with the peptide E2 alone. Conclusion:The interaction between the E1 and E2 domains of gp41 increased the efficacy of mAb 2F5 binding to its E2 epitope. Such an interaction may occur after gp41 folding into a six-helix bundle. Antigens containing both domains might be used to induce broadly neutralizing 2F5-like antibodies.

Neutralization of porcine endogenous retrovirus by antibodies against the membrane-proximal external region of the transmembrane envelope protein

Immunization of different species including goats, rats, hamsters and guinea pigs with the recombinant ectodomain of the transmembrane envelope (TM) protein p15E of porcine endogenous retrovirus (PERV) has been shown to result in the production of virus-neutralizing antibodies. The sera recognize two groups of epitopes, one located in the fusion peptide-proximal region (FPPR) and the second in the membrane-proximal external region (MPER) of p15E. Most interestingly, the epitopes in the MPER are similar to epitopes in the TM protein gp41 of human immunodeficiency virus type 1 (HIV-1) recognized by mAbs 2F5 and 4E10, which broadly neutralize HIV-1. To study which epitope and which antibody population are involved in the process of neutralization of PERV, this study generated a new antiserum in a goat using an elongated ectodomain of p15E. The immune serum neutralized PERV at a higher titre and recognized broader epitopes in the FPPR and MPER of p15E. For the first time, antibody subpopulations were isolated from this serum using affinity chromatography with immobilized proteins and peptides corresponding to the FPPR and MPER of p15E. Only the affinity-purified antibodies specifically binding the MPER neutralized PERV, indicating that, as in the case of HIV-1, the MPER is an important target of neutralizing activity.

Investigation of membrane protein—protein interactions using correlative FRET-PLA

Fluorescence resonance energy transfer (FRET) analysis and the recently developed proximity ligation assay (PLA) are widely used to study protein-protein interactions in situ. We have developed correlative FRET-PLA to monitor interactions between membrane proteins that frequently cause problems in confirmatory co-immunoprecipitation assays. Correlative FRET-PLA is particularly aimed at delivering robust and reliable results and is useful for investigating protein-protein interactions.

Characterization of a monoclonal anti-capsid antibody that cross-reacts with three major primate lentivirus lineages

Mouse monoclonal antibodies with varying specificities against the Gag capsid of simian and human immunodeficiency virus (SIV/HIV) were generated by immunizing mice with whole inactivated SIVagmTYO-1. Monoclonal antibody AG3.0 showed the broadest reactivity recognizing the Gag capsid protein (p24-27) and Gag precursors p38, p55, and p150 of HIV-1, HIV-2, SIVmac, and SIVagm. Using overlapping peptides, the AG3.0 epitope was mapped in capsid to a sequence (SPRTLNA) conserved among HIV-1, HIV-2, SIVrcm, SIVsm/mac, and SIVagm related viruses. Because of its broad cross-reactivity, AG3.0 was used to develop an antigen capture assay with a lower detection limit of 100 pg/ml HIV-1 Gag p24. Interestingly, AG3.0 was found to have a faster binding on/off rate for SIVagmVer and SIVmac Gag than for SIVagmSab Gag, possibly due to differences outside the SPRTLNA motif. In addition, the ribonucleic acid (RNA) coding for AG3.0 was sequenced to facilitate the development of humanized monoclonal antibodies.

Improved split-ubiquitin screening technique to identify surface membrane protein-protein interactions

Yeast-based methods are still the workhorse for the detection of protein-protein interactions (PPIs) in vivo. Yeast two-hybrid (Y2H) systems, however, are limited to screening for a specific group of molecules that interact in a particular cell compartment. For this reason, the split-ubiquitin system (SUS) was developed to allow screening of cDNA libraries of full-length membrane proteins for protein-protein interactions in Saccharomyces cerevisiae. Here we demonstrate that a modification of the widely used membrane SUS involving the transmembrane (TM) domain of the yeast receptor Wsc1 increases the stringency of screening and improves the selectivity for proteins localized in the plasma membrane (PM).

P19-31. Neutralizing antibodies specific for the transmembrane envelope proteins of gammaretroviruses and of HIV-1 (2F5, 4E10): comparison of their action

Background Antibodies broadly neutralizing HIV-1 such as 2F5 and 4E10 recognize one epitope in the membrane-proximal external region (MPER) of the HIV transmembrane envelope (TM) protein gp41 MPER of gp41 (ELDKWA, NWF(D/N)IT, respectively). We reported previously, that immunizing with the TM protein of different gammaretroviruses such as the porcine endogenous retrovirus, PERV, and the feline leukemia virus, FeLV, neutralizing antibodies were obtained, that recognized two epitopes. One epitope was located in the fusion peptide-proximal region (FPPR) (epitope 1, E1), the other in the MPER of p15E (E2). E2 of gammaretroviruses was similarly located as NWF(D/N)IT (E2) of HIV-1 and despite the evolutionary separation of HIV and gammaretroviruses, an unexpected sequence homology was observed in this region (epitope FEGWFN) (Fiebig et al., Virology, 307,406, 2003; Langhammer et al., Immunology, 117, 229, 2005; Vaccine, 23, 3341, 2005). We also reported identification of an E1 region in gp41 of HIV-1 located similarly as E1 of gammaretroviruses (Fiebig et al., AIDS, 23, 887, 2009) and enhancing the binding of 2F5 and 4E10 to their epitopes in E2.