José Menezes

Biological differences between Epstein-Barr virus (EBV) strains with regard to lymphocyte transforming ability, superinfection and antigen induction.

Abstract EB virus (EBV) preparations derived from various producing lymphoblastoid cell lines (LCL) differed in their biological properties, as judged by the following four tests: (1) cord blood lymphocyte (CBL) transformation into EBV-carrying LCL; (2) early antigen (EA) induction in Raji cells; (3) inhibition of Raji cell growth; (4) induction of the EBV-determined nuclear antigen (EBNA) in CBL. B95-8 virus transformed and induced EBNA in CBL but did not induce EA in Raji cells, nor did it inhibit their growth. P3HR-1 virus did not transform CBL, induced no EBNA or EA in CBL, but induced EA in Raji cells and inhibited their growth. EBV isolated from the QIMR-WIL, 833L, F137 and cb-8-7 LCL resembled the B95-8 virus with regard to its biological activity (CBL transformation, EA induction in and growth inhibition of Raji cells). Transformation of CBL as contrasted to EA induction in, and growth inhibition of Raji cells thus appear as mutually exclusive viral functions.

Innate Immune Response of the Human Host to Exposure with Herpes Simplex Virus Type 1: In Vitro Control of the Virus Infection by Enhanced Natural Killer Activity via Interleukin-15 Induction

ABSTRACT Infections with herpes simplex virus type 1 (HSV-1) in humans and in animal models are accompanied by enhanced natural killer (NK) activity. In vitro, HSV-1 also enhances the NK activity of human peripheral blood mononuclear cells (PBMC). The molecular basis of this enhanced NK activity, however, is not well characterized. We investigated the role of human interleukin-15 (IL-15) in this phenomenon and report here that HSV-1-mediated enhanced NK activity was abrogated by neutralizing antibodies for IL-15 but not for other cytokines (i.e., IL-2, IL-12, gamma interferon [IFN-γ], tumor necrosis factor alpha, or IFN-α). Anti-CD122 antibodies which block signaling through IL-2 receptor β chain, and therefore neutralize the effects of IL-15 (and IL-2), also abrogated this enhancement. Furthermore, HSV-1 increased the levels of IL-15 mRNA and the production of IL-15 in HSV-1-infected PBMC cultures. The neutralization of IL-15 in cocultures of PBMC with HSV-1-infected cells significantly increased HSV-1 production. These results strongly suggest a role for IL-15 in the HSV-1-mediated in vitro enhancement of NK activity and in the PBMC-mediated suppression of HSV-1 replication.

Infection of EBV-Genome-Negative and – Positive Human Lymphoblastoid Cell Lines with Biologically Different Preparations of EBV

Epstein-Barr virus (EBV) derived from the B95-8 line has transforming activity for cord blood cells, whereas virus derived from the P3HR-1 line lacks such activity. When the two viral preparations were compared for their ability to infect the same EBV-genome-negative lymphoblastoid cell line, BJA-B, they induced approximately the same number of EBV-determined nuclear antigen (EBNA)-positive cells. EBNA is compatibile with continued cell proliferation. No early antigen (EA)-positive cells appeared in the B95-8 virus-infected cultures, whereas P3HR-1 virus-infected cells went on to express EA. EA signals the entry of the cell into the lytic cycle. No late viral antigen (VCA) appeared and, as a consequence, the P3HR-1 virus infection became abortive. In contrast, the EBNA-positive cells induced by the B95-8 virus continued to divide over several weeks. These findings show that different EBV isolates may differ in their biological activity, probably due to their having different degrees of viral dependence on restrictive host cell controls.

Epstein—Barr virus receptor expression on human CD8+ (cytotoxic/suppressor) T lymphocytes

In 1977 we showed that cells of a human lymphocytic leukaemia-derived T line (Molt-4) have receptors for Epstein-Barr virus (EBV). More recently, EBV-positive human T cell lymphomas have been recognized and human T cell lines containing the EBV genome have been established in vitro. To understand better the interaction of EBV with T cells, we decided to determine first whether human peripheral blood T lymphocytes express receptors for EBV. Using flow cytometry we examined the binding of both lymphocyte-transforming (B95-8) and non-transforming (P3HR-1) strains of EBV to T lymphocyte subpopulations, using a double labelling technique with T cell-specific phycoerythrinated monoclonal antibodies (Leu 2a) and fluoresceinated viral preparation. Our results suggest that, in general, about 50% of the CD8+ (or suppressor/cytotoxic) T cell subpopulation from both EBV-seropositive and -seronegative individuals can bind EBV. EBV receptor expression on these T cells was about 10 and 51 times less than that on Molt-4 and Raji (an EBV receptor-positive B cell line) cells, respectively. The specificity of this binding was demonstrated by the inhibition of attachment of viral preparations preincubated with a monoclonal antibody directed against the viral ligand (gp240/350), and by preincubating these target T cells with unlabelled virus. We were unable to detect EBV-induced antigens in infected T cells, suggesting that, as in Molt-4 cells, virus internalization may not occur in fresh T cells and/or that the virus receptor may not be completely functional. We were also unable to detect C3d (or CR2) receptors on these T cells, or to inhibit virus attachment by treating the targets with an anti-CR2 monoclonal antibody (OKB7), suggesting that the EBV receptor on CD8+ peripheral blood lymphocytes is different from that on B cells.

