Immunoglobulin g antibody-mediated enhancement of measles virus infection can bypass the protective antiviral immune response

Antibodies to viral surface glycoproteins play a crucial role in immunity to measles by blocking both virus attachment and subsequent fusion with the host cell membrane. Here, we demonstrate that certain immunoglobulin G (IgG) antibodies can also enhance the entry of measles virus (MV) into monocytes and macrophages. Antibody-dependent enhancement of infectivity was observed in mouse and human macrophages using virions opsonized by a murine monoclonal antibody against the MV hemagglutinin (H) glycoprotein, polyclonal mouse anti-MV IgG, or diluted measles-immune human sera. Neither H-specific Fab fragments nor H-specific IgM could enhance MV entry in monocytes or macrophages, indicating involvement of a Fc γ receptor (FcγR)-mediated mechanism. Preincubation with an anti-fusion protein (anti-F) monoclonal antibody or a fusion-inhibitory peptide blocked infection, indicating that a functional F protein was required for viral internalization. Classical complement pathway activation did not promote infection through complement receptors and inhibited anti-H IgG-mediated enhancement. In vivo, antibody-enhanced infection allowed MV to overcome a highly protective systemic immune response in preimmunized IfnarKo-Ge46 transgenic mice. These data demonstrate a previously unidentified mechanism that may contribute to morbillivirus pathogenesis where H-specific IgG antibodies promote the spread of MV infection among FcγR-expressing host cells. The findings point to a new model for the pathogenesis of atypical MV infection observed after immunization with formalin-inactivated MV vaccine and underscore the importance of the anti-F response after vaccination.     

Suppression of cell-mediated tumor cell lysis and complement-induced cytotoxicity by trypan blue.

Different forms of cell-mediated cytotoxicity were suppressed in the presence of trypan blue. The systems affected included lysis of antibody-coated tumor cells by normal and C. parvum-stimulated mouse peritoneal cells and lysis of allogeneic targets by immune effector cells. The inhibition, measured in a 4-hr 51Cr release assay, was reversible and did not occur in the presence of 30% fetal calf serum or albumin. Binding between effector and target cells through Fc receptors was not affected, and lysis of allogeneic cells was inhibited at the lytic step rather than at the binding step. In contrast, lysis of sensitized erythrocytes was not inhibited by trypan blue, suggesting that lysis of these targets may not involve the steps required in tumor cell lysis. Trypan blue blocked the function of antibody before binding to target cells and also suppressed complement-induced cytolysis. Most individual complement components were susceptible to the inhibitory action of trypan blue. These results reveal an affinity of trypan blue for proteins in general that may be responsible for many of its biologic actions.     

Neuraminidase reversal of resistance to lysis of herpes simplex virus-infected cells by antibody and complement.

The susceptibility to lysis by antibody and complement was examined in four human cell lines. The cells were infected with herpes simplex virus type 1 and lysis was assessed by the 51Cr release test by using antibodies to herpes simplex virus and guinea pig serum as a source of complement. The four cell lines were found to differ in their susceptibility to lysis, although virus replication was readily demonstrated in the different cell lines. By indirect immunofluorescence, no differences in the expression of virus antigens at the surface of the cells could be found between the different cell lines. Treatment of cells with neuraminidase markedly enhanced the sensitivity of the cells which were relatively insensitive to lysis. The enhancement of susceptibiltiy to lysis by neuraminidase occurred if cells were treated before reaction of the cells with antibody and if the cells were reacted with antibody before treatment with the enzyme. No enhancement was observed when cells were reacted with antibody and complement before neuraminidase treatment. Neuraminidase treatment did not seem to enhance appreciably the quantity of antibody which reacted at the cell surface. The observations suggest that surface properties of certain cells render the cells resistant to lysis by antibody and complement and that the resistance to lysis can be abrogated by treating the cells with neuraminidase.     

