RESUMEN
With the rapid spread of human immunodeficiency virus (HIV) infection worldwide it is clear that effective strategies for mucosal vaccination against lentiviruses are urgently required. The aim of the present study is to determine whether protective immune responses against a mucosal challenge by feline immunodeficiency virus (FIV) can be elicited by targeting the immunization to the medial iliac lymph nodes--the principal site of migration of cells from the genital and rectal mucosa. Cats were challenged with homologous FIV via the rectal route. Targeted lymph node immunization was found to be an effective route of immunization eliciting both humoral and proliferative responses to peptide-based and fixed cell vaccines. Vaccination with fixed virus infected cells elicited protection against a cell-free mucosal FIV challenge. In addition, some cats vaccinated with fixed uninfected cells also remained uninfected following a cell-associated FIV challenge.
Asunto(s)
Antígenos Virales/administración & dosificación , Síndrome de Inmunodeficiencia Adquirida del Felino/prevención & control , Glicoproteínas/administración & dosificación , Virus de la Inmunodeficiencia Felina/inmunología , Ganglios Linfáticos/inmunología , Vacunación/veterinaria , Proteínas del Envoltorio Viral/administración & dosificación , Vacunas Virales/administración & dosificación , Administración Rectal , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/inmunología , Antígenos Virales/química , Antígenos Virales/inmunología , Gatos , Células Cultivadas/trasplante , Células Cultivadas/virología , Evaluación de Medicamentos , Síndrome de Inmunodeficiencia Adquirida del Felino/inmunología , Productos del Gen gag/inmunología , Glicoproteínas/química , Glicoproteínas/inmunología , Virus de la Inmunodeficiencia Felina/fisiología , Inyecciones Intralinfáticas , Datos de Secuencia Molecular , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Proyectos Piloto , Linfocitos T/trasplante , Linfocitos T/virología , Vacunación/métodos , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunología , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/inmunología , Vacunas Virales/inmunologíaRESUMEN
Feline immunodeficiency virus (FIV) is a natural lentiviral pathogen of cats which can be experimentally transmitted via rectal and vaginal routes--the major routes of human immunodeficiency virus type 1 transmission in man. An important objective for lentiviral research is the development of vaccine strategies which generate good mucosal immune responses capable of giving protection from a mucosal virus challenge. The experimental vaccines employed in this study were based on (a) a peptide from the third variable region of the FIV envelope glycoprotein and (b) fixed whole FIV, Glasgow-8 strain. Adjuvants used were Quil A and cholera toxin for mucosal administration and incomplete Freund's adjuvant and immune stimulating complexes for subcutaneous injection. Mucosal immunization was given by rectal and intranasal routes. Both antibody and proliferative responses were elicited by mucosal immunization and cholera toxin was found to be a good mucosal adjuvant. The addition of a lipo thioester to the FIV peptide improved IgG and IgA responses upon parenteral administration. However, no protection from a rectal FIV challenge was achieved.
