Transmembrane Envelope Protein p15E Research Articles

Induction of neutralizing antibodies specific for the envelope proteins of the koala retrovirus by immunization with recombinant proteins or with DNA

BackgroundThe koala retrovirus (KoRV) is the result of a transspecies transmission of a gammaretrovirus with fatal consequences for the new host. Like many retroviruses, KoRV induces lymphoma, leukemia and an immunodeficiency that is associated with opportunistic infections in the virus-infected animals. We recently reported the induction of neutralizing antibodies by immunization with the recombinant ectodomain of the transmembrane envelope protein p15E of KoRV. Since the neutralization titers of the p15E-specific sera were only moderate, we investigated the use of the surface envelope protein gp70 to induce neutralizing antibodies.FindingsWe immunized rats and goats with the recombinant gp70 protein of the KoRV, an unglycosylated protein of 52kD (rgp70/p52) or with the corresponding DNA. In parallel we immunized with recombinant rp15E or with a combination of rp15E and rgp70/p52. In all cases binding and neutralizing antibodies were induced. The gp70-specific sera had titers of neutralizing antibodies that were 15-fold higher than the p15E-specific sera. Combining rp15E and rgp70/p52 did not significantly increase neutralizing titers compared to rgp70/p52 alone. High titers of neutralizing antibodies specific for gp70 were also induced by immunization with DNA. Since KoRV and PERV are closely related, we investigated cross-neutralization of the antisera. The antisera against p15E and gp70 of PERV and KoRV inhibited infection by both viruses.ConclusionThe envelope proteins of the KoRV may therefore form the basis of an effective preventive vaccine to protect uninfected koalas from infection and possibly an immunotherapeutic treatment for those already infected.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-015-0296-2) contains supplementary material, which is available to authorized users.

Novel neutralising antibodies targeting the N-terminal helical region of the transmembrane envelope protein p15E of the porcine endogenous retrovirus (PERV)

Previously, immunising different species with the transmembrane envelope protein p15E of the porcine endogenous retrovirus (PERV), neutralising antibodies were induced which recognised epitopes in the fusion peptide proximal region (FPPR) and in the membrane-proximal external region (MPER). Only the MPER-specific antibodies were shown to neutralise and these antibodies targeted epitopes in the MPER similarly localised as the epitopes recognised by antibodies broadly neutralising HIV-1 such as 2F5 and 4E10. To study whether neutralising antibodies could be induced immunising with subunits of p15E, recombinant proteins corresponding to the N-terminal, the C-terminal helical region (NHR, CHR) and a p15E with a mutation in the Cys-Cys loop were produced. Whereas none of these antigens induced MPER-specific neutralising antibodies, the animals immunised with the FPPR/NHR subunit and the mutated p15E produced neutralising antibodies binding to the NHR. Therefore, for the first time, antibodies specific for the NHR and neutralising PERV were described.

Increased titers of neutralizing antibodies after immunization with both envelope proteins of the porcine endogenous retroviruses (PERVs)

Despite enormous difficulties to induce antibodies neutralizing HIV-1, especially broadly neutralizing antibodies directed against the conserved membrane proximal external region (MPER) of the transmembrane envelope protein, such antibodies can be easily induced in the case of gammaretroviruses, among them the porcine endogenous retroviruses (PERVs). In addition to neutralizing antibodies directed against the transmembrane envelope protein p15E, neutralizing antibodies were also induced by immunization with the surface envelope protein gp70. PERVs represent a special risk for xenotransplantation using pig tissues or organs since they are integrated in the genome of all pigs and infect human cells and a vaccine may protect from transmission to the recipient. To investigate the effect of simultaneous immunization with both proteins in detail, a study was performed in hamsters. Gp70 and p15E of PERV were produced in E. coli, purified and used for immunization. All animals developed binding antibodies against the antigens used for immunization. Sera from animals immunized with p15E recognized epitopes in the MPER and the fusion peptide proximal region (FPPR) of p15E. One MPER epitope showed a sequence homology to an epitope in the MPER of gp41 of HIV-1 recognized by broadly neutralizing antibodies found in HIV infected individuals. Neutralizing antibodies were detected in all sera. Most importantly, sera from animals immunized with gp70 had a higher neutralizing activity when compared with the sera from animals immunized with p15E and sera from animals immunized with gp70 together with p15E had a higher neutralizing activity compared with sera from animals immunized with each antigen alone. These immunization studies are important for the development of vaccines against other retroviruses including the human immunodeficiency virus HIV-1.