Soluble factors from rabbit spleen cells kill and lyse Treponema pallidum in vitro

Antibody and complement immobilize (kill) Treponema pallidum in vitro. Recent evidence also documents immobilization by soluble factors released by activated macrophages and lymphocytes. Immune-mediated lysis of treponemes, however, has not been reported. The findings in this paper focus on apparent treponemal lysis by rabbit splenic cell preparations. Using cells from animals infected testicularly for 9 to 12 days, unfractionated splenic preparations, as well as adherent and nonadherent preparations, killed and lysed T. pallidum. Phagocytosis alone could not explain the detrimental effects of adherent cells. When cytochalasin B was used to block phagocytosis, decreases in treponemal numbers were still detected. In related studies, immune rabbit sera did not enhance treponemicidal activity of the adherent cells. To assess the specificity of these reactions, T. pallidum was incubated with two monocyte-like cell lines (human U937 and mouse P388D1). Neither cell line was detrimental, and treponemal numbers were not lowered. The soluble nature of the treponemicidal factors from adherent and nonadherent preparations was shown by physically separating these cells from the organisms and demonstrating treponemal killing and lysis. In summary, clearance of T. pallidum from infected tissues is probably at least partially attributed to macrophage phagocytosis. Our findings suggest another mechanism involving lytic factors secreted by activated adherent and nonadherent cells.     

Studies on the mechanism of lymphocyte-mediated cytolysis. VI. A reappraisal of the requirement for protein synthesis during T cell-mediated lysis.

The role of protein synthesis in the mechanism of T cell-mediated cytolysis has been re-investigated. Cytolytically active (C57BL/L anti-DBA/2) spleen cells treated with pactamycin (10-7 M to 10-6 M) exhibited suppressed protein synthesis (100 +/- 5%), but unimpeded lytic activity. Drug-treated effector cells, incubated for prolonged (up to 24 hr) periods of time in the presence and absence of antigen, showed no siginificant diminution of lytic activity although the incorporation of 3H-leucine into protein was totally ablated. Studies with emetine, another irreversible inhibitor of protein synthesis, gave identical results. These findings are difficult to reconcile with the hypothesis that effector T cells lysis via a soluble protein mediator.     

Replication of measles virus in Vero cells at elevated temperatures

In one-step growth experiment of measles virus (MV) in Vero cells at 39 C, the appearance of MV infectivity was delayed for 24 hr and the maximum titer was reduced by approximately 1,000-fold, when compared with those at 35 C. MV infectivity was thermolabile at the high temperature. Penetration was rather enhanced at 39 C. By Northern blot hybridization, viral RNAs including 50S genome-sized RNA and mRNAs were first detectable 24 hr post-infection (PI) at 35 C and 36 hr PI at 39 C, respectively. Rapid degradation of viral mRNAs was not observed in the infected cells at 39 C. The synthesis of N, F, and M proteins was relatively reduced at the high temperature and appearance of the other viral protein was delayed, in agreement with the time course of viral RNA synthesis. All these data suggest that less efficient synthesis of viral RNA, restriction of synthesis of N, F, and M proteins at translational level and thermolability of infectivity are all involved in the suppressed MV production in Vero cells at 39 C.     

Differential effects of protein synthesis inhibition on CTL and targets in cell-mediated cytotoxicity

The reactions that lead to target cell lysis by cytotoxic T cells (CTL) are despite intensive investigations poorly understood. To examine the relative roles effectors and targets play in the lytic reaction, protein synthesis in either CTL or targets was inhibited before assay of lysis. We show, in agreement with previous results, that de novo protein synthesis is not necessary in either effectors or targets during the cytolytic reaction. However, activation of CTL requires protein synthesis. Activated CTL respond to protein synthesis inhibitors with a cycling of activity, a result that is interpreted to be consistent with a stimulus secretion mechanism. Treatment of targets with protein synthesis inhibitors prior to incubation with CTL leads to a very rapid and irreversible loss of lytic susceptibility. It is shown that the decrease in lysability is not due to lack of proper CTL target interaction: MHC class I antigens are expressed on drug-treated targets and these cells serve as cold targets in competitive inhibition experiments. Moreover, drug-treated targets trigger transient Ca2+ mobilization and generation of inositol phosphates in CTL. It is therefore concluded that drug-treated targets are able to trigger CTL function but lack a component that is required for their successful lysis.     

