Long-term protection of chimpanzees against high-dose HIV-1 challenge induced by immunization.

A combination AIDS vaccine approach consisting of priming with adenovirus-HIV-1MN gp160 recombinants followed by boosting with HIV-1SF2 gp120 was evaluated in chimpanzees. Long-lasting protection, requiring only three immunizations, was achieved against a low-dose challenge with the SF2 strain of HIV-1 and a subsequent high-dose SF2 challenge administered 1 year later without an intervening boost. Notably, neutralizing antibody responses against both clinical and laboratory isolates developed in three chimpanzees and persisted until the time of high-dose challenge. The possibility that cytotoxic T-lymphocytes contribute to low-dose protection of a chimpanzee lacking neutralizing antibodies is suggested. Our results validate the live vector priming/subunit booster approach and should stimulate interest in assessing this combination vaccine approach in humans.

Comparison between murine natural antibodies and natural killer cells: recognition of separate target structures as revealed by differential in vitro expression and dependence on glycosylation.

The specificity of complement-fixing, cytotoxic antibodies against the YAC lymphoma in sera of normal young adult (A X C57BL)F1 mice was studied. In vivo-maintained, immunoselected sublines of the YAC lymphoma expressed low amounts of the natural antibody (NAb) target structure. These cell lines were also resistant to natural killer (NK) cell-mediated lysis. After 2-3 weeks of in vitro culture the immunoselected cell lines became NK sensitive, but they remained resistant to NAb. When several independently derived variants selected for low NK sensitivity were tested for their ability to absorb NAb, the degree of absorption varied considerably among the variants. NAb could be inhibited by purified C-type virus particles and also by bacterial sonicates and various glycoprotein preparations. Treatment of target cells with tunicamycin, an inhibitor of asparagine-linked glycosylation, decreased the sensitivity to NAb lysis but had no impact on NK sensitivity. Thus the results indicated that a) NAb and NK cells recognized separate target structures and b) the target structure(s) for NAb but not for NK cells were saccharides of the N-glycosidic type.

Quantitative measurement of fusion between human immunodeficiency virus and cultured cells using membrane fluorescence dequenching.

Human immunodeficiency virus (HIV) was purified by sucrose gradient centrifugation and labeled with octadecylrhodamine B-chloride (R-18) under conditions resulting in 90% quenching of the fluorescence label. Incubation of R-18-labeled HIV (R-18/HIV) with CD4-positive CEM and HUT-102 cells, but not with CD4-negative MLA-144 cells, resulted in fluorescence dequenching (DQ, increase in fluorescence) of 20-25%. Similar level of DQ was observed upon incubation of CEM cells with R-18-labeled Sendai virus. DQ was observed when R-18/HIV was incubated with CD4+ cells at 37 degrees C, but not at 4 degrees C. Most of the increase in fluorescence occurred within 5 min of incubation at 37 degrees C and was independent of medium pH over the range of pH 5-8. Preincubation of cells with the lysosomotropic agent NH4Cl had no inhibitory effect on DQ. Complete inhibition was observed when target cells were fixed with glutaraldehyde prior to R-18/HIV addition. Our results demonstrate application of membrane fluorescence dequenching method to a quantitative measurement of fusion between HIV and target cell membranes. As determined by DQ, HIV penetrates into target cells by a rapid, pH-independent, receptor-mediated and specific process of fusion between viral envelope and cell plasma membrane, quite similar to that observed with Sendai virus.

Pattern of Epstein-Barr virus (EBV)-specific proteins synthesized during primary lytic infection of mouse lymphocytes by EBV.

Normal mouse lymphocytes were implanted with EBV receptors and exposed to the virus of P3HR-1 strain. 5% of the cells expressed early (EA) and viral capsid (VCA) antigens as assayed by immunofluorescence 24 h after the infection. Only 0.1% of cells expressed nuclear-like antigen (EBNA) 48 h post-infection. When labelled metabolically with [35S]methionine, extracted, immunoprecipitated with EBV-positive sera, and analyzed by SDS-gel electrophoresis and autoradiography, about 20 EBV-determined proteins ranging from 19 to 165 kd were detected. Their pattern and relative quantitative expression differed from those in P3HR-1 virus superinfected Raji cells. Polypeptides of approximate molecular size 78, 72, 65, 48 and 26.5 kd were predominant in EBV-infected mouse lymphocytes. In contrast, 130, 98, 59, 50.5 and 36 kd proteins were predominant in the induced Raji cells. Our results demonstrate that rodent lymphocytes can be used for the direct biochemical analysis of EBV-translational products during primary lytic infection in normal cells.