Asunto(s)
Anticuerpos Antivirales/sangre , Virus de la Inmunodeficiencia Felina/inmunología , Activación de Linfocitos , Recto/virología , Vacunas Virales/inmunología , Administración Intranasal , Administración Rectal , Secuencia de Aminoácidos , Animales , Gatos , Inmunización , Inmunoglobulina A/sangre , Inmunoglobulina G/sangre , Datos de Secuencia Molecular , Linfocitos T/inmunología , Vacunas Virales/administración & dosificaciónRESUMEN
Feline immunodeficiency virus (FIV) infection is a naturally occurring lentiviral infection of cats which progresses to immunodeficiency in a manner strikingly similar to that observed in HIV infection in man. The rectal and cervico-vaginal mucosae are common routes of transmission of HIV and it has been shown that the gastrointestinal tract is an important site of HIV infection and primary pathology. Although biting is the principle mode of transmission for FIV, we have shown that it is possible to reliably infect cats via both the rectal and vaginal routes. Using a biotin-streptavidin linked immunoperoxidase technique we have detected FIV core and envelope proteins in the colonic follicle associated epithelial cells, cells within the lymphoid follice and occasional cells in the lamina propria. Further, in the intestine we have detected FIV RNA and proviral DNA in epithelial cells, colonic lymphoid aggregates and isolated lamina propria cells. We have studied a group of asymptotic cats which have been rectally infected with FIV for 1 year or longer and shown an increase in the number of lamina propria CD8+ cells and greater levels of IL-2, IL-6, IL-10 and gamma-IFN mRNA. Since these cats remained clinically healthy these results might suggest that both local antibody and class I restricted cytotoxic lymphocytes (CTLs) may play a role in control of viral replication. We have investigated a range of vaccination regimes for their ability to generate responses which would protect from rectal challenge with virulent virus. Cats have been immunized with whole virus (FIV-pet, FIV-GLA-8), V3, V3MAP or C2 with cholera toxin (CT), or Quil A based adjuvants via rectal, intra-nasal, parenteral or targeted lymph node routes, and challenged rectally with ten mucosal cat infectious doses (MCID) of FIV-GLA-8. We have shown that the adjuvant effects of cholera toxin and Quil A are not influenced by the route of delivery (intraperitoneal (i.p.) versus rectal) with CT more effective in stimulating humoral and Quil A more effective in stimulating cellular responses to FIV antigens. However we have shown that, quantitatively, CT is more effective when used as an adjuvant via the intra-nasal than the rectal route. Recently, we have begun to investigate if the promising results obtained with targeted lymph node (TLN) vaccination in monkeys could be reproduced in the cat. We have shown that TLN was more effective than rectal immunisation in stimulating both humoral and proliferative responses. In a preliminary study we have also been able to detect FIV specific CTLs and have observed protection from rectal challenge in four out of four cats.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida del Felino/prevención & control , Virus de la Inmunodeficiencia Felina/inmunología , Animales , Biotecnología , Gatos , Citocinas/inmunología , Síndrome de Inmunodeficiencia Adquirida del Felino/inmunología , Síndrome de Inmunodeficiencia Adquirida del Felino/virología , Femenino , Humanos , Inmunidad Mucosa , Virus de la Inmunodeficiencia Felina/patogenicidad , Membrana Mucosa/virología , Vacunas Virales/farmacologíaRESUMEN
The cytotoxic responses of peripheral blood lymphocytes from cottontop tamarins to in vitro restimulation with autologous lymphoblastoid cell lines (LCL) were assayed. Lymphocytes from immune tamarins that had recovered from EBV challenge developed potent cytotoxicity for natural killer (NK) cell targets and for autologous LCL. The cytotoxicity for LCL targets was EBV-specific, as B cell blasts uninfected with EBV were not killed. The cell lines could be maintained by repeated stimulation with LCL and the addition of IL-2. Flow cytometry showed that they were T cell lines expressing CD2, CD3, CD4, CD8 and CD25. Dual-colour flow cytometry revealed two subpopulations, one CD4+ CD8+ population and the other CD4- CD8+. After separation by magnetic cell sorting both subpopulations were shown to be cytotoxic and the CD4+ CD8+ fraction was also shown to be MHC class II-restricted; the MHC restriction of the CD8+ subpopulation could not be determined. The unseparated T cells and both the subpopulations were able to inhibit LCL outgrowth in vitro. In contrast, PBL from naive tamarins stimulated by autologous LCL developed less NK cell cytotoxicity and little cytotoxicity for LCL. The cytotoxic response was enhanced at higher levels of LCL stimulation, but the cells were unable to inhibit LCL outgrowth in vitro. We conclude that cytotoxic responses capable of inhibiting LCL growth in vitro correlate with in vivo immunity in the tamarin model and provide a basis for understanding the mechanism of vaccine-induced immune protection.