Subacute sclerosing panencephalitis virus dominantly interferes with replication of wild-type measles virus in a mixed infection: implication for viral persistence.

Interaction between the Edmonston or Nagahata strain of acute measles virus (MV) and the defective Biken strain of MV isolated from a patient with subacute sclerosing panencephalitis (SSPE) was examined by a cell fusion protocol. Biken-CV-1 cells nonproductively infected with Biken strain SSPE virus were fused with neomycin-resistant CV-1 cells. All the fused cells selected with the neomycin analog G418 expressed Biken viral proteins, as determined by an immunofluorescence assay. This procedure enabled the transfer of Biken viral genomes into cells previously infected with MV. In the fused cells coinfected by Biken strain SSPE virus and Edmonston or Nagahata strain MV, early MV gene expression was suppressed, as determined by immunoprecipitation with strain-specific antibodies. Maturation of Edmonston strain MV was also suppressed. When the coinfected fused cells were selected with G418, Biken viral proteins remained at a constant level for up to 7 weeks. Wild-type MV proteins gradually decreased to a barely detectable level after 4 weeks and became undetectable after 7 weeks. Immunofluorescence studies showed a steady decline in cells expressing wild-type MV proteins in the coinfected cultures. These results suggest that Biken strain SSPE virus dominantly interferes with the replication of wild-type MV. The possible mechanisms of dominant interference and the implication for evolution of a persistent MV infection are discussed.     

TPA induction of EL4 resistance to macrophage-released TNF: role of ADP-ribosylation in tumoricidal activities of TNF and other factors

Activated macrophages synthesize and release numerous tumoricidal soluble factors that can be divided into receptor- or nonreceptor-dependent agents. Tumor necrosis factor (TNF) would be an example of the former. In our experimental model the killing of EL4 thymoma cells by syngeneic activated macrophages involves, but not exclusively, TNF. Our results show that approximately 50% of the anti-EL4 activity expressed by macrophages can be specifically inhibited with rabbit anti-mouse TNF antibody. EL4 variants resistant to the lytic activity of TNF were still susceptible to macrophage-mediated lysis. A tumor-promoting phorbol ester, TPA, rendered TNF-sensitive and -insensitive EL4 cells resistant to M phi-mediated lysis. However, TPA down-regulated TNF-specific binding sites on both TNF-sensitive and -resistant cell surface membranes, suggesting that resistance to TNF involves postligand:receptor events. Tumor cell G-protein involvement (ADP-ribosylation), as a result of TNF-TNF receptor interactions, was investigated. The results showed that pertussis toxin was cytotoxic against TNF-sensitive and -resistant EL4 cells but not against TPA-treated target cells. Inhibitors of ADP-ribosyltransferase inhibited pertussis toxin cytotoxicity and macrophage-mediated lysis but did not interfere with recombinant TNF lytic activity.     

Human receptor for measles virus (CD46) enhances nitric oxide production and restricts virus replication in mouse macrophages by modulating production of alpha/beta interferon

Complement regulatory protein CD46 is a human cell receptor for measles virus (MV). In this study, we investigated why mouse macrophages expressing human CD46 restricted MV replication and produced higher levels of nitric oxide (NO) in response to MV and gamma interferon (IFN-gamma). Treatment of MV-infected CD46-expressing mouse macrophages with antibodies against IFN-alpha/beta blocked NO production. Antibodies against IFN-alpha/beta also inhibited the augmenting effect of MV on IFN-gamma-induced NO production in CD46-expressing mouse macrophages. These antibodies did not affect NO production induced by IFN-gamma alone. These data suggest that MV enhances NO production in CD46-expressing mouse macrophages through action of IFN-alpha/beta. Mouse macrophages expressing a human CD46 mutant lacking the cytoplasmic domains were highly susceptible to MV. These cells produced much lower levels of NO and IFN-alpha/beta upon infection by MV, suggesting the CD46 cytoplasmic domains enhanced IFN-alpha/beta production. When mouse macrophages expressing tailless human CD46 were exposed to culture medium from MV-infected mouse macrophages expressing intact human CD46, viral protein synthesis and development of cytopathic effects were suppressed. Pretreating the added culture medium with antibodies against IFN-alpha/beta abrogated these antiviral effects. Taken together, these findings suggest that expression of human CD46 in mouse macrophages enhances production of IFN-alpha/beta in response to MV infection, and IFN-alpha/beta synergizes with IFN-gamma to enhance NO production and restrict viral protein synthesis and virus replication. This novel function of human CD46 in mouse macrophages requires the CD46 cytoplasmic domains.     