Infection of human epithelial cells by Epstein-Barr virus (EBV). II. Biochemical characterization of EBV-determined proteins synthesized in epithelial cells.

Primary cultures of epithelial cells were grown from tonsils of patients with diseases not related to EBV. The cells were implanted with EBV receptors and exposed to EBV of the transforming (B95-8, AG-876) and nontransforming (P3HR-1) strains. The EBV-infected and control cells were pulsed with [35S]methionine at 18-24 h after infection, and cell extracts were prepared for immunoprecipitation with anti-EBV sera and analysis by gel electrophoresis and autoradiography. About 20 EBV-determined proteins ranging from 22 to 185 kDa were detected in P3HR-1 virus-infected epithelial cells. Only a few polypeptides were detected in extracts of cells infected with AG-876 virus while no EBV-specific proteins were immunoprecipitated from extracts of B95-8 virus-infected cells. These results demonstrate that the system of EBV receptor-implanted normal human epithelial cells can be used for direct biochemical analysis of EBV infection in the epithelial tissue.

The detection of human minor alloantigens following restriction determinant implantation.

The recognition of minor alloantigens by cytotoxic T lymphocytes (CTL) serves as a model for the recognition of tumor and viral antigens. Progress in this area has been limited, however, since CTL recognize minor alloantigens only in association with self-class I antigens. Thus, experiments designed to study minor alloantigens are limited to target cells that share HLA determinants with the CTL. We raised CTL lines that recognized human minor alloantigens. In order to circumvent the problem that only target cells which expressed the appropriate restriction determinants could be tested for minor antigens. Sendai virus mediated fusion was used to integrate appropriate HLA antigens into cells that did not express them naturally. The target cells were then tested in CML for their expression of minor antigens. The results of experiments demonstrated that, following class I implantation, the detection of minor antigens on certain restriction determinant negative cells was possible. Furthermore, the restriction determinant was able to associate with the minor antigen in a manner appropriate for recognition by the T-cell receptor.

Necrotizing lymphoid vasculitis in X-linked lymphoproliferative syndrome.

An 8-year-old maternally related relative of three boys who had developed agammaglobulinemia associated with Epstein-Barr virus (EBV)-induced infectious mononucleosis was studied for X-linked lymphoproliferative syndrome (XLP) in 1979. At that time, he demonstrated no striking immunologic aberrations and was seronegative for EBV. Subsequently, immunologic abnormalities including failure to switch from IgM to IgG antibody synthesis after secondary immunization with bacteriophage phi X174 were detected. In 1983, he experienced episodic intracerebral hemorrhages, with the second being fatal. At autopsy, necrotizing vasculitis and aneurysms involving arteries of the central nervous system were observed. Studies of blood obtained immediately before and after death failed to show antibodies to EBV. However, EBV genome was demonstrated in tissues obtained at autopsy by DNA hybridization studies. Fatal lymphoid vasculitis in this patient is unique among boys with XLP in the registry. These findings probably extend the phenotypic expressions of XLP.

Interaction of a noncytopathic human immunodeficiency virus type 1 (HIV-1) with target cells: efficient virus entry followed by delayed expression of its RNA and protein.

Recently described noncytopathic human immunodeficiency viruses type-1 (HIV-1) form a new category within the HIV-1 family due to their unique biological properties and predominant occurrence in symptom-free individuals. To study the mechanism of noncytopathic HIV-1 infection, we compared the infectivity and life cycles of two closely related HIV-1 clones with noncytopathic (N1T-E) or cytopathic (N1T-A) properties. N1T-E virus exhibited slow kinetics of infection in T cells and monocytes. Slow infection was not due to defective virus entry, because N1T-E and N1T-A exhibited equally efficient virus-cell fusion activity and nucleocapsid internalization. Kinetic studies of N1T-E genome expression revealed low levels of viral RNA, structural proteins, and Tat protein during the first 7 days after virus entry. In contrast, cells infected with the same dose of cytopathic N1T-A virus began to express high levels of genomic RNA, structural proteins, and Tat protein within 48 hr of infection; the expression peaked on Day 5, followed by complete cell lysis. No delay in N1T-E replication, as compared to N1T-A, was observed after transfection of cloned N1T-E proviral DNA. N1T-E virus had intact Tat, Rev, and fusion functions and replicated well in chronically infected cells. These results suggest that delayed processing or expression of HIV-1 genome during the early phase of the virus replicative cycle is an important determinant in noncytopathic infection.

HTLV-III/LAV antibodies and virus in hemophilia patients in Nebraska: survey and initiation of a prospective study.

