RESUMEN
BACKGROUND: Canadian-born children of South Asian [SA] ethnicity develop inflammatory bowel disease [IBD] at similar rates to those among Caucasian children. We evaluated the variation in phenotypic spectrum of IBD in SA and Caucasian children in a national paediatric inception cohort of new-onset IBD. METHODS: Patients aged <17 years, enrolled in a Canadian nationwide inception cohort study, were included. Baseline demographic and IBD phenotypic features were compared between SA and Caucasian children. Longitudinal outcomes through 18 months of follow-up were compared matched by propensity scores. RESULTS: Of 1156 children enrolled over 2014 to 2019, 623 were Caucasian [98% and 88% parents Canadian born] and 114 SA [79% Canadian born, 87% parents SA born]. Fewer SAs have a first-degree relative with IBD, 6% vs 19% in Caucasians, p = 0.002. SAs present at a younger age, median age 11.4 years (interquartile range [IQR] 9.2-14.3) vs 13 years [IQR 10.9-15 years], p = 0.03 and more commonly with a UC/IBD-U [ulcerative colitis/IBD-unclassified] subtype [ratio of UC/IBD-U to CD 1.2:1 vs 1:1.8 for Caucasians, p <0.001]. Additionally, a greater proportion of SA CD patients present with colonic-only disease [colonic-only CD/UC/IBD-U in SAs 67% vs 57% for Caucasians, p = 0.001], and among those with CD, colonic CD in SAs 31% vs 23% in Caucasians, p = 0.20]. Perianal fistulising disease was also numerically more common in SAs (14 [27%] vs 64 [18%], p = 0.06]. Adjusting for differences in phenotypic presentation, anti-tumour necrosis factor [TNF] exposure, and time to initiation was similar, and two-thirds of children, whether anti-TNF exposed or naïve, were in corticosteroid-free clinical remission at 18 months irrespective of ethnicity. CONCLUSIONS: The phenotypic spectrum of new-onset IBD in SA children differs from that of Caucasian children, but treatment and clinical course are similar within phenotypic subgroups.
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Colitis Ulcerosa , Enfermedad de Crohn , Enfermedades Inflamatorias del Intestino , Adolescente , Canadá/epidemiología , Niño , Estudios de Cohortes , Colitis Ulcerosa/terapia , Enfermedad de Crohn/terapia , Etnicidad , Humanos , Estudios Prospectivos , Inhibidores del Factor de Necrosis TumoralRESUMEN
Zaire ebolavirus (EBOV) is a highly pathogenic filovirus which can result in Ebola virus disease (EVD); a serious medical condition that presents as flu like symptoms but then often leads to more serious or fatal outcomes. The 2013-16 West Africa epidemic saw an unparalleled number of cases. Here we show characterisation and identification of T cell epitopes in surviving patients from Guinea to the EBOV glycoprotein. We perform interferon gamma (IFNγ) ELISpot using a glycoprotein peptide library to identify T cell epitopes and determine the CD4+ or CD8+ T cell component response. Additionally, we generate data on the T cell phenotype and measure polyfunctional cytokine secretion by these antigen specific cells. We show candidate peptides able to elicit a T cell response in EBOV survivors and provide inferred human leukocyte antigen (HLA) allele restriction. This data informs on the long-term T cell response to Ebola virus disease and highlights potentially important immunodominant peptides.
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Ebolavirus/inmunología , Epítopos de Linfocito T/inmunología , Glicoproteínas/inmunología , Fiebre Hemorrágica Ebola/inmunología , Linfocitos T/inmunología , África Occidental/epidemiología , Linfocitos T CD4-Positivos , Linfocitos T CD8-positivos , Ebolavirus/genética , Ensayo de Immunospot Ligado a Enzimas , Epidemias , Glicoproteínas/genética , Fiebre Hemorrágica Ebola/epidemiología , Humanos , Inmunidad Celular , Interferón gamma , SobrevivientesRESUMEN
Suboptimal vitamin D (vitD) status and reduced lean body mass are highly prevalent in pediatric inflammatory bowel diseases (IBD). The study objective was to determine sarcopenia prevalence and associations with vitD status in newly diagnosed pediatric IBD. Children with Crohn's disease (CD; n = 58) and ulcerative colitis (UC; n = 27) were included. Primary outcomes included body composition (total/regional/percent fat mass (FM), fat-free mass (FFM), skeletal muscle mass (SMM)), and vitD status (serum 25(OH)D). Sarcopenia was defined as SMM-z < -2. Additional variables measured included serum CRP, ESR, anthropometric, Pediatric Crohn's Disease Activity Index (PCDAI), and the Pediatric Ulcerative Colitis Disease Activity index (PUCAI). Sarcopenia and suboptimal 25(OH)D levels (< 50 nmol/l) were found in 23.5% (n = 20) and 52% (n = 44) of children, respectively. Younger children (< 13 years) with CD with suboptimal 25(OH)vitD (< 50 nmol/l) had the greatest frequency of sarcopenia (57.1%) (p = 0.004). Sarcopenia was prevalent in newly diagnosed, young children with CD with vitD deficiency.