Complement reversal of immunosuppression induced with plasma of rats infected with Trypanosoma brucei rhodesiense

Suppression of antibody production by splenic lymphocytes from rats immunized with sheep red blood cells (SRBC) after incubation with plasma from rats infected with Trypanosoma brucei rhodesiense was confirmed. Suppressive activity became evident in plasma after the sixth day of infection and was manifested by reduction in the number of hemolytic Jerne plaques produced by the treated cells. The activity was temporally associated with increased amounts of soluble immune complex (SIC) reduced titers of lytic complement, elevated titers of immunoconglutinin (IK) and anemia. Treatment of suppressive plasma with hemolysin sensitized SRBC alexinated with horse complement to reduce IK did not reduce suppressive activity, and the activity appeared to have been enhanced when the plasma was heated to inactivate the remaining complement (C'). When fresh rat C' was added to the treated cells, the suppression was largely, though not completely, reversed. Treatment of spleen cells with SIC prepared in vitro from bovine serum albumin (BSA) and rabbit antiBSA also suppressed the plaque forming capacity of the cells. Complexes of BSA-antiBSA-C' and complexes of BSA-antiBSA-C'-IK were equally suppressive. Again, addition of fresh C' to cells treated with these complexes largely, though not completely, reversed the suppressive effect on the cells. From the results it is suggested that immunosuppression associated with experimental T. b. rhodesiense infection may be in part a suppression of the capacity of induced lymphocytes to produce antibody. It is possible that the suppression was mediated by SIC present in the plasma of the infected rats and this effect was probably enhanced by reduced levels of complement in the suppressive plasma.     

Enhancement of murine T cell I-J expression by limited proteolysis

I-J-encoded structures on peripheral T cells and thymocytes appear normally to be blocked or shielded by material that is susceptible to proteolysis. Limited proteolysis with trypsin, papain, pronase, or chymotrypsin increased the number of peripheral T cells and thymocytes lysed by anti-I-Jk serum and complement. Proteolysis did not induce I-Jk expression on B cells or on negative strain T cells. Increased lysis was enzyme concentration and time dependent and was not due to increased susceptibility of protease-treated cells to lysis by antibody plus complement; proteolysis rendered T cells and thymocytes less susceptible to lysis by anti-H-2Kk, anti-H-2Dd, and anti-Lyt-2 antibodies. Absorption experiments showed that I-Jk determinant density was increased in the protease-treated T cell population. The I-Jk determinants detected are proteins or glycoproteins; extended proteolysis removed these molecules from the T cell surface. Treatment of T cells or thymocytes with activated macrophage culture supernatant containing proteolytic activity produced a small but reproducible increase in I-Jk expression. Proteolysis of lymphocyte membranes, possibly mediated by macrophages, may have a role in cellular differentiation and immune activation.     

Target Cells of Cytotoxic T Lymphocytes Directed to the Individual Structural Proteins of Rabies Virus

Target cells of cytotoxic T lymphocytes (CTL) directed to the individual structural proteins (except for the large polymerase (L) protein) of rabies virus were established by expressing only the respective protein in murine neuroblastoma (NA) and murine macrophage (J774-1) cell lines. Mice infected with the ERA strain of rabies virus developed CTL responses to all of these rabies virus proteins. The cytotoxic activity was abrogated by pretreatment of the effector cells with anti-CD8 monoclonal antibody (MAb) and complement but not with anti-CD4 MAb. Cell lysis by CTL was blocked in the presence of anti-major histocompatibility complex (MHC) class 1 antibodies in J774-1 cell lines. Rabies virus-infected cells express these proteins at the surface, which can be recognized and lysed by the respective CTL. Mice immunized with β-propiolactone-inactivated virus induced a CTL response against glycoprotein but not against internal viral components. This assay system might be useful for further analysis of the possible contribution of these proteins in the cell-mediated immune protection against rabies.     