Asunto(s)
Infecciones por Herpesviridae/inmunología , Herpesvirus Humano 4/inmunología , Saguinus/inmunología , Linfocitos T Citotóxicos/inmunología , Infecciones Tumorales por Virus/inmunología , Animales , Antígenos CD/inmunología , Células Cultivadas , Citotoxicidad Inmunológica , Citometría de Flujo , Inmunofenotipificación , Saguinus/virología , Linfocitos T Citotóxicos/patologíaRESUMEN
32 monoclonal antibodies reactive with human CD antigens were tested against tamarin peripheral blood lymphocytes, ConA blasts and lymphoblastoid B cell lines derived from tamarin cells. Reagents that cross-react with MHC class I and II, B cells (CD20, -21 and -23), monocytes (CD14) and NK cells (CD16, -56) have been identified. In addition monoclonals that cross-react with T cells (CD2, CD3), the CD4/CD8 subsets of T cells and the IL-2 receptor (CD25) are reported. A monoclonal against the beta chain of LFA-1 (CD18) cross-reacted strongly, but there was only a very poor cross-reaction with a monoclonal against the alpha chain of CD11a. Two monoclonals tested against ICAM-1(CD54) were negative.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos CD/inmunología , Linfocitos/inmunología , Saguinus/inmunología , Animales , Células Cultivadas , Concanavalina A/farmacología , Reacciones Cruzadas/inmunología , Citometría de Flujo , Humanos , Activación de Linfocitos/inmunologíaRESUMEN
The association of Epstein-Barr virus (EBV) with a growing number of human malignancies underlines the importance of efforts aimed at preventing the infection with this potential carcinogen and of establishing animal models for human virus-associated tumors. Cottontop tamarins have been used in EBV vaccine studies because virus infection regularly induces lymphomas similar to those seen in human immunocompromised individuals. In recent years, several vaccines based on the gp340/220 envelope protein of EBV have been developed and shown to prevent the development of EBV-associated lymphomas in this model. Using in situ hybridization and immunohistology, we have characterized EBV infection in one nonimmunized and three immunized animals after challenge with a standard tumorigenic dose of EBV. In the nonimmunized animal, EBV-infected lymphoid cells were detected in numerous tissues showing no obvious lymphoma infiltration. Surprisingly, variable numbers of virus-carrying cells were also found in all three immunized animals that were protected against the development of virus-associated lymphoma. This observation demonstrates that vaccination does not induce sterilizing immunity against EBV infection in this model. Double labeling suggested a B cell phenotype of the majority of these cells. EBV infection of nonlymphoid cells was not observed. Analysis of viral gene expression in immunized animals suggested a restricted form of virus latency different from that seen in EBV-driven lymphomas in nonimmunized cottontop tamarins. These results raise the possibility that immunized cottontop tamarins protected against the development of EBV-driven lymphoma or animals exposed to a sublymphomagenic dose of virus may serve as a model for EBV infection in humans.
Asunto(s)
Linfoma de Burkitt/microbiología , Linfoma de Burkitt/veterinaria , Herpesvirus Humano 4 , Enfermedades de los Monos/microbiología , Saguinus , Animales , Linfoma de Burkitt/patología , Modelos Animales de Enfermedad , Herpesvirus Humano 4/aislamiento & purificación , Humanos , Tejido Linfoide/microbiología , Tejido Linfoide/patología , Enfermedades de los Monos/patologíaRESUMEN
The Epstein-Barr virus (EBV) is associated with a range of life-threatening diseases in humans. Development of an effective vaccine has therefore been an important objective. One problem in the development of a subunit vaccine for human administration is the selection of a satisfactory adjuvant since the only one currently licensed for human use is alum, although this is not considered to be very effective. The present study demonstrated that a subunit vaccine composed of the EBV envelope glycoprotein gp340 with alum as the adjuvant did elicit protective immunity against EBV-induced lymphoma in three out of five cottontop tamarins. Furthermore, rabbits immunized with gp340/alum developed the same range of antibody responses as rabbits immunized with gp340/SAF-1, an experimental adjuvant claimed to be more effective than alum. Therefore, these results indicate that alum should be evaluated as an adjuvant as part of a human trial of a gp340-based subunit vaccine.
