Herpes simplex type1:lacZ recombinant viruses. II. Microtiter plate-based colorimetric assays for the discovery of new antiherpes agents and the points at which such agents disrupt the viral replication cycle.

A panel of microtiter plate-based colorimetric assays for monitoring HSV-1 growth has been made. The panel consists of 4 different HSV-1 (strain KOS) lacZ recombinant viruses which express beta-galactosidase under the control of different HSV-1 promoters derived from each class of herpes simplex gene expression: immediate-early (ICP4), early (TK), delayed early (gD) and late (gC). Inhibitors of HSV-1 growth were evaluated using differential effects on each of the reporter viruses as a measure of which points in the viral replication cycle an inhibitor was acting. Aphidicolin (DNA synthesis inhibitor) was studied as a model compound. At an m.o.i. of 0.05, at 24 h postinfection (h p.i.), aphidicolin inhibited 80% of viral growth at 1 micrograms/ml, as determined by a reduction in ICP4-driven activity within the second cycle of infection. At m.o.i. 5, within the first infectious cycle, aphidicolin had no effect on the signals from either the ICP4 or TK viruses at 3 micrograms/ml, while largely suppressing gD and fully inhibiting gC-driven signals at 2 micrograms/ml. This profile is consistent with the behavior expected of a DNA synthesis inhibitor. Five inhibitors of unknown mechanism were evaluated. Two compounds inhibited ICP4-driven activity within the first infectious cycle and were classified as potential inhibitors of viral entry, uncoating or IE gene expression (XF884, BT318). One compound inhibited gD and gC-driven activity without inhibiting signal from the ICP4 and TK viruses, and was classified as a potential DNA synthesis inhibitor (DS810). Two compounds (S5193, ER622) had effects on gD- and gC-driven activity which were somewhat different from aphidicolin and DS810, but which could be interpreted as inhibition of viral assembly and/or egress. The potency of XF884 varied with the time postinfection at which it was added to cells (IC50 3.7 to > 10 micrograms/ml) while the effects of BT318 were independent of time of addition (IC50 11.4 micrograms/ml). These results suggest XF884 inhibits viral entry while BT318 is acting after viral entry, possibly as a direct inhibitor of ICP4 gene expression. Together, these results suggest the panel of recombinant herpes viruses has utility in aiding in the identification of the points in the herpes life cycle at which antiherpes drug candidates, of unknown mechanisms, are acting.

Pharmacokinetics and metabolism of rimantadine hydrochloride in mice and dogs.

We studied the pharmacokinetics and metabolism of rimantadine hydrochloride (rimantadine) following single-dose oral and intravenous administration in mice and dogs. Absorption of the compound in mice was rapid. Maximum concentrations in plasma occurred at less than 0.5 h after oral administration, and the elimination half-life was 1.5 h. Peak concentrations in plasma following oral administration were markedly disproportional to the dose (274 ng/ml at 10 mg/kg, but 2,013 ng/ml at 40 mg/kg). The bioavailability after an oral dose of 40 mg/kg was 58.6%. Clearance was 4.3 liters/h per kg, and the volume of distribution was 7.6 liters/kg at 40 mg/kg. In contrast to the results observed in mice, absorption of the compound in dogs was slow. Maximum concentrations in plasma occurred at 1.7 h after oral administration, and the elimination half-life was 3.3 h. A further difference was that peak concentrations in plasma were approximately proportional to the dose. Following administration of a single oral dose of 5, 10, or 20 mg/kg, maximum concentrations in plasma were 275,800, and 1,950 ng/ml, respectively. The bioavailability after an oral dose of 5 mg/kg was 99.4%. The clearance was 3.7 liters/h per kg, and the volume of distribution was 13.8 liters/kg at 5 mg/kg. Mass balance studies in mice, using [methyl-14C]rimantadine, indicated that 98.7% of the administered dose could be recovered in 96 h. Less than 5% of the dose was recovered as the parent drug in dog urine within 48 h. Finally, gas chromatography-mass spectrometry studies, done with mouse plasma, identified the presence of two rimantadine metabolites. These appeared to be ring-substituted isomers of hydroxy-rimantadine.

Assessment of host immune status during progressive growth and after excision of DNA virus (simian virus 40) tumors in hamsters: comparison of tumor-specific and tumor-unrelated parameters of immune responsiveness.

The kinetics of cell-mediated immunity to simian virus 40 (SV40) tumor-specific transplantation antigen (TSTA) were compared to the kinetics of tumor-unrelated parameters of immune responsiveness in the assessment of the immune statuses of inbred MHA/SsLAK hamsters during the course of progressive syngeneic SV40 tumor growth and after tumor excision. With the use of the tumor cell neutralization test in vivo and the macrophage migration inhibition assay in vitro, specific cellular immunity to SV40 TSTA was detected by 4 days after tumor cell inoculation, when the tumor was small. This tumor-specific immune response was no longer detected at 7 days after tumor cell inoculation, when the tumor had reached a mean diameter of 12.5 mm, but it returned by 14 days after surgical excision of the tumor. The patterns of host responsiveness to mitogens in spleen cells derived from tumor-bearing animals or from tumor-excised animals generally showed little or no correlation with the kinetics of tumor-specific cellular immunity. The kinetics of the humoral immune response to murine erythrocytes, as determined by hemagglutination assays, correlated much more closely with the kinetics of tumor-specific immunity than did the responses to mitogens. IgG antibody (T-dependent) responses were more affected by progressive tumor growth than were IgM antibody (T-independent) responses. The data suggest that results of tests with the use of tumor-unrelated parameters of immune responsiveness for the assessment of the immune status of cancer patients should be interpreted with caution.