Measles virus exploits dendritic cells to suppress CD4+ T-cell proliferation via expression of surface viral glycoproteins independently of T-cell trans-infection

Dendritic cells (DC) have been proposed to play a pivotal role in transient immune suppression induced by measles virus (MV) infection. In the present study, we show that DC-induced suppression of T-cell proliferation was not mediated by IL-10 or IFNalpha/beta, which are released following infection of DC, but required cell contacts between MV-infected DC and T cells. Human sera containing neutralizing anti-MV antibodies, as well as anti-MV hemagglutinin (HA) or fusion protein (F) mAbs, were found (i) to reverse suppression and (ii) to restore DC allostimulatory capacity. Interestingly, DC-induced T-cell suppression was associated with both phenotypic and functional DC maturation, as demonstrated by IL-12 production and chemotaxis to MIP-3beta. These data suggest that MV infection turns on the maturation program of DC allowing migration to draining lymph nodes, where potent T-cell immune suppression might be achieved via cell surface expression of HA and F glycoproteins, independently of T cell trans-infection.     

High major histocompatibility complex-unrestricted lysis of simian immunodeficiency virus envelope-expressing cells predisposes macaques to rapid AIDS progression

Before the development of virus-specific immune responses, peripheral blood mononuclear cells (PBMC) from uninfected rhesus monkeys and human beings have the capacity to lyse target cells expressing simian immunodeficiency virus (SIV) or human immunodeficiency virus-1 (HIV) envelope (gp130 and gp120) antigens. Lysis by naive effector cells does not require major histocompatibility complex (MHC)-restricted antigen presentation, is equally effective for allogeneic and xenogeneic targets, and is designated MHC-unrestricted (UR) lysis. UR lysis is not sensitive to EGTA and does not require de novo RNA or protein synthesis. Several kinds of envelope-expressing targets, including cells that poorly express MHC class I antigens, can be lysed. CD4(+) effectors are responsible for most of the lytic activity. High lysis is correlated with high expression of HIV or SIV envelope, specifically, the central one-third of the gp130 molecule, and lysis is completely inhibited by a monoclonal antibody against envelope. Our work extends observations of human lymphocytes expressing HIV gp120 to the SIV/rhesus monkey model for AIDS. Additionally, we address the relevance of UR lysis in vivo. A survey of PBMC from 56 uninfected rhesus monkeys indicates that 59% of the individuals had peak UR lytic activity above 15% specific lysis. Eleven of these monkeys were subsequently infected with SIV. Animals with UR lytic activity above 15% specific lysis were predisposed to more rapid disease progression than animals with low UR lytic activity, suggesting a strong correlation between this form of innate immunity and disease progression to AIDS.     

Establishment of a Vero cell line persistently infected with African swine fever virus.

A Vero cell line persistently infected with African swine fever virus was established by infecting the cells in the presence of 10 mM NH4Cl (Vero-P cell line). The virus derived from the Vero-P cultures infected Vero cells, and virus titers were comparable to those obtained in Vero cells acutely infected with African swine fever virus. The structural proteins of the virus from Vero-P cells were similar to those of the virus produced in lytic infections. Virus production was low when the Vero-P cells were growing logarithmically and increased considerably in confluent cultures when lysis appeared in a fraction of the cell population.     