Thirty-nine patients from the Nebraska Regional Hemophilia Center were studied for the prevalence and titers of antibodies to HTLV-III/LAV, and for clinical symptoms of possible progression toward the acquired immunodeficiency syndrome (AIDS). Fourteen of 26 (54%) patients with hemophilia A were positive for HTLV-III/LAV antibodies as determined by immunofluorescence and Western blotting. Retrovirus was detected in cultured lymphocytes of two out of three seropositive individuals by reverse transcriptase assay and molecular hybridization with cloned HTLV-III/LAV probe. Of the twelve factor VIII-deficient patients who were seronegative, one had received only heat-treated factor VIII concentrates, six only cryoprecipitate or fresh frozen plasma, two prothrombin complex concentrates, one had not been transfused since 1983, and two had never been transfused. None of the patients treated only with factor IX concentrates, volunteer donor plasma, or cryoprecipitate had HTLV-III/LAV antibodies. In 12 of 14 seropositive hemophiliacs, titers of serum antibodies to HTLV-III/LAV ranged from 1:1,280 to 1:10,240, indicating a strong immune response against HTLV-III/LAV antigens and/or persistent infection with the virus. In spite of the possible exposure to HTLV-III/LAV during the past four years, none of the patients in this group had AIDS. Whether or not this group of patients has developed immunity to the AIDS retrovirus remains to be determined.

Transfection of lymphoblastoid cells using DNA-loaded reconstituted Sendai virus envelopes: expression of transfected DNA and selection of transfected cells.

The American Burkitt's lymphoma cell line Loukes was cotransfected with cloned BamHI K fragment of EBV DNA and a vector pSV2neo. Reconstituted Sendai virus envelopes (RSVE) loaded with DNA were used for efficient gene transfer. Two cell lines have been obtained following culture in the presence of geneticin sulfate (G-418). Messenger RNA from both transfected DNAs was expressed during the whole period of observation, 42 days after transfection. This method provides a relatively simple and efficient means for selection of lymphoblastoid cells expressing a transfected gene.

Host cell range of adult T-cell leukemia virus. I. Viral infectivity and binding to various cells as detected by flow cytometry.

Adult T-cell leukemia virus is the member of a human type-C retrovirus family (HTLV) found to be associated with adult T-cell leukemia (ATL) in Japan. In our study, HTLV was isolated from the MT-2 cell line, purified on sucrose gradient and labelled with fluorescein-isothiocyanate (FITC-HTLV). The protein pattern of the virus was determined by SDS-gel electrophoresis and assured by Western blotting using ATL patient serum. Fresh human lymphocytes, separated B and T cells, mouse and rabbit lymphocytes, mouse fibroblasts, and 13 different tumor cell lines were tested in parallel for binding of FITC-HTLV and infectability by the virus. Virus binding to cell receptors was assayed by flow cytometry. Successful infection was monitored by following the expression of HTLV-determined antigen (HTLA). Most of the cells bound FITC-HTLV at levels ranging from 5% to 130% of the MT-2 cell binding. Only fresh human T, mouse and rabbit lymphocytes were infectable by cell-free virus preparations. The results demonstrate that HTLV receptors are present on different types of cells of both human and animal origin, and that infection by the virus is restricted to fewer host cells but not limited to a specific class of human lymphocytes.

Antibodies to HTLV-III/LAV among aboriginal Amazonian Indians in Venezuela.

Serum samples from 224 aboriginal Amazonian Indians were tested for antibodies to HTLV-III/LAV by an indirect immunofluorescence (IF) assay. 9 individuals (4%), 5 of them female, were seropositive by IF and by confirmatory western blotting and radioimmunoprecipitation tests. 3 of the positive sera were collected in 1968. HTLV-III/LAV seropositivity rates varied among the ethnic groups and ranged from 13.3% among the Pemon Indians to 3.3% among the Yanoama tribe. The titres of HTLV-III/LAV antibodies ranged from 1/40 to 1/320. All individuals tested were apparently healthy at the time of the study. None of 211 randomly chosen, healthy blood donors from Venezuelan cities had antibodies to HTLV-III/LAV. The prevalence of specific antibodies among Amazonian Indians suggests the HTLV-III/LAV or a closely related cross-reactive virus may be endemic in this area. The findings also indicate that this virus is indigenous in non-negroid Latin American and negroid tropical populations.

Prevalence of AIDS-associated retrovirus and antibodies among male homosexuals at risk for AIDS in Greenwich Village.