Asunto(s)
Antígenos Virales/inmunología , Herpesvirus Humano 4/inmunología , Proteínas de la Matriz Viral/inmunología , Vacunas Virales/administración & dosificación , Adyuvantes Inmunológicos/administración & dosificación , Compuestos de Alumbre/administración & dosificación , Animales , Anticuerpos Antivirales/biosíntesis , Antígenos Virales/administración & dosificación , Femenino , Infecciones por Herpesviridae/prevención & control , Humanos , Inmunización , Masculino , Conejos , Saguinus , Infecciones Tumorales por Virus/prevención & control , Vacunas Sintéticas/administración & dosificación , Proteínas de la Matriz Viral/administración & dosificaciónRESUMEN
A replication-defective recombinant adenovirus (Ad) expressing the full length Epstein-Barr virus (EBV) major envelope glycoprotein gp340/220 was tested for its ability to protect against EBV-induced lymphoma in the cottontop tamarin. Antibody responses against Ad capsid proteins and EBV gp340/220 were observed but these antibodies did not neutralize EBV in vitro. However, all immunized animals were protected against challenge following three intramuscular doses of the recombinant Ad. These data indicate that the recombinant Ad is potentially a useful vector for vaccination.
Asunto(s)
Antígenos Virales/inmunología , Herpesvirus Humano 4/inmunología , Linfoma/prevención & control , Infecciones Tumorales por Virus/prevención & control , Proteínas de la Matriz Viral/inmunología , Adenoviridae , Animales , Anticuerpos Antivirales/biosíntesis , Antígenos Virales/biosíntesis , Vectores Genéticos , Linfoma/microbiología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Saguinus , Vacunas Sintéticas/inmunología , Proteínas de la Matriz Viral/biosíntesis , Vacunas Virales/inmunología , Replicación ViralRESUMEN
Inoculation of the cottontop tamarin with Epstein-Barr virus (EBV) gives rise to the development of mono- and/or oligoclonal large cell malignant lymphoma. A cDNA library was generated with the RNA extracted from an EBV-induced tamarin lymphoma biopsy in order to study the transcripts expressed in the tumour tissue. Fifteen EBV-specific cDNA clones were localized in the corresponding viral genomic fragments. Among them, two correspond to the EBNA-2 gene, and two others to the latent membrane protein gene. The majority of the cDNA clones were localized in the BamHI A fragment which has not been associated with latent expression. Furthermore, cDNAs were also found from the BamHI D and I fragments. Sequence analysis of the cDNAs localized in BamHI A showed that they correspond to a rightward transcript in the BALF-3 region, with the one clone that was sequenced containing four exons and three introns. The above results were confirmed by testing three different biopsies with the rapid amplification of cDNA ends-PCR method.
Asunto(s)
ADN Viral/genética , Genes Virales/genética , Herpesvirus Humano 4/genética , Linfoma de Células B Grandes Difuso/microbiología , Transcripción Genética/genética , Infecciones Tumorales por Virus/microbiología , Animales , Secuencia de Bases , Biopsia , Biblioteca de Genes , Datos de Secuencia Molecular , ARN Viral/genética , SaguinusRESUMEN
Inoculation of the cottontop tamarin with Epstein-Barr virus (EBV) invariably gives rise to mono- or oligoclonal large cell lymphoma occurring at multiple sites, and which resembles to a certain extent B cell lymphoma that occurs in the immunodeficient patient. The viral transcriptional pattern in tamarin tumour biopsies and in the corresponding tumour cell lines was investigated by means of the synthesis of radioactive single-stranded cDNA. It was found that the EBV transcripts came mainly from the fragments BamH1-H, BamH1-S, BamH1-A and EcoR1-Dhet. Transcripts from a few other early or late genes, namely BARF1, BSLF1/BMLF1, BBLF-4, BLLF1 and BXLF2, were also detected in one of the three biopsies tested. It would be important to characterize the transcripts that originate from the region where viral latent expression has not previously been observed. Our results also revealed that there is a sharp increase in EBV transcription in the tumour cell lines derived from the tamarin lymphomas. Simultaneously, the copy number of the viral genome was found to be amplified. Such a significant change in viral activity might be indicative of a close virus-host cell interaction in vivo.
