Lysis of herpes simplex virus-infected cells early in the infectious cycle by human antiviral antibody and complement.

Chang liver (CL) cells and human embryonic lung fibroblasts (MRC-5) were infected with type 1 herpes simplex virus (HSV), ant the time postinfection at which these cells became susceptible to lysis by antiviral antibody and complement of human origin was determined in a 51Cr release assay. Using a 1:2 dilution of fresh HSV antibody-positive human serum, we initially detected specific lysis by 4 h postinfection in HSV-infected CL cells and by 3 h postinfection in HSV-infected MRC-5 cells in suspension. MRC-5 cells were more completely lysed than CL cells. Protein inhibition studies with cycloheximide showed that all of the HSV-infected CL cells and most (83%) of the HSV-infected MRC-5 cells injured early in the infectious cycle were attacked because of newly synthesized viral surface antigens rather than because of adherent input virus. Suspension cells early in the infectious cycle were less completely lysed and required higher concentrations of both antiviral antibody and complement for lysis than cells that were in the later stages of infection (18 h postinfection). Guinea pig serum was inferior to human serum as a complement source for lysis of early infectious cycle cells. Lysis early in the infectious cycle was directly proportional to the multiplicity of infection and inversely proportional to the cell concentration. Infected cells in monolayers were lysed less readily and about 1 to 2 h later in the infectious cycle than infected cells in suspension. This difference was pronounced for CL cells, but modest for MRC-5 cells. These studies demonstrate that, despite previously held notions, HSV-infected tissue culture cells can be lysed by antiviral antibody and complement early in the infectious cycle before the initial production of progeny virus particles. The demonstration of lysis was highly dependent on experimental conditions, however, including cell type, suspension versus monolayer culture, cell density, and concentration of antibody and complement as well as the source of complement.

Analysis of the lytic step in the herpes simplex virus antibody-dependent cellular cytotoxicity system.

An antibody-dependent cellular cytotoxicity (ADCC) system in which herpes simplex virus-infected Chang liver cells are used was assessed for its dependency on cellular energy, ribonucleic acid and protein synthesis, and cytoskeletal structures such as microfilaments and microtubules. The cytotoxic reaction was only slightly inhibited when glycolysis was blocked in a glucose-free medium containing 10(-2) M 2-deoxy-d-glucose. It was more substantially inhibited when respiration was blocked with 10(-2) M sodium azide. The reaction was totally ablated, however, only when both glycolysis and respiration were suppressed. This inhibitory effect of energy deprivation was mediated solely at the level of the effector cell. Ribonucleic acid synthesis or protein synthesis by the effector cells was not required, as shown by the fact that neither actinomycin D, cycloheximide, nor emetine significantly inhibited ADCC. The ADCC reaction was partially inhibited by cytochalasin B, whose inhibitory effect was rapidly reversible, and was completely and irreversibly inhibited by cytochalasin A. Cytochalasin A acted on the effector cells rather than the target cells. The reaction was also partially inhibited by colchicine, whose inhibitory effect was directed solely against the effector cells and was slowly reversible. The inhibitory effects of cytochalasin B and colchicine, when used in tandem at submaximal inhibitory concentrations, were slightly more than additive. The results suggest a cooperative role for effector cell microfilaments and microtubules in mediating ADCC. Kinetic studies of ongoing herpes simplex virus ADCC reactions after initial centrifugation showed that the lytic step requires expenditure of metabolic energy as well as intact function of both microfilaments and microtubules. These findings, in concert with previous data, indicate that the ADCC process against herpes simplex virus-infected Chang liver cells can be resolved into adhesion and lytic steps. The lytic step can be readily distinguished from the adhesion step by its increased sensitivity to low ambient temperature or metabolic energy deprivation, its sensitivity to thermal inactivation, its requirements for extracellular divalent cations, and its dependence on normal function of both microfilaments and microtubules.

Participation of three major glycoprotein antigens of herpes simplex virus type 1 early in the infectious cycle as determined by antibody-dependent cell-mediated cytotoxicity.