Peripheral Blood Cytotoxic γδ T Lymphocytes from Patients with Human Immunodeficiency Virus Type 1 Infection and AIDS Lyse Uninfected CD4+ T Cells, and Their Cytocidal Potential Correlates with Viral Load

ABSTRACT Progression of human immunodeficiency virus type 1 (HIV-1) infection in humans is marked by declining CD4+-T-cell counts and increasing virus load (VL). Cytotoxic T lymphocytes (CTL) play an important role in the lysis of HIV-infected cells, especially during the early phase of asymptomatic infection. CTL responses in the later phase of disease progression may not be as effective since progressors with lower CD4+-T-cell counts have consistently higher VL despite having elevated CTL counts. We hypothesized that, apart from antiviral effects, some CTL might also contribute to AIDS pathogenesis by depleting CD4+ T cells and that this CTL activity may correlate with the VL in AIDS patients. Therefore, a cross-sectional study of 31 HIV-1-infected patients at various clinical stages was carried out. Purified CTL from these donors as well as HIV-seronegative controls were used as effectors against different human cell targets by using standard 51Cr release cytolytic assays. A direct correlation between VL and CTL-mediated, major histocompatibility complex (MHC)-unrestricted lysis of primary CD4+-T-cell, CEM.NKR, and K562 targets was observed. CD4+-T-cell counts and duration of infection also correlated with MHC-unrestricted cytolytic activity. Our data clearly show that γδ CTL are abnormally expanded in the peripheral blood of HIV-infected patients and that the Vδ1 subset of γδ T cells is the main effector population responsible for this type of cytolysis. The present data suggest that γδ CTL can contribute to the depletion of bystander CD4+ T cells in HIV-infected patients as a parallel mechanism to HIV-associated immunopathogenesis and hence expedite AIDS progression.

Epstein-Barr virus (EBV)-lymphoid cell interactions. I. Quantification of EBV particles required for the membrane immunofluorescence assay and the comparative expression of EBV receptors on different human B, T and null cell lines.

We report data on the number of Epstein-Barr virus (EBV) particles required to detect virus binding to target cells (Raji or BJA-B) by means of membrane immunofluorescence (MIF). After determining the optimum conditions for the MIF assay the following aspects of EBV-lymphoid cell interactions were examined: (i) binding of two different strains of EBV to various types of human lymphoid cell lines, (ii) expression of receptors for both EBV and complement on these lines and (iii) induction of EBV-induced nuclear antigen (EBNA) in the different target cells used. The results showed that a minimum of about 2.7 x 10(3) enveloped virus particles/cell were required for an optimum visualization of EBV binding to the target cells tested, and that a lymphoid cell may bear receptors for one prototype strain of EBV but not for the other. A number of cell lines, particularly those of T and null type which express EBV receptors, did not synthesize EBNA, thus indicating that these lines were resistant to EBV infection. Several of these lines, although expressing cell surface EBV receptors, lacked complement receptors.

Persistence of both human cytomegalovirus and Epstein-Barr virus genomes in two human lymphoblastoid cell lines.

By DNA-DNA reassociation kinetic analysis, less than one genome equivalent per cell of human CMV-DNA was found in two lymphoblastoid cell lines, one derived from the peripheral blood of a congenitally infected male infant at the age of 21 months (D4 cell line), the other obtained by co-cultivation of lethally X-irradiated cells from the 9-month lymphoblastoid cell line previously described by Joncas et al. (1975) with cord blood leukocytes of a female newborn (M1 cell line). Human CMV antigens could not be detected and virus could not be rescued from these cells by co-cultivation with fully permissive human fibroblasts. It may be that the CMV-DNA is defective. Epstein-Barr virus DNA as well as EBNA and EBV-EA antigens were present in these cell lines. Both lines express surface markers characteristic of thymus-independent, B lymphocytes. The CMV-DNA of the CMV-DU strain, isolated from this infants urine five times successively from the age of 1 day to 30 months, appears to be closely related to the DNA of the AD-169 strain by reciprocal hybridization and by electrophoretic pattern analysis of the restriction enzyme cleavage products. Experimental attempts to transform cord blood leukocytes with this urine strain of CMV before or after u.v. irradiation have so far failed.

Studies on the Induction of IgG-Fc Receptors and Synthesis of IgM in Primary and Chronically-Infected Lymphoid (Raji) Cells by Herpes Simplex Virus

The present paper reports on the induction of two cell surface markers on human lymphoid cells following herpes simplex virus (HSV) infection. While both primary and chronic infections of human lymphoid cells led to the induction of receptors for the Fc region of 7S IgG, chronic HSV infection was also characterized by the induction of surface-bound IgM. Surface and intracellular Fc receptors were detected in the human lymphoid cell line, Raji, infected with HSV types 1 and 2. Under optimal conditions with a multiplicity of infection (m.o.i.) of 50 to 100 p.f.u. per cell, this marker was inducible in only about 53% of the infected cells. Kinetic studies revealed the appearance of these receptors at around 5 h following HSV infection and they reached a plateau 16 to 18 h p.i. Interestingly, this Fc receptor expression (i.e. percentage of positive cells) was found to be similar in primary and chronically HSV-infected Raji cells. Both human leukocyte interferon and phosphonoacetic acid (PAA), an inhibitor of herpesvirus DNA polymerase activity, effectively inhibited Fc receptor synthesis during primary HSV-infection and these two agents suppressed its induction in chronically HSV-infected Raji (Raji-HSV) cells. This inhibitory or suppressive effect, particularly of PAA, suggests that this HSV-induced Fc receptor may represent a late virus function in the infected cell. Unlike primary HSV infection, about 80% of the chronically HSV-infected Raji cells were found to express surface-bound IgM. This IgM induction was suppressed by long-term interferon treatment but not with PAA-treatment. Superinfection studies of interferon and PAA-treated Raji-HSV cells indicate that only the former would develop Fc receptors suggesting a protective role of this IgM against superinfection by HSV.