Asunto(s)
Herpesvirus Humano 4/genética , Linfoma/microbiología , Infecciones Tumorales por Virus/microbiología , Animales , ADN Viral/genética , Modelos Animales de Enfermedad , Amplificación de Genes , Expresión Génica , Genes Virales , Herpesvirus Humano 4/patogenicidad , Linfoma/etiología , Sistemas de Lectura Abierta , Saguinus , Transcripción Genética , Células Tumorales Cultivadas/microbiología , Infecciones Tumorales por Virus/etiologíaRESUMEN
Inoculation with Epstein-Barr virus (EBV) induces malignant lymphomas in the cottontop tamarin (Saguinus oedipus oedipus). This provides an experimental animal model for assessing the efficacy of candidate EBV vaccines which are intended to reduce the incidence of human tumours associated with EBV infection. Previous work has shown that experimental vaccines based on the major virus envelope glycoprotein gp340 prepared from the membranes of EBV-infected cells are effective in protecting cottontop tamarins against EBV-induced disease. However, not all purified gp340 preparations induce protective immunity against EBV lymphoma in the tamarin. In this work, cottontop tamarins were immunized with recombinant gp340, produced using a bovine papillomavirus (BPV) expression vector, and a threonyl muramyl dipeptide adjuvant formulation. Although the recombinant-derived gp340 lacked the membrane anchor sequence of authentic gp340 and was expressed in mouse cells, it was immunogenic and induced virus-neutralizing antibodies. Healthy vaccinated tamarins were protected against EBV-induced disease. The demonstration that a recombinant gp340 product is able to elicit protective immunity in the cottontop tamarin is a significant step in the development of an EBV vaccine because previously it had not been clear whether a recombinant product would have the exact tertiary structure, including the necessary carbohydrate components, to induce protective immunity. A recombinant gp340 vaccine offers various advantages over production of the authentic molecule by laborious biochemical separation, including lower cost and the absence of potentially oncogenic EBV DNA. Therefore, recombinant gp340 produced using the BPV expression vector is suitable for development as a candidate EBV vaccine for a human Phase I trial and beyond.
Asunto(s)
Antígenos Virales/inmunología , Linfoma de Burkitt/prevención & control , Herpesvirus Humano 4/inmunología , Proteínas del Envoltorio Viral/inmunología , Proteínas de la Matriz Viral , Vacunas Virales , Acetilmuramil-Alanil-Isoglutamina , Adyuvantes Inmunológicos , Animales , Anticuerpos Antivirales/biosíntesis , Antígenos Virales/genética , Modelos Animales de Enfermedad , Regulación Viral de la Expresión Génica , Vectores Genéticos , Inmunización , Papillomaviridae/genética , Saguinus , Vacunas Sintéticas , Proteínas del Envoltorio Viral/genéticaRESUMEN
The Epstein-Barr virus (EBV) major envelope glycoprotein gp340 is the subject of current efforts to develop an EBV subunit vaccine. The importance of gp340-specific humoral immunity has been highlighted by studies of natural infection in humans and gp340 immunization of experimental animals. The former studies have demonstrated the presence of gp340-specific serum antibodies which mediate EBV neutralization, complement fixation, and antibody-dependent cellular cytotoxicity. The latter studies have often shown a correlation between the induction of gp340-specific EBV-neutralizing antibodies and protection from virus challenge. We have used a series of bacterial beta-galactosidase-gp340 fusion proteins and overlapping synthetic peptides from the gp340 open reading frame to map the positions of B-cell epitopes within the gp340 primary amino acid sequence. The data reported here indicate the presence of B-cell epitopes within the carboxy-terminal third of the gp340 polypeptide chain. These epitopes could not be detected with a peptide enzyme-linked immunosorbent assay, thereby suggesting that they are discontinuous. Affinity purification of antibodies with a gp340 fusion protein from the carboxy terminus of the gp340 polypeptide chain has been used to show that these antibodies are not EBV neutralizing in vitro. The consequences of these findings for future EBV vaccine development are considered.