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Enfermedades Inflamatorias del Intestino , Sarcopenia , Deficiencia de Vitamina D , Vitamina D/sangre , Adolescente , Niño , Preescolar , Estudios de Cohortes , Humanos , Enfermedades Inflamatorias del Intestino/complicaciones , Enfermedades Inflamatorias del Intestino/epidemiología , Estado Nutricional , Factores de Riesgo , Sarcopenia/complicaciones , Sarcopenia/epidemiología , Deficiencia de Vitamina D/complicaciones , Deficiencia de Vitamina D/epidemiologíaRESUMEN
Here, we present the complete genome sequences of two Zika virus (ZIKV) strains, EcEs062_16 and EcEs089_16, isolated from the sera of febrile patients in Esmeraldas City, in the northern coastal province of Esmeraldas, Ecuador, in April 2016. These are the first complete ZIKV genomes to be reported from Ecuador.
RESUMEN
Crimean-Congo Hemorrhagic Fever (CCHF) is a severe tick-borne disease, endemic in many countries in Africa, the Middle East, Eastern Europe and Asia. Between 15-70% of reported cases are fatal with no approved vaccine available. In the present study, the attenuated poxvirus vector, Modified Vaccinia virus Ankara, was used to develop a recombinant candidate vaccine expressing the CCHF virus nucleoprotein. Cellular and humoral immunogenicity was confirmed in 2 mouse strains, including type I interferon receptor knockout mice, which are susceptible to CCHF disease. Despite the immune responses generated post-immunisation, the vaccine failed to protect animals from lethal disease in a challenge model.
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Anticuerpos Antivirales/sangre , Virus de la Fiebre Hemorrágica de Crimea-Congo/inmunología , Fiebre Hemorrágica de Crimea/prevención & control , Inmunogenicidad Vacunal/inmunología , Nucleoproteínas/inmunología , Vacunas Sintéticas/inmunología , Proteínas Virales/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Antivirales/inmunología , Línea Celular , Embrión de Pollo , Chlorocebus aethiops , Cricetinae , Fiebre Hemorrágica de Crimea/inmunología , Humanos , Inmunización , Ratones , Ratones Noqueados , Receptor de Interferón alfa y beta/genética , Células Vero , Carga Viral/inmunologíaRESUMEN
Tuberculosis (TB) is a reemerging disease. The only available vaccine, Mycobacterium bovis BCG, is delivered intradermally and confers highly variable efficacy against pulmonary disease. There is an urgent need for improved vaccination strategies. Murine studies suggest that immunizations delivered directly to the respiratory mucosa might be a more effective route of vaccination. This study compared the immunogenicity of a leading candidate tuberculosis (TB) vaccine, modified vaccinia virus Ankara expressing antigen 85A (MVA85A), in rhesus macaques, delivered either as an aerosol or as an intradermal boost immunization 12 weeks after an intradermal BCG prime vaccine. Aerosol vaccination was well tolerated. MVA85A delivered by aerosol or by intradermal injection induced antigen-specific immune responses in the periphery and the lung, with a trend toward the highest response when the compartment and route of delivery were matched. The ability of poxvirus-vectored vaccines delivered by the systemic route to induce responses in the mucosal immune compartment in macaques is in contrast to the independent compartmentalization of mucosal and systemic immune systems described in mice. Unlike intradermal vaccination, aerosol vaccination did not induce a detectable serum anti-vector antibody response. The delivery of vaccines to the lungs might provide an immunization strategy that limits the induction of systemic anti-vector immunity, which would be extremely useful in the development of improved vaccine strategies. This is the first study to show a recombinant MVA-vectored vaccine to be highly immunogenic when delivered by the aerosol route to nonhuman primates. These results provide important safety and proof-of-concept data for further evaluation of this route of immunization for use in human clinical trials.