Tissue culture cells infected with herpes simplex virus type 1 synthesize three major glycoprotein antigens (Ag-11, Ag-8, and Ag-6), which have been characterized by crossed immunoelectrophoresis. The three viral antigens have been identified as a mixture of gA and gB (Ag-11), gD (Ag-8), and gC (Ag-6). Recent findings have shown that antibodies directed to each of the three antigens individually are able to mediate antibody-dependent cell-mediated cytotoxicity when tissue culture cells late in the infectious cycle (18 h postinfection) are used. In this work, antibody-dependent cell-mediated cytotoxicity was applied to study the time postinfection at which the individual viral antigens first made their appearance at the cell surface. All three viral antigens (Ag-11, Ag-8, and Ag-6) could be demonstrated as newly synthesized from 3 to 4 h postinfection, and the quantities of the antigens at the surfaces of the infected cells increased with time postinfection. The use of cycloheximide and ultraviolet-inactivated virus demonstrated that input virus could be detected by antibody-dependent cell-mediated cytotoxicity during the first 2 h postinfection, but the cytotoxicity caused by input virus remained constant with time postinfection. In conclusion, these observations demonstrate the participation of individual herpes simplex virus surface antigens in antibody-dependent cell-mediated cytotoxicity attack on cells early in infection.

Herpes simplex virus glycoproteins: participation of individual herpes simplex virus type 1 glycoprotein antigens in immunocytolysis and their correlation with previously identified glycopolypeptides.

Tissue culture cells infected with herpes simplex type 1 virus express virus-specified glycoprotein antigens on the plasma membrane. Three of these have been previously identified and have been designated as Ag-11, Ag-8, and Ag-6. In the present study, immunoglobulins to each of the antigens were shown to be capable of mediating immunocytolysis in the presence of either complement (antibody-dependent complement-mediated cytotoxicity) or peripheral blood mononuclear cells (antibody-dependent cell-mediated cytotoxicity [ADCC]). Two herpes simplex virus type 1 strains, VR-3 and F, reacted similarly in the ADCC test in the presence of immunoglobulins to Ag-11, Ag-8, and Ag-6 in both infected Chang liver cells and HEp-2 cells. Anti-Ag-6, however, produced a lower ADCC reaction in HEp-2 cells than in Chang liver cells, suggesting differences in the Ag-6 surface expression in, or release from, these cells. Chang liver and HEp-2 cells infected with the MP mutant strain of herpes simplex virus type 1 showed reduced ADCC in the presence of anti-Ag-11 and anti-Ag-8, but no reactivity at all with anti-Ag-6. Crossed immunoelectrophoretic analysis showed that MP-infected cell extracts contain Ag-11 and Ag-8, but lack Ag-6. Polypeptide analysis of herpes simplex virus type 1 strains F, VR-3, and MP showed that Ag-11 consists of the glycoproteins gA and gB, that Ag-8 consists of gD, and that Ag-6 consists of gC. In conclusion, the present study demonstrates that either one of the glycoproteins (gC, gD, and a mixture of gA and gB) can function as a target for immunocytolysis and that the antibody preparation to gC (Ag-6) does not cross-react with any of the other glycoproteins.

Analysis of the adhesion step in the herpes simplex virus antibody-dependent cellular cytotoxicity system.

The lysis of herpes simplex virus-infected tissue culture cells by antibody-dependent cellular cytotoxicity (ADCC) requires a preliminary step in which effector cells adhere to the immunoglobulin G antibody-coated targets. To study the adhesion step, we made use of two observations: (i) some of the mononuclear cells in human blood form rosettes with antibody-coated target cells, and (ii) most ADCC effector cells can be removed by allowing mononuclear cells to adhere to monolayers of antibody-sensitized tissue culture cells. The effect of various experimental conditions on the adhesion step was assessed in ADCC cultures both at unit gravity and after centrifugation. At unit gravity both rosette formation and monolayer adhesion were partially reduced at 4 degrees C as compared to 37 degrees C. Both were also partially inhibited in glucose-free medium containing sodium azide and 2-deoxyglucose but were unaffected in glucose-free medium containing only one of these energy inhibitors. In contrast, after centrifugation neither reaction was inhibited at 4 degrees C or in glucose-free medium with sodium azide and 2-deoxyglucose. Cytochalasin B but not colchicine suppressed both reactions. Inhibition by cytochalasin B could not be reversed by centrifugation. Both reactions were independent of extracellular Ca(2+) and Mg(2+) and were unaffected by rendering mononuclear cells cytotoxically inactive by brief heat shock. These findings indicate that the adhesion step in ADCC directed against virus-infected or uninfected tissue culture cells is only modestly dependent on effector cell energy generation, that centrifugation greatly reduces this dependence, and that microfilaments but not microtubules are necessary. The modest ambient temperature and energy requirements, independence of extracellular divalent cations, lack of sensitivity to colchicine, and relative resistance to supraphysiological temperature serve to distinguish the adhesion step from the lytic step in ADCC.