Asunto(s)
Anticuerpos Antivirales/análisis , Antígenos Virales/inmunología , Linfocitos B/inmunología , Epítopos/análisis , Herpesvirus Humano 4/inmunología , Proteínas de la Matriz Viral , Vacunas Virales/inmunología , Secuencia de Aminoácidos , Técnica del Anticuerpo Fluorescente , Infecciones por Herpesviridae/inmunología , Humanos , Inmunoglobulina G/análisis , Péptidos/síntesis química , Péptidos/inmunología , Valores de Referencia , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
In EBV-immortalized lymphoblastoid cell lines (LCLs) a small number of "latent" proteins are expressed. These are the EBV nuclear antigens, EBNAs 1-6, and a latent membrane protein, LMP. We have investigated the expression of these proteins in a variety of EBV-associated tumours and cell lines. Whereas transplant and B-cell lymphomas from cotton-top tamarins appear to express the full range of antigens found in LCLs, we and others have found that in Burkitt's lymphomas (BL) and a nasopharyngeal carcinoma (NPC) isolate, EBNA expression is restricted to EBNA-I. (In NPC, but not in BL, LMP may also be expressed). In order to ask what restricts the expression of EBNA 2-6 in NPC and BL cells it seemed reasonable to consider the possibility that the DNA sequences normally regulating expression of these antigens could be chemically modified. In this analysis, a tight inverse correlation between methylation of CpG dinucleotides in the 5' flanking region of the EBNA-2 gene and the expression of EBNAs 2-6 has been revealed. In the NPC tumour, CpG methylation within the gene is also observed, as is specific methylation over the EBNA-I region I and II binding sites (in oriP). The significance of these observations is considered.
Asunto(s)
ADN Viral/metabolismo , Fosfatos de Dinucleósidos/metabolismo , Infecciones Tumorales por Virus/metabolismo , Antígenos Virales/genética , Southern Blotting , Línea Celular , ADN Viral/análisis , Fosfatos de Dinucleósidos/análisis , Antígenos Nucleares del Virus de Epstein-Barr , Regulación Viral de la Expresión Génica/fisiología , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/inmunología , Humanos , Immunoblotting , Metilación , Mapeo Restrictivo , Células Tumorales Cultivadas/metabolismo , Infecciones Tumorales por Virus/genéticaRESUMEN
The efficacy of a new vaccine preparation against Epstein-Barr (EB) virus was investigated in cotton-top tamarins. The vaccine consists of fast protein liquid chromatography-purified EB virus membrane antigen glycoprotein of 340 Kd (MA gp340) mixed with a synthetic muramyl dipeptide adjuvant emulsified in squalane containing a pluronic polymer; it is suitable for both scaled-up batch production and eventual administration to man. Vaccinated tamarins rapidly developed ELISA detectable high titre antibodies to MA gp340, and their sera became strongly EB virus-neutralising. After challenge with a massive 100% carcinogenic dose of EB virus, the vaccinated tamarins had a strikingly low level of circulating EB virus-carrying mononuclear cells, in contrast to a control animal, and remained entirely free of tumours. This first-generation vaccine has thus been validated in experimental animals and the way opened for a phase I human trial.
