Preparation of Membrane Fraction from Herpes Simplex Virus‐Infected Cells which Induce Cytotoxic T Lymphocytes

Abstract

The immunogenic capacity of herpes simplex virs (HSV)-infected cells and their subcellular membrane fractions was investigated by assessing the anti-HSV cytotoxic T lymphocyte (CTL) response in cultures of spleen lymphocytes from HSV-primed BALBJc mice. Methylchloranthrane-induced fibrosarcoma (Meth A) cells infected with HSV (HSV-Meth A) were fixed either with glutaraldehyde or by heating at 56 C to preserve their immunogenic competence and then used as a stimulator. Microsomes and plasma membranes were prepared from HSV-Meth A and their immunogenic activities were determined. Though the recovery of stimulatory activity in the plasma membrane fraction was half of that in the microsome fraction, the activity in the former was much more stable than in the latter and the plasma membrane fraction proved to be well qualified as an immunogen for anti-HSV CTL induction. Upon purification, the specific activity of the membrane fraction, on the basis of protein concentration, increased 43-fold.Anti-herpes simplex virus cytotoxic T lymphocytes (anti-HSV CTL) are presumed to suppress HSV growth in the early phase of infection and to prevent the secondary outgrowth of HSV in the recurrent disease (12). In fact, enhanced activity of anti-HSV CTL has been noted in post-herpetic patients (17,18,20), as well as in the animal model system of HSV infection (5, 9, 22). For induction of CTL, inoculation oflive virus has been employed widely (25), but it is not feasible to use live HSV because of the oncogenic potency of the HSV genome (1, 6). It is therefore desirable to develop a simplified artificial immunogen specific for CTL induction. To this end, the attempt to reconstitute the immunogen by using HSV antigens and histocompatible membrane antigens appears to be promising (8), as described for other virus systems (4, 7).By virtue of the method previously devised for in vitro assay of anti-HSV CTL (22), it is now feasible to identity the immunogenic molecules of HSV and the requirements of major histocompatibility complex (MHC)-related molecules. In the present study, we reevaluated the immunogenicity of HSV-infected methyl-757

Immuno-viral therapy of brain tumors by combination of viral therapy with cancer vaccination using a replication-conditional HSV

“…As stimulators, 1Â10 6 CT26 or G207 -infected Meth A cells were added to the medium after inactivation with 200 g /mL mitomycin C. Before the coculture, the Meth A cells were infected with G207 at an MOI of 10 pfu / cell overnight, and the G207 was inactivated by incubation at 568C for 15 minutes. 26 Effector cells were harvested after 7-8 days of in vitro culture.…”

“…For the preparation of HSV-infected target cells, Meth A cells were infected with G207 at an MOI of 10 overnight and the viruses were inactivated by heating to 568C for 15 minutes. 26 The target cells labeled with 50 Ci of Na 51 CrO4 ( 51 Cr ) were mixed with effector cells for 4 hours at the effector-totarget ratios indicated. The amount of 51 Cr release was determined with a -counter, and the percent -specific lysis was calculated from triplicate samples as follows: [ (experimental cpm À spontaneous cpm )/ (maximum cpm À spontaneous cpm ) ]Â100.…”