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Vacunas contra la Tuberculosis , Tuberculosis/inmunología , Tuberculosis/prevención & control , Administración por Inhalación , Animales , Antígenos Bacterianos/inmunología , Líquido del Lavado Bronquioalveolar/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Inmunidad Mucosa/inmunología , Inmunización Secundaria , Macaca , Mycobacterium bovis/inmunología , Vacunas contra la Tuberculosis/administración & dosificación , Vacunas contra la Tuberculosis/efectos adversos , Vacunas contra la Tuberculosis/inmunología , Vacunación , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/efectos adversos , Vacunas Atenuadas/inmunología , Vacunas de ADN , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/efectos adversos , Vacunas Sintéticas/inmunologíaRESUMEN
Following a suspected case of hantavirus in a patientsuffering from acute kidney injury, rodents fromthe patient's property in Yorkshire and the Humber,United Kingdom (UK) were screened for hantaviruses.Hantavirus RNA was detected via RT-PCR in two Rattusnorvegicus. Complete sequencing and phylogeneticanalysis established the virus as a Seoul hantavirus,which we have provisionally designated as strainHumber. This is the first hantavirus isolated from wildrodents in the UK and confirms the presence of a pathogenicSeoul virus in Europe.
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Lesión Renal Aguda/diagnóstico , Anticuerpos Antivirales/sangre , Infecciones por Hantavirus/epidemiología , ARN Viral/análisis , Virus Seoul/aislamiento & purificación , Lesión Renal Aguda/virología , Animales , Enfermedades Transmisibles Emergentes/virología , Reservorios de Enfermedades , Infecciones por Hantavirus/diagnóstico , Infecciones por Hantavirus/virología , Humanos , Masculino , Datos de Secuencia Molecular , Filogenia , ARN Viral/genética , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Enfermedades de los Roedores , Virus Seoul/genética , Análisis de Secuencia de ADN , Reino Unido/epidemiologíaRESUMEN
OBJECTIVE: To determine the frequency of and risk factors for major adverse drug reactions (MADRs) associated with anti-tuberculosis treatment at a tuberculosis (TB) referral hospital in the Republic of Korea. METHODS: Data from an ongoing natural history cohort study were analyzed for permanent regimen changes due to adverse drug reactions and confirmed by chart review. RESULTS: Among 655 subjects, there were 132 MADRs in 112 (17%) subjects. The most common MADRs were gastrointestinal (n = 53), musculoskeletal (n = 22), psychiatric (n = 10), visual (n = 9) and peripheral neuropathic (n = 8). MADRs were more frequent in subjects being treated with second-line regimens (16%) compared to first-line regimens (2.5%). Drugs frequently associated with MADRs were amikacin (3/10, 30%), linezolid (8/29, 28%), para-aminosalicylic acid (47/192, 24%), pyrazinamide (31/528, 5.8%), macrolides (2/44, 4.5%) and cycloserine (12/272, 4.4%). Fluoroquinolones accounted for a single MADR (1/377, 0.003%), despite widespread usage. In multivariate analysis, infection with multi- or extensively drug-resistant disease and previous history of anti-tuberculosis treatment were risk factors for MADR, with adjusted hazard ratios of respectively 2.2 (P = 0.02) and 1.6 (P = 0.04). CONCLUSION: MADRs are common during anti-tuberculosis chemotherapy in this population, occurring in more than one in six subjects. New and less toxic agents to treat drug-resistant TB are urgently needed.
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Antituberculosos/efectos adversos , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Tuberculosis/tratamiento farmacológico , Adulto , Anciano , Antituberculosos/uso terapéutico , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Análisis Multivariante , República de Corea , Factores de Riesgo , Resultado del Tratamiento , Adulto JovenRESUMEN
SETTING: A tuberculosis (TB) referral hospital in South Korea. OBJECTIVE: To evaluate predictors of treatment outcomes and survival among non-human immunodeficiency virus (HIV) infected patients with extensively drug-resistant TB (XDR-TB). DESIGN: Patients who were diagnosed with XDR-TB at the National Masan Tuberculosis Hospital from January 2001 to December 2005 were included in this study. We conducted a retrospective review of their medical records and mortality data. RESULTS: A total of 176 non-HIV-infected patients with XDR-TB were included. TB-related mortality was 48% (84/