Asunto(s)
Acetilmuramil-Alanil-Isoglutamina/análogos & derivados , Adyuvantes Inmunológicos , Herpesvirus Humano 4/inmunología , Infecciones Tumorales por Virus/prevención & control , Vacunas Virales , Animales , Callitrichinae , Evaluación Preclínica de MedicamentosRESUMEN
Inoculation of cottontop tamarins with a large dose of Epstein-Barr virus (EBV) leads to the induction of multiple EBV genome-positive lymphomas. These tumors have been characterized as oligoclonal or monoclonal large-cell malignant lymphomas that closely resemble the EBV genome-positive B-cell lymphomas that arise in human allograft recipients. The expression of latent and lytic EBV-encoded proteins was investigated in these virus-induced tamarin lymphomas and in derived cell lines. The tamarin tumors were found to express EBV nuclear antigen 1 (EBNA 1), EBNA 2, EBNA leader protein, and the latent membrane protein (LMP) as determined both by immunohistochemical staining and by immunoblotting. However, within the limits of the immunoblotting assays, no expression of the EBNA 3a protein family could be detected. Assays for lytic-cycle proteins by using both polyclonal human sera and monoclonal antibodies against viral capsid antigen, early antigen, and membrane antigen (gp340/220) showed minimal, if any, expression of these antigens in the lymphoma biopsies. In contrast, the cell lines derived from these lymphomas, even in early passage, expressed abundant levels of the lytic-cycle antigens and also expressed the EBNA 3a protein as well as EBNA 1, EBNA 2, EBNA leader protein, and LMP. This finding suggests that the virus-lymphoma cell interaction, in particular the switch to lytic cycle, is subject to some form of host control in vivo. The expression of EBNA 2 and LMP in these tamarin lymphomas strengthens their resemblance to posttransplant lymphomas in humans, since these human tumors are also EBNA 2 and LMP positive (L. S. Young, C. Alfieri, K. Hennessy, H. Evans, C. O'Hara, K. Anderson, A. Rickinson, E. Kieff, and J. I. Cohen, submitted for publication). Since both proteins are known to be important effector molecules of virus-induced B-cell growth transformation in vitro, their expression in these lymphomas constitutes the best evidence for a direct oncogenic role for EBV in vivo.
Asunto(s)
Callitrichinae/microbiología , Herpesvirus Humano 4/genética , Leucemia Linfocítica Crónica de Células B/genética , Animales , Antígenos Virales/genética , Linfocitos B , Biopsia , Callitrichinae/genética , Línea Celular , Antígenos Nucleares del Virus de Epstein-Barr , Regulación de la Expresión Génica , Glicoproteínas/genética , Immunoblotting , Hibridación de Ácido Nucleico , Proteínas Virales/genéticaRESUMEN
We have previously reported that continuous in vitro passage in the presence of 3T3 feeders of a non-tumorigenic adenoma-derived epithelial cell line, designated PC/AA, resulted in its becoming immortal. At early passage PC/AA was normal diploid, whereas every cell of PC/AA late passage had an isochromosome 1(q) which led us to suggest that abnormalities of chromosome 1 may be involved in tumour progression. We now report the isolation of a 3T3-feeder-independent variant of early-passage PC/AA, designated PC/AA/FI, which was immortal in vitro and remained non-tumorigenic. Each cell of PC/AA/FI again has an isochromosome 1(q), like the late-passage PC/AA. However, with PC/AA/FI it is the other chromosome 1 of the homologous pair which is involved in the formation of the isochromosome 1(q). This is possible to determine because of the polymorphic centromeric heterochromatin on chromosome 1 of the early-passage PC/AA. With the late-passage PC/AA (grown with 3T3 feeders) the homologue with the large C-band has given rise to an isochromosome 1(q) whereas with PC/AA/FI it is the other homologue with the smaller C-band which has given rise to this isochromosome. Both the immortal PC/AA/FI and the immortal PC/AA late passage, therefore, have independent abnormalities involving chromosome 1. These results indicate that chromosome 1 may be involved in in vitro immortalization.