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.

Increased Neutralizing Antibody Response after Simultaneous Immunization with Leucogen and the Feline Leukemia Virus Transmembrane Protein

To develop improved vaccination strategies against feline leukemia virus (FeLV), rats were immunized with the transmembrane envelope protein p15E of FeLV alone or in combination with the commercial vaccine Leucogen® comprising the nonglycosylated FeLV surface envelope protein. Binding and neutralizing antibodies were induced in both groups and in the group immunized with Leucogen alone. Higher titers of antibodies neutralizing FeLV were induced by simultaneous immunization with Leucogen and p15E compared to the responses using Leucogen or p15E alone, suggesting that combination vaccines should be used in the future. Epitope mapping of p15E-specific antibodies induced by simultaneous immunization with Leucogen and p15E revealed the same pattern of response as obtained after immunization with p15E alone: one epitope was localized in the membrane-proximal external region (MPER) and the other in the fusion peptide-proximal region, and they are related to the epitopes detected after immunization with p15E of the porcine endogenous retrovirus and the koala retrovirus. The data indicate that these epitopes in the MPER are an effective target for neutralization and that antigens containing them may therefore prove to be a useful component of vaccines against retroviruses, including HIV-1.

Antibodies neutralizing feline leukaemia virus (FeLV) in cats immunized with the transmembrane envelope protein p15E

The feline leukaemia virus (FeLV) vaccines that are currently in wide use are generally poor inducers of virus-neutralizing antibodies, although such antibodies appear after recovering from challenge. However, the presence of neutralizing antibodies in cats recovering from natural FeLV infection clearly correlates with resistance to subsequent infection and passive transfer of antibodies can protect other animals. After demonstrating the induction of neutralizing antibodies in rats and goats immunized with the transmembrane envelope protein p15E of FeLV, cats were immunized with the same antigen. High titres of neutralizing antibodies specific for FeLV were induced and epitope mapping revealed a pattern of recognition similar to that seen following immunization of rats and goats. These epitopes are highly related to epitopes recognized after immunization with porcine endogenous retrovirus (PERV) p15E and to epitopes recognized by neutralizing antibodies in patients infected with human immunodeficiency virus type 1. The ability of p15E to induce neutralizing antibodies in cats suggests that it should be included in the next generation of vaccines. In contrast, sera from FeLV-infected animals usually fail to recognize the neutralization-relevant epitopes in p15E. Since homologous epitope sequences are present in feline endogenous retroviruses, it appears that tolerance against these sequences is not induced.

Neutralising antibodies against the transmembrane protein of feline leukaemia virus (FeLV)

Neutralising antibodies specific for feline leukaemia virus (FeLV) were induced by immunisation with recombinant FeLV transmembrane envelope protein p15E. Epitope mapping revealed two epitopes located in similar regions to those previously identified for the porcine endogenous retrovirus (PERV). One of the epitopes has partial homology and both are located in regions corresponding to epitopes recognised by neutralising antibodies in patients infected with HIV-1.

Neutralizing antibodies against conserved domains of p15E of porcine endogenous retroviruses: basis for a vaccine for xenotransplantation?