Human polymorphonuclear leucocytes as mediators of antibody-dependent cellular cytotoxicity to herpes simplex virus-infected cells.

Human polymorphonuclear leucocytes (PML) were able to mediate antibody-dependent cellular cytotoxicity (ADCC) against target cells acutely infected with type 1 Herpes simplex virus. The reaction mediated by PML occurred more slowly and required higher concentrations of immune serum than that mediated by human mononuclear cells (MC). At the same ratio of effector cells to target cells, PML-mediated ADCC was less than MC-mediated ADCC. The observed relationship between the number of effector cells added, and the number of target cells lysed, showed that cytolysis mediated by both PML and MC was consistent with 'one hit' probability predictions. This suggested that target cell death resulted from an interaction with a single effector cell. The calculated frequency of effector cells in PML preparation was similar to that in MC, approximately 3-5%. Preliminary examination of the nature of the effector cells suggested that they did not comprise a morphologically distinct subclass of PML. These experiments demonstrate a possible new role for PML in host defence against viral infections.

Human monocyte-macrophage-mediated antibody-dependent cytotoxicity to herpes simplex virus-infected cells.

Human peripheral blood mononuclear cells which mediate antibody-dependent cellular cytotoxicity (ADCC) against herpes simplex virus- (HSV) infected target cells consist of both adherent (MA) and nonadherent (MNA) effector cell populations. These two cell populations can be distinguished by their different phagocytic properties and morphologic appearance, their requirement for antibody in the ADCC reaction, and the rapidity with which they lyse target cells in the presence of immune serum. The MA cells are predominantly phagocytic and have the morphologic characteristics of monocyte-macrophages, whereas the MNA cells are nonphagocytic and appear to be small to medium-sized lymphocytes. Optimal expression of ADCC by MA cells requires higher concentrations of immune serum than does MNA cell-mediated ADCC. MA-mediated cell killing is first detectable by 8 hr and reaches completion after 24 hr of incubation. In contrast, MNA-mediated ADCC produces target cell damage by 2 hr and reaches completion at 8 hr of incubation. Unlike MNA effector cells, the MA effector cells are profoundly inhibited after preincubation with either latex or silica particles. The HSV immune status of the donor had no effect on the ability of either cell population to mediate ADCC. These data demonstrate the participation of both nonadherent mononuclear cells, presumably K cells, and monocyte-macrophages, in ADCC directed against HSV-infected target cells.

The mononuclear cell in human blood which mediates antibody-dependent cellular cytotoxicity to virus-infected target cells. II. Identification as a K cell.

Studies were carried out to determine whether the mononuclear cell in human blood which mediates antibody-dependent cellular cytotoxicity (ADCC) to herpes simplex virus (HSV)-infected target cells has surface Fc receptors which participate in the reaction. The F (ab')2 fragment of human IgG antibody was inactive both in ADCC and in complement-mediated cytolysis, but retained the capacity to neutralize infectious virus, to agglutinate erythrocytes coated with viral antigens, and to bind to the surface of virus-infected cells. Treatment of sensitized virus-infected target cells with staphylococcus protein A, which has affinity for the Fc epitope of IgG, strongly reduced their susceptibility to lysis by ADCC in a dose-dependent relationship. These findings indicate that the Fc portion of IgG antibody to the virus is necessary for cytotoxicity. Treatment of blood mononuclear cells with either heat-aggregated gamma-globulin or HSV immune complexes inhibited effector cell activity. The presence of "third party" cellular immune complexes also strongly inhibited ADCC. Adsorption of mononuclear cells to plastic surfaces coated with soluble third party immune complexes resulted in a significant reduction in effector cell activity. These findings demonstrate that the ADCC effector cell possesses surface Fc receptors which are utilized in the ADCC reaction. The presence of Fc receptors on the surface of the effector cell indicates that it is a K cell rather than a null cell.