Asunto(s)
Adenoma/genética , Poliposis Adenomatosa del Colon/genética , Cromosomas Humanos Par 1 , Neoplasias Colorrectales/genética , Adenoma/ultraestructura , Poliposis Adenomatosa del Colon/patología , Animales , Línea Celular , Aberraciones Cromosómicas/genética , Aberraciones Cromosómicas/patología , Trastornos de los Cromosomas , Células Clonales/ultraestructura , Neoplasias Colorrectales/ultraestructura , Humanos , Cariotipificación , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Células Tumorales CultivadasRESUMEN
Two new epithelial cell lines from sporadic human colorectal adenomas designated S/AN and S/RG are reported. S/AN was from a villous adenoma and S/RG from a tubular adenoma. Both cell lines have extended growth capacities in vitro reaching passages 18 and 15, respectively, so far and show no signs of senescence. S/AN and S/RG have retained in vitro the ability to form mucin-producing goblet-like cells. Every cell of S/AN has a deletion on the short arm of chromosome 1 and one normal copy of chromosome 1. S/AN is also monosomic for chromosome 18. The majority of cells of S/RG only have one normal copy of chromosomes 6, 7, 14, 17, 18, and 22. S/RG also has several marker chromosomes. Although aneuploid S/AN and S/RG are nontumorigenic in athymic nude mice, these cytogenetic abnormalities are insufficient for the fully tumorigenic phenotype. The common abnormality for S/AN and S/RG is monosomy for chromosome 18, indicating that this is a central and important step in colorectal carcinogenesis. Our cytogenetic analysis of the adenoma cell lines suggests at least two possible routes by which premalignant colonic cells can develop and progress to malignancy. S/RG, unlike most other adenoma cell lines, is clonogenic. Aneuploidy, clonogenicity, and extended in vitro growth capacity may therefore be useful in vitro markers for adenoma cell lines with a relatively high malignant potential.
Asunto(s)
Adenoma/genética , Aberraciones Cromosómicas , Neoplasias Colorrectales/genética , Humanos , Cariotipificación , Células Tumorales CultivadasRESUMEN
Experimental induction of malignant lymphomas can be achieved in the cottontop tamarin by inoculation with Epstein-Barr (EB) virus. This system provides an animal model for assessing the efficacy of vaccine protection against the virus which is intended to reduce the incidence of human tumours associated with EB virus infection, namely endemic Burkitt's lymphoma and undifferentiated nasopharyngeal carcinoma. Cottontop tamarins have been vaccinated with the major envelope glycoprotein of EB virus, gp340, incorporated into immune-stimulating complexes (iscoms) and were thereby protected against a 100% lymphomagenic dose of virus. The gp340 iscoms are highly immunogenic, requiring only a few micrograms of immunogen to induce protective immunity and thus would be a strong candidate for further development as an EB virus vaccine for use in man.
Asunto(s)
Linfoma de Burkitt/prevención & control , Herpesvirus Humano 4/inmunología , Proteínas del Envoltorio Viral/inmunología , Vacunas Virales , Adyuvantes Inmunológicos , Animales , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Glicoproteínas/inmunología , Microscopía Electrónica , Neoplasias Nasofaríngeas/prevención & control , Saguinus , VacunaciónRESUMEN
In the course of developing an effective Epstein-Barr (EB) virus vaccine, the immune responses in cotton-top tamarins to a tumourigenic dose of EB virus were studied. Cell mediated responses were measured using a tissue culture 'growth inhibition' assay where peripheral blood lymphocytes were tested for their ability to inhibit the outgrowth of autologous EB virus transformed lymphoblastoid cells. This system has previously been recognized as a very sensitive assay for detecting cell-mediated responses to EB virus in man. Using this assay no cell-mediated immunity was detected up to the time of death in two tamarins following injection with a tumourigenic dose of EB virus. However, two other animals which had recovered from tumours induced by a first dose of EB virus 18 months previously when subsequently re-stimulated with a second tumourigenic dose did exhibit cell-mediated responses. These latter animals remained healthy following the re-challenge and did not show evidence of EB virus-induced disease.