Identification, functional characterization and partial purification of thymus-derived lymphocytes in inbred hamsters.

Antiserum specific for hamster thymus-derived lymphocytes, prepared by immunization of rabbits with brain tissue from MHA/ssLAK hamsters, was, in the presence of guinea-pig complement, cytotoxic to hamster thymocytes greater than lymph node cells greater than spleen cells, while virtually unreactive against bone marrow cells. This antiserum markedely inhibited spleen cell response to the T cell mitogen, Concanavalin A, while the response to the B the T cell mitogen, pokeweed, was much less inhibited. These in vitro effects of the anti-hamster T cell serum were confirmed by utilizing lymphoid cells from thymectomized, lethally-irradiated, bone marrow-reconstituted hamsters. Lymph node cells from such animals were killed by the anti-T cell serum only to the same extent as bone marrow cells, while spleen cells from these animals gave a good response to pokeweek mitogen but were virtually unresponsive to Concanavalin A. Passage of hamster spleen cells over nylon wool columns yielded effluent populations highly enriched in T lymphocytes. The eluted cells were fully capable of T cell functions, as determined by their blastogenic response to various T and B cell mitogens in vitro and their ability to inhibit the growth of syngeneic SV40 tumours in vivo.

Thymus-derived lymphocyte-dependent rejection of syngeneic papovavirus (SV40) and methylcholanthrene tumors in inbred hamsters.

Treatment of specifically sensitized MHA hamster lymphoid cells with rabbit antisera specific for hamster thymus-derived lymphocytes, in the presence of C, eliminated those cells capable of inhibiting the growth of syngeneic SV40 and methylcholanthrene tumors in vivo. Thymectomized, lethally-irradiated, bone marrow-reconstituted hamsters, shown to be devoid to T cell function, were, after attempted specific sensitization to the two syngeneic tumor cell lines, unable to reject either tumor by direct challenge in vivo. In addition, lymphocytes from such animals were incapable of inhibiting the growth of either tumor cell line in normal syngeneic recepients in the tumor cell neutralization assay. These data strongly support the conclusion that specifically sensitized thymus-derived lymphocytes are required for the rejection of syngeneic SV40 and methylcholanthrene tumors in inbred hamsters.

Induction of hamster T cell-specific antiserum in rabbits using hamster brain tissue.

Antiserum specific for hamster thymus-derived lymphocytes, prepared by immunizing rabbits with hamster brain tissue, was cytotoxic to hamster thymocytes greater than lymph node cells greater than spleen cells, while virtually non-reactive against bone marrow. This antiserum inhibited spleen cell response to the T cell mitogen, phytohemagglutinin, but not to lipopolysaccharide, a B cell mitogen.

Restoration of specific immunity against SV40 tumor-specific transplantation antigen to lymphoid cells from tumor-bearing mice.

Specific cell-mediated immunity to SV40 tumor-specific transplantation antigen (TSTA) in BALB/c mice undergoing progressive tumorigenesis by syngeneic SV40-transformed cells (VLM) was investigated in vivo using a tumor-cell neutralization test. Specific cellular reactivity to SV40 TSTA was not detected in BALB/c mice bearing large tumors (10-15 mm mean diameter) but was demonstrable after tumor excision. Specific cytotoxic reactivity against syngeneic SV40-transformed cells in vivo could be restored to lymphoid cells from VLM tumor-bearing mice either by culturing the lymphoid cells in vitro or by treating them with papain or trypsin. Enzyme-treated lymphoid cells from MCA tumor-bearing BALB/c mice had no cytotoxic reactivity against VLM cells. These studies suggest that tumor-bearing hosts possess lymphocytes which are sensitized to the TSTA of the tumor but that the reactivity of these lymphocytes is blocked.

In vitro studies on the cellular immune response of tumor-bearing mice to SV40-transformed cells.

Specific cell-mediated immunity to SV 40 tumor-specific transplantation antigen (TSTA) in mice undergoing tumorigenesis by syngeneic SV 40-transformed BALB/C cells was investigated by the macrophage migration inhibition (MMI) and transplantation rejection tests. Specific cellular reactivity to SV40 TSTA could be demonstrated in BALB/c mice early after tumor cell inoculation. This activity was no longer detectable during the later stages of tumor growth but was again demonstrable 2 weeks after tumor excision. Addition of an equal number of non-reactive peritoneal exudate (PE) cells from tumor-bearing mice to PE cells from mice immune to SV40 TSTA specifically abrogated the reactivity of the latter cells to soluble SV40 TSTA. When lymphoid cells with blocking activity were cultured in vitro they not only lost their blocking capacity but also regained their reactivity to SV40 TSTA in the MMI test. These findings indicate that tumor-bearing hosts possess lymphocytes specifically sensitized to the TSTA of the tumor and that the specific reactivity of these cells can be regained after culture in vitro.