Free Access
Vet. Res.
Volume 31, Number 1, January-February 2000
Page(s) 136 - 137
How to cite this article Vet. Res. (2000) 136-137
Vet. Res. 31 (2000) 136-137

Antibody-induced endocytosis of viral glycoproteins, expressed on pseudorabies virus-infected monocytes protects these cells from complement-mediated lysis

H.W. Favoreela, H.J. Nauwyncka, P. Van Oostveldtb and M.B. Pensaerta

a  Laboratory of Virology, Faculty of Veterinary Medicine
b  Laboratory of Biochemistry and Molecular Cytology, Faculty of Agricultural and Applied Biological Sciences, University of Gent, Salisburylaan 133, 9820 Merelbeke, Belgium

Abstract - Pseudorabies virus (PrV) can cause abortion in sows with an immune system activated by vaccination. Virus-carrying blood monocytes are essential for the spread of the virus from the respiratory tract to the pregnant uterus. Two major adaptive immune effector mechanisms should normally be capable of eliminating PrV-infected monocytes. First, newly synthesised viral proteins may be processed and coupled to the major histocompatibility complex class I (MHC I) which then is transported to the plasma membrane. This MHC I-antigen-complex can be recognised by cytotoxic T-lymphocytes (CTLs). Second, specific antibodies are capable of binding to newly synthesised viral envelope glycoproteins, which become expressed in the plasma membrane of the infected cell. Antibodies in association with complement or phagocytes may then result in the lysis of the infected cell. Addition of virus-specific antibodies to PrV-infected swine kidney cells in vitro is known to induce a redistribution of the plasma membrane-anchored viral glycoproteins. This redistribution finally leads to the release of the viral glycoproteins into the surrounding medium, leaving viable cells without visually detectable levels of viral glycoproteins on their plasma membrane. In the present study it was examined whether a similar phenomenon occurs in the natural carrier of the virus, the blood monocyte, in order to evaluate if this process may be significant to the immune evasion of the virus. Blood was collected from the vena jugularis from PrV-negative pigs and blood mononuclear cells were separated on Ficoll-Paque (Pharmacia Biotech AB, Uppsala, Sweden). Blood monocytes were purified by plastic adhesion, and were cultivated for 24 h. Afterwards, the cells were inoculated with PrV strain 89V87 or Kaplan and incubated at $37\,^\circ$C with 5% CO 2 for 13 h. After washing of the cells, FITC-labelled virus-specific antibodies were added (0.1 mg IgG/ml), and the cells were incubated at $37\,^\circ$C for different time periods (0, 5, 10, 30 and 60 min) before fixation with 0.4% formaldehyde and analysis by fluorescence microscopy and/or confocal laser scanning microscopy. Shortly after the addition of antibodies, viral plasma membrane glycoproteins become aggregated (patches). These patches are then internalised by the cell, leaving an infected cell with no visually detectable levels of viral glycoproteins on its plasma membrane. Antibody-induced endocytosis is a fast and efficient process. Endocytosis started at 10 min post-antibody addition, and was completed in 65% of the infected cells at 1 h post-antibody addition. Furthermore, only very few quantities of viral glycoproteins on the plasma membrane (reached after 7 h PI) and very low concentrations of antibodies (0.04 mg IgG/mL) were needed to induce endocytosis. Genistein, a specific inhibitor of tyrosine kinase activity, was found to be a very efficient inhibitor of viral glycoprotein internalisation (100% inhibition at 50  $\mu$g/mL). We also evaluated the effect of viral glycoprotein internalisation on complement-mediated lysis of the infected monocytes. Monocytes were infected for 10 h, and incubated with virus-specific antibodies for 2 h ( $\pm 100\%$ of the infected cells displayed internalised viral glycoproteins at this time point). The control cells were incubated with antibodies in the presence of $50\,\mu$g/mL genistein, or were incubated without antibodies. Afterwards, the cells were washed and incubated with different concentrations of guinea pig complement (0-10 IU) for 1 h. Afterwards, 20  $\mu$g/mL of the DNA-staining fluorochrome, propidium iodide, was added for 5 min. Propidium iodide specifically stains dead cells which allows to determine the percentage of dead cells by flow cytometry. Compared relatively to the viability of the cells incubated without either antibodies or the complement, viability of the cells, incubated with the complement for 1 h decreased slightly to 79% $\pm$ 12% for cells incubated without antibodies (no activation of the complement), and to 84% $\pm$ 4% for cells incubated with antibodies (internalised viral glycoproteins and antibodies). The viability dropped to 24% $\pm$ 11% for cells incubated with antibodies and genistein (there was no internalisation of viral glycoproteins and antibodies), which was not caused by toxic effects of genistein. We can therefore state that antibody-induced endocytosis of viral glycoproteins protects PrV-infected cells from complement-mediated lysis. When performing double labelling experiments, we observed that the MHC I co-aggregates and undergoes co-endocytosis with the viral glycoproteins. This may indicate that the addition of virus-specific antibodies to PrV-infected monocytes can hide these cells from both humoral and cellular immune responses. To investigate this hypothesis, we are currently constructing an in vitro assay to evaluate the effect of MHC I co-endocytosis on the capacity of cytotoxic T-lymphocytes to eliminate PrV infected monocytes. Furthermore, we are examining whether the observed processes also occur in vivo. Preliminary experiments, consisting of the injection of colostrum-free piglets with biotinylated PrV-specific antibodies, followed by PrV-inoculation, already showed that endocytosis of antibodies occurs in vivo in infected cells, e.g. in alveolar macrophages.

Corresponding author: H.J. Nauwyncka Tel.: (32) 9 264 73 73; fax: (32) 9 264 74 95;

© INRA, EDP Sciences 2000