Natural cytotoxic cells and tumor necrosis factor activate similar lytic mechanisms

The lytic activity of natural cytotoxic (NC) cells has several characteristics which clearly distinguish it from other cell-mediated lytic activities and from most soluble cytolytic factors. An exception is the lytic activity mediated by tumor necrosis factor (TNF). In this paper, we report a detailed comparison of NC and TNF lysis of target cells which are used as prototype NC targets or TNF targets, and show that the two cytolytic activities have very similar, if not identical, lytic mechanisms. We present data showing that target cells which are NC-sensitive are also TNF-sensitive and that target cells which are NC-resistant are also TNF-resistant. Moreover, cells selected either in vivo or in vitro for NC resistance are selected for TNF resistance, and cells selected for TNF resistance are selected for NC resistance. The analysis of the kinetics of 51Cr release mediated by NC cells or by TNF show that both activities affect similar kinetics, in that there is no cell lysis for several hours after targets and effectors first interact. However, NC and TNF lytic activities can be distinguished. By using the cell lines 10ME or B/C-N as targets, it can be shown that whereas NC-mediated lysis is dependent on protein synthesis, TNF-mediated lysis is not. We also show that targets which are resistant to NC-mediated lysis because they express a protein synthesis-dependent resistance mechanism also require protein synthesis to resist TNF-mediated lysis, suggesting that the same resistance mechanism protects cells against both NC cells and TNF. Together, these data strongly support the hypothesis that NC cells and TNF activate the same lytic mechanism within target cells and that TNF may mediate the lytic activity of NC effector cells.     

Antibody-dependent cellular cytotoxicity against murine leukemia viral antigens: studies with human lymphoblastoid cell lines and human peripheral lymphocytes as effector cells comparing rabbit, goat, and mouse antisera.

A thymic lymphoblastoid cell line derived from a New Zealand Black mouse produces murine leukemia virus (MuLV) and was used as a target in model systems for the in vitro study of antibody-dependent cellular cytotoxicity (ADCC). Several human lymphoblastoid cell lines were investigated as potential effector cells. The most promising (Raji cells) bound to antibody-coated target cells but caused only modest levels of ADCC at 25:1 effector-to-target cell ratio with substantial lysis in the absence of antiserum. Human peripheral lymphocytes were active as effector cells in ADCC at a 5:1 ratio and produced no lysis in the absence of antibody. These cells were used to demonstrate that high dilutions of rabbit antisera to MuLV antigens p30, p15, p12, and p10 were capable of mediating lysis of MuLV-producing target cells but not of a virus-negative murine cell line. A murine antiserum to Thy 1.2 and three caprine antisera to MuLV antigens that were active in complement-mediated cytotoxicity functioned poorly in inducing ADCC; however, rabbit antisera to similar antigens were 16- to 512-fold more efficient in cell-mediated than in complement lysis. The inefficiency of goat antisera was not due to shedding of cell surface antigens or generation of blocking factors but rather to lack of lytic interaction of antibody-coated targets with the effector cells.     

Adenovirus gene function required for induction of nuclear acidic protein synthesis: binding of these proteins to adenovirus DNA.

Two different viral DNA-defective temperature-sensitive mutants of adenovirus 12 (H 12) were defective in their ability to induce the synthesis of various molecular weight classes of nuclear acidic proteins, both virion and nonvirion components, after lytic infection of human embryo kidney (HEK) cells at the restrictive temperature. This finding indicates that the induction of nuclear acidic protein synthesis is an adenovirus gene function(s). Treatment of infected cells with actinomycin D at an early stage of virus maturation suppressed the synthesis of an acidic virion protein (hexon), but allowed the synthesis of other classes of nuclear nonvirion acidic proteins during the subsequent late maturation period, suggesting that different mechanisms control virion and nonvirion polypeptide synthesis. The interaction of the nuclear acidic proteins isolated from H 12-infected cells with native-labeled H 12 DNA was studied using the membrane filter technique. Measurements of the ability of different DNA preparations to inhibit the H 12 DNA-acidic protein complex formation suggest that the nuclear acidic proteins bound to native H 12 or HEK cell DNA with much higher affinity than to native calf thymus DNA. Moreover, native H 12 DNA was able to bind the acidic proteins more efficiently than did denatured H 12 DNA. The acidic proteins isolated from the cytoplasm of H 12-infected cells bound approximately 100-fold less to native H 12 DNA than did the nuclear proteins. Furthermore, the H 12 DNA binding affinity of the nuclear acidic proteins from uninfected cells, or from H 12-infected and 1-beta-D-arabinofuranosylcytosine-treated cells, was somewhat lower than that of the nuclear proteins from infected (untreated) cells.