Porcine xenotransplants may offer a potential solution to the problem posed by the limited supply of allotransplants. However, xenotransplantation may be associated with the risk of transmission of microorganisms, in particular of porcine endogenous retroviruses (PERVs) that are an integral part of the porcine genome and able to infect human cells in vitro. Possible strategies to prevent virus transmission include the development of PERV knockout animals or of effective vaccines. When antisera prepared against the main structural proteins of PERV were screened, a goat antiserum against the recombinant ectodomain of the transmembrane envelope protein p15E was found to neutralize PERV infectivity. Epitope mapping using overlapping peptides revealed two epitopes, E1 (GPQQLEK) and E2 (FEG WFN ). These sequences are identical for all PERVs and are highly conserved among all gammaretroviruses. Interestingly, antibodies isolated from AIDS patients and specific for sequences of HIV-1 partially homologous with E2 (Mab4E10, LWN WFN ) or located in close proximity to E2 (Mab2F5, ELDKWA) are known to neutralize several strains of HIV-1. It is the first report showing epitope mapping of gammaretrovirus-specific neutralizing antibodies and demonstrating similarity to corresponding epitopes in HIV. These domains of the transmembrane proteins of different retroviruses are an effective target for neutralizing antibodies and may be a useful antigen to create an antiretroviral vaccine.

Comparison of retroviral p15E-related factors and interferon alpha in head and neck cancer.

Head and neck squamous cell carcinomas (HNscc) produce low-molecular-mass factors (low-M(r) factors, M(r) < or = 25,000), which are antigenically related to the immunosuppressive retroviral transmembrane envelope protein p15E. These P15E-related tumour factors are thought to be responsible for some immunological impairments found in these patients (particularly the defective monocyte chemotaxis). A sequential and functional homology has been reported to exist between a bioactive fragment of interferon alpha (IFN alpha) and the putative immunosuppressive region of retroviral p15E (CKS-17). In this study we investigated (a) a possible functional and structural relationship between p15E and IFN alpha, and (b) the presence of and the relationship between p15E-related low-M(r) factors and IFN alpha in HNscc patients. We report the following results. (a) Recombinant human (rhu) IFN alpha was able to inhibit monocyte chemotaxis. (b) The anti-p15E antibodies crossreacted with rhuIFN alpha in a dot-blot technique; however, the anti-IFN alpha antibodies did not crossreact with disrupted murine leukaemia virus (p15E source). (c) Low-M(r) factors (n = 8-11) prepared from the sera of HNscc patients, which inhibit the monocyte chemotactic responsiveness, could be adsorbed by the anti-p15E antibodies as well as by the anti-IFN alpha antibodies. However, the abilities of the factors to adsorb to the two categories of antibodies (namely, anti-p15E and anti-IFN alpha) did not correlate. (d) Immunohistochemically we found IFN alpha-related epitopes, in almost all HNscc specimens studied (17/18), in locations distinctive from those of p15E-related factors. The anti-IFN alpha antibodies used in this study mainly reacted with basal epithelial cells close to the basal membrane, the prickle and granular cells of the squamous cell carcinomas. The anti-p15E antibodies mainly reacted with corneal layers, the granular and prickle cells, and did not react with basal epithelial cells. Our findings suggest that the immunosuppressive factors produced by HNscc cells are heterogeneous and p15E- and/or IFN alpha-related.

Inhibition of Lymphocyte Proliferation by a Synthetic Peptide Homologous to Retroviral Envelope Proteins

The retroviral transmembrane envelope protein p15E is immunosuppressive in that it inhibits immune responses of lymphocytes, monocytes, and macrophages. A region of p15E has been conserved among murine and feline retroviruses; a homologous region is also found in the transmembrane envelope proteins of the human retroviruses HTLV-I and HTLV-II and in a putative envelope protein encoded by an endogenous C-type human retroviral DNA. A peptide (CKS-17) was synthesized to correspond to this region of homology and was examined for its effects on lymphocyte proliferation. CKS-17 inhibited the proliferation of an interleukin-2-dependent murine cytotoxic T-cell line as well as alloantigen-stimulated proliferation of murine and human lymphocytes. Four other peptides, representing different regions of virus proteins, were inactive. These results suggest that the immunosuppressive portion of retroviral transmembrane envelope proteins may reside, at least in part, in a-conserved sequence represented by the CKS-17 peptide.