LAV/HTLV-III has been closely linked to the acquired immunodeficiency syndrome (AIDS). We have studied and correlated the prevalence of AIDS-associated retrovirus and retroviral antibodies in several groups of male homosexuals from Greenwich Village. Retrovirus was detected in cultured peripheral blood lymphocytes by testing for reverse transcriptase (RT) and confirmed by establishment of virus-producer cell lines, and electron microscopy. Seventy-six percent of patients with AIDS, 93% with AIDS-related complex (ARC), 69% with generalized lymphadenopathy (LAS), and 35% of asymptomatic homosexuals were positive for virus in the RT assay. Transmission of the virus from RT-positive lymphocytes into the CEM cell line was successful in 10 of 11 randomly chosen cases. No virus isolates were obtained from lymphocytes of 8 heterosexual individuals. Serum antibodies against AIDS-associated virus were detected by indirect immunofluorescence assay and confirmed by Western blotting, using an LAV/HTLV-III-producer cell line, LAV-N1, which we established. LAV/N1 virus was purified by ultracentrifugation through sucrose gradient and the pattern of its proteins was determined by SDS-gel electrophoresis and Western blotting using sera from an AIDS patient. The major polypeptides of LAV/HTLV-III (19, 25-27, 32, 42 and 54 kilodalton) were present. These proteins did not react in Western blots with sera positive for Adult T cell leukemia virus (ATLV). thus, LAV-N1 and ATLV were not antigenically related. In our assay for LAV/HTLV-III antibodies, 18 (100%) of patients with AIDS, 13 (100%) of patients with ARC, 24 (69%) of 35 patients with LAS and 9 (39%) of 23 asymptomatic homosexuals were sero-positive. Heterosexual controls were negative. All IF-positive sera tested by Western blot contained antibodies against specific viral proteins. High titers (greater than or equal to 1:1280) of serum antibodies against LAV/HTLV-III virus were detected in 71% of AIDS patients, 62% with ARC, 38% LAS and 13% among asymptomatic homosexuals. Our data show that the presence of LAV/HTLV-III antibodies correlates with the presence of infectious virus. Antibody titers may also correlate with progression toward AIDS.

Establishment of retrovirus-, Epstein-Barr virus-positive B-lymphoblastoid cell lines derived from individuals at risk for acquired immune deficiency syndrome (AIDS).

Peripheral blood lymphocytes from male individuals at risk for AIDS were cultured in the presence of interleukin-2. Approximately 90% of cultures originating from pre-AIDS and AIDS patients were retrovirus-positive as detected by the reverse transcriptase (RT) assay and confirmed by electron microscopy. Prolonged incubation of the retrovirus-positive cells resulted in the establishment of several interleukin-2-independent B-lymphoblastoid cell lines. These cells were positive for Epstein-Barr virus (EBV)-specific antigens and contained EBV particles. When irradiated cells from the new lines were cocultivated with an RT-negative T-cell line CEM, an efficient transmission of retrovirus was detected. The supernatants from cocultivated cells had 5-10 fold higher levels of RT activity as compared with the supernatant from the cell line alone. Type-C retroviral particles and budding structures similar to those of human T cell leukemia virus type III (HTLV-III) and lymphadenopathy-associated virus (LAV) were found by electron microscopy. HTLV-III/LAV-specific polypeptides were detected by immunoprecipitation with sera from lymphadenopathy and AIDS patients, but not with sera from healthy individuals. Our data suggest that EBV-infected B lymphocytes from individuals at risk for AIDS may serve as a biological reservoir for the AIDS-associated retrovirus.

Effects of differentiation inducing chemicals on in vivo malignancy and NK susceptibility of metastatic lymphoma cells.

In the present investigation we have determined the effects of the differentiation-inducing chemicals dimethyl sulfoxide (DMSO) and sodium butyrate on the growth and tumorigenicity of a highly malignant/metastatic cell line (RAW117-H10) and its less tumorigenic parental lymphoma cell line (RAW117-P). Both of these agents at doses shown to be nontoxic slowed the growth of these cells in suspension culture and significantly lengthened the doubling time while reducing colony formation in the agar tumor stem cell assay. Corresponding to these observations, the in vivo tumorigenicity of the highly malignant RAW117-H10 line was reduced by both chemicals but particularly by butyrate treatment. In addition, both agents increased the expression of some glycoproteins and the glycolipid asialo GM1. There was also a corresponding increase in the NK susceptibility of the normally NK resistant RAW117-H10 cells. To determine if the decreased malignancy of the highly malignant cells following treatment with the chemical agents was primarily due to the alteration in cell surface glycoconjugates or merely due to concomitant decreased growth potential, we transplanted highly glycosylated membrane fragments from normal syngeneic thymocytes to both RAW117-P and RAW117-H10 cells using a Sendai virus mediated membrane fusion technique. The in vivo tumorigenicity of the membrane altered RAW117-H10 cells was significantly decreased. These results strongly suggest that the decreased in vivo malignancy of RAW117-H10 cells resulting from treatment with chemical differentiation agents is caused by their increased susceptibility to NK cell mediated lysis which in turn results from cell surface changes involving altered, primarily increased, expression of certain glycosylated surface molecules. The cell surface glycoconjugates, such as receptors for certain lectins and glycolipid asialo GM1, can be used as markers for malignant potential and NK sensitivity of malignant lymphoid cells.

