Stefan Langhammer

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.

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.

Immunization with the transmembrane protein of a retrovirus, feline leukemia virus: Absence of antigenemia following challenge

A major challenge in the development of vaccines against retroviruses is the induction of neutralizing antibodies since they prevent infection of the cells where the virus may persist. The transmembrane envelope (TM) protein contains highly conserved domains and seems to be a suitable target. To study whether vaccinating with a TM protein of a retrovirus could protect from infection in vivo, cats were immunized with the TM protein p15E of feline leukemia virus (FeLV) and subsequently challenged. For the first time we show that immunization with a retroviral TM protein prevented antigenemia. The level of neutralizing antibodies after the boost immunization correlated with the outcome of FeLV infection.

Inhibition of poxvirus spreading by the anti-tumor drug Gefitinib (Iressa™)

The threat of smallpox virus as a bioterrorist weapon is raising international concerns again since the anthrax attacks in the USA in 2001. The medical readiness of treating patients suffering from such infections is a prerequisite of an effective civil defense system. Currently the only therapeutic option for the treatment of poxvirus infections relies on the virostatic nulceosid analog cidofovir, although severe side effects and drug resistant strains have been described. A growing understanding of poxvirus pathogenesis raises the possibility to explore other appropriate targets involved in the viral replication cycle. Poxvirus encoded growth factors such as the Vaccinia Growth Factor (VGF) stimulate host cells via the Epidermal Growth Factor Receptor (EGFR) and thereby facilitate viral spreading. In this study we could visualize for the first time the paracrine priming of uninfected cells for subsequent infection by orthopoxviruses directly linked to EGFR phosphorylation. Since EGFR is a well known target for anti-tumor therapy small molecules for inhibition of its tyrosine kinase (TK) activity are readily available and clinically evaluated. In this study we analyzed three different EGFR receptor tyrosine kinase inhibitors for inhibition of orthopoxvirus infection in epithelial cells. The inhibitor shown to be most effective was Gefitinib (Iressa) which is already approved as a drug for anti-tumor medication in the USA and in Europe. Thus Gefitnib may provide a new therapeutic option for single or combination therapy of acute poxvirus infections, acting on a cellular target and thus reducing the risk of viral resistance to treatment.

LDH-A influences hypoxia-inducible factor 1α (HIF1 α) and is critical for growth of HT29 colon carcinoma cells in vivo

Serum lactate dehydrogenase (LDH) is a well-known clinical surrogate parameter. A high activity of LDH is associated with a poor prognosis in different tumor types. Here we demonstrate by a gene silencing approach that LDH-A is critical for in vivo but not in vitro growth of HT29 colon carcinoma cells. We provide evidence that the suppression of the LDH-A gene leads to an increased level of hypoxia inducible factor 1α (HIF1α) but in consequence not to an increase of HIF1 regulated proteins such as carbonic anhydrase IX (CAIX), vascular endothelial growth factor (VEGF), prolyl-hydroxylase 2 (PHD2), and factor-inhibiting HIF (FIH) in cell cultures and tumor lysates. This effect is independent of LDH activity in vivo. We conclude that LDH-A has an influence on the activity of HIF1α and thus on the adaptation of cells to a hypoxic tumor microenvironment in HT29 colon cells. We suggest the use of LDH-M as a potential therapeutic target for anticancer treatment.

Induction of HIV-neutralising Antibodies of the 2F5/4E10 Type

Neutralising antibodies recognising membrane proximal epitopes of gp41 have been isolated from HIV-infected patients. Since these epitope domains are highly conserved, the corresponding antibodies 2F5 and 4E10 neutralise a broad range of HIV strains. Numerous attempts by several laboratories to generate 2F5/4E10-like antibodies by vaccination have failed, obviously because the conformation of the domain is difficult to reproduce. Recently we reported induction of neutralising antibodies against the porcine endogenous retrovirus (PERV) and the feline leukaemia virus (FeLV) by immunisation with their transmembrane envelope (TM) proteins p15E. In both retroviral TM proteins two epitope regions were identified, one located in the N-terminal part (designated E1) and the other located in the C-terminal part of p15E (E2). E2 is related to the 2F5/4E10-epitope, and is located opposite E1 when the TM envelope protein has folded. An E1 domain was identified in the C-terminal part of gp41 and two strategies were developed to induce neutralising antibodies against HIV. First, immunisation was performed with a hybrid protein based on p15E of PERV, containing the E1 domain and the E2 (2F5/4E10 epitope) domain of gp41 of HIV-1. Second, immunisation was performed with a hybrid containing the N-terminal backbone of p15E of FeLV and only the E2 (2F5/4E10 epitope) domain of gp41. With both strategies antibodies neutralising laboratory and primary strains of HIV-1 were induced. These strategies may be used to generate a vaccine inducing broadly neutralising antibodies to prevent or curtail HIV infection. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005

A novel three-dimensional cell culture method enhances antiviral drug screening in primary human cells

Abstract Gefitinib is a specific inhibitor of the epidermal growth factor receptor (EGFR) and FDA approved for treatment of non‐small cell lung cancer. In a previous study we could show the in vitro efficacy of gefitinib for treatment of poxvirus infections in monolayer (2D) cultivated cell lines. Permanent cell lines and 2D cultures, however, are known to be rather unphysiological; therefore it is difficult to predict whether determined effective concentrations or the drug efficacy per se are transferable to the in vivo situation. 3D cell cultures, which meanwhile are widely distributed across all fields of research, are a promising tool for more predictive in vitro investigations of antiviral compounds. In this study the spreading of cowpox virus and the antiviral efficacy of gefitinib were analyzed in primary human keratinocytes (NHEK) grown in a novel 3D extracellular matrix‐based cell culture model and compared to the respective monolayer culture. 3D‐cultivated NHEK grew in a polarized and thus a more physiological manner with altered morphology and close cell–cell contact. Infected cultures showed a strongly elevated sensitivity towards gefitinib. EGFR phosphorylation, cell proliferation, and virus replication were significantly reduced in 3D cultures at gefitinib concentrations which were at least 100‐fold lower than those in monolayer cultures and well below the level of cytotoxicity. Our newly established 3D cell culture model with primary human cells is an easy‐to‐handle alternative to conventional monolayer cell cultures and previously described more complex 3D cell culture systems. It can easily be adapted to other cell types and a broad spectrum of viruses for antiviral drug screening and many other aspects of virus research under more in vivo‐like conditions. In consequence, it may contribute to a more targeted realization of necessary in vivo experiments. HighlightsOrthopoxvirus infection was studied in an innovative and easy‐to‐handle 3D cell culture system with primary human cells.Comparison of primary human cells in 3D and monolayer culture revealed significant differences in cell physiology.Expression of the cowpox virus EGF homologue CGF was more than 10‐fold increased in 3D cell culture infection.Cowpox virus infection strongly increased EGFR phosphorylation in infected NHEK independent of the cell culture model.CPXV infection in 3D cultured NHEK was 50‐fold more sensitive to antiviral treatment with the EGFR inhibitor gefitinib.