The mononuclear cell in human blood which mediates antibody-dependent cellular cytotoxicity to virus-infected target cells. I. Identification of the population of effector cells.

Mononuclear cells (MC) from human blood were fractionated by a variety of physical and immunologic techniques, and the cellular subpopulations generated were assessed for their capacity to lyse herpes simplex virus (HSV)-infected target cells in the presence of IgG antibody to HSV. Latex phagocytosis and surface marker studies were performed in parallel in order to identify the major effector cells by their phagocytic properties and their possession of surface immunoglobulin and receptors for either sheep erythrocytes, C3, or the Fc fragment of IgG. Cytotoxic effector cell activity was unaffected or slightly enhanced after the removal of plastic-adherent or carbonyl iron-adherent MC, indicating that the major effector cell is not a classical monocyte. Similar results were obtained after removal of more than 90% of the T cells by depletion of rosette-forming cells. Likewise, effector cell activity was generally unchanged when more than 95% of the B cells were removed by filtering MC on nylon wool columns. Effector cell function was also found to be normal in three patients with B cell-deficient X-linked agammaglobulinemia. These observations strongly suggest that the effector cells are not T cells or B cells. A 4- to 5-fold enrichment in effector cells, however, was consistently found in a subpopulation, consisting of 5% of the unfractionated MC, that was dramatically enriched both for nonphagocytic cells with only Fc receptor (K cells) and for nonphagocytic cells with no detectable surface markers (null cells). Since, as is demonstrated in the accompanying report, effector surface Fc receptors play a critical role in the mediation of antibody-dependent cellular cytotoxicity directed at HSV-infected target cells, the major mononuclear effector cell in human blood is a K cell.

Antibody-dependent cellular cytotoxicity to target cells infected with herpes simplex viruses: functional adequacy in the neonate.

The functional adequacy of antibody-dependent cellular cytotoxicity (ADCC) in the human neonate was evaluated in a 51Cr release assay which employs tissue culture cells acutely infected with type 1 or type 2 herpes simplex virus (HSV) as targets. Two aspects of ADCC were assessed: cytotoxic effector activity in cord blood mononuclear cells (MC) and the ability of the antibody mediating ADCC to pass the placenta. Although effector cell activity was readily detected in all 13 cord blood specimens tested, cord blood MC showed moderately reduced cytotoxic activity when compared with blood MC from normal adults at the same effector cell:target cell ratio. This finding suggests that effector cells in cord blood make up a reduced proportion of the total circulating MC population and may be of relevance to the newborn's increased susceptibility to HSV infection. On the other hand, the number of MC in cord blood was found to be almost twice that of adult blood, suggesting that the absolute number of ADCC effector cells in cord blood was within the adult range. The antibody mediating ADCC to HSV-infected cells was shown to be transferred quantitatively across the placenta, providing further evidence that it is an IgG immunoglobulin.

Detection by indirect immunofluorescence of Fc receptors in cells acutely infected with Herpes simplex virus.

In attempting to use the indirect fluorescent antibody test (IFA) to measure antibodies to herpes simplex virus (HSV), we found that all human sera gave a positive reaction with Chang liver cells infected with type 1 (HSV). All sera gave equivalent titers of 320-640 for acetone-fixed cells and about 40 for live cells (membrane fluorescence) in the presence of fluorescein-labeled antisera to human Ig; none of the sera reacted with uninfected cells. The fluorescence seen in fixed cells was primarily cytoplasmic; some cells showed a diffuse fluorescence, obscuring the demarcation between the nucleus and cytoplasm. Purified IgG from antibody-negative human sera and a purified Fc fragment of IgG were positive both for cytoplasmic and membrane fluorescence, whereas F(ab')2, IgM and IgA were unreactive. The reaction was also seen when an antiserum conjugate specific for the Fc fragment of IgG was used. The reactive IgG was present in freshly prepared plasma and serum; it could not be removed from serum either by ultracentrifugation or by serial absorption with HSV-infected cells. These findings suggest that the nonspecificity of the IFA results from the formation of low-avidity bonds between the large mass of native serum IgG and an Fc receptor on the plasma membrane and in the cytoplasm of cells infected with HSV. The results also suggest that extreme caution be exercised in attempting to use the IFA in the serodiagnosis of infections with HSV and perhaps the other human herpes-viruses.