Testing for antibodies to AIDS-associated retrovirus (HTLV-III/LAV) by indirect fixed cell immunofluorescence: specificity, sensitivity, and applications.

Seropositivity to the AIDS-associated retrovirus, HTLV-III/LAV, has profound implications. Simple and reliable tests are needed to detect such antibodies. A rapid, sensitive indirect immunofluorescence assay (IFA) on acetone-fixed virus-producing CEM/LAV-N1 cells was adapted for detection of human antibodies to HTLV-III/LAV. Specific and nonspecific patterns of of immunofluorescent reactivity were easily distinguished, and results paralleled those obtained by Western blotting and radioimmunoprecipitation (RIP), indicating that there is no need to confirm IFA positivity. In contrast, the commercial enzyme-linked immunosorbent assay (ELISA) was less reliable. False positives occurred with sera from seven hemophiliacs that were negative on Western blots, and false-negative reactions were observed on two occasions. These involved low-titer AIDS-patients' sera that were positive on Western blots, and from one of which virus was successfully isolated. Our results emphasize the requirement for confirmatory assays when the ELISA test is used for primary screening of sera for antibodies to HTLV-III/LAV. The IFA method is especially well-suited to quantitative analysis of serum antibody levels. Our data suggest that serum antibody titers rise as disease progression occurs, ultimately falling as severe complications ensue. It is suggested that in laboratories where the demand for HTLV-III/LAV antibody testing is not excessive (1,000-2,000 sera/month), IFA could serve as the only serological assay for both screening and epidemiological purposes.

Antibodies to acquired immune deficiency syndrome (AIDS)-associated virus (HTLV-III/LAV) in Venezuelan populations.

Serum samples from 850 individuals from Venezuela were tested for the presence of antibodies to HTLV-III/LAV virus, the probable etiological agent of acquired immune deficiency syndrome (AIDS). At the time of the study, none of the individuals tested had symptoms indicative of AIDS or related disorders. Viral antibodies were assayed by indirect immunofluorescence (IF) assay, using a chronically infected, HTLV-III/LAV producer cell line CEM/LAV-NIT established in our laboratory. Twenty individuals (2.5%), 8 of them (40%) female, were seropositive by IF and by confirmatory Western blotting and radioimmunoprecipitation assays. The seropositivity rate ranged from 2.4% (11 of 465) in the general healthy population, 4% (2 of 50) among patients with Chagas' disease, and up to 29.2% (7 of 24) among patients with acute malaria infection. The titers of HTLV-III/LAV antibodies ranged from 1:40 to 1:640. In addition, 2 of 36 patients with hemophilia A (5.5%) also had antibodies to HTLV-III/LAV. Two of 7 patients with acute malaria had specific antibodies both to HTLV-III/LAV and HTLV-I, as determined by IF and Western blotting. None of over 169 randomly chosen, healthy blood donors from seven major Venezuelan cities, as well as none of 99 patients with leukemia/lymphoma, had antibodies to HTLV-III/LAV. The presence of specific antibodies among various Venezuelan populations indicates that HTLV-III/LAV, or a closely related cross-reactive virus, is indigenous in Latin American subjects as was previously indicated for tropical populations of central Africa. Isolation and characterization of this virus will help to understand the origin and etiology of AIDS.

Acellular pertussis vaccine in children with perinatal human immunodeficiency virus-type 1 infection.

The immunogenicity of an acellular pertussis vaccine containing genetically detoxified pertussis toxin, filamentous haemoagglutinin and pertactin was studied in 12 children [median age: 45 (6-107) months] with perinatal human immunodeficiency virus-type 1 (HIV-1) infection. Antibody response to all antigens was observed in six cases and another children 3 reacted to two or one antigen(s), but titres were lower than those from healthy controls. Antibody titre fold-rise correlated with preimmunization CD4-positive cell counts. Significant titres were still detectable 4 months after the third dose. The acellular vaccine is immunogenic in a portion of children with perinatal HIV-1 infection but early vaccination might be more effective, taking advantage of still adequate CD4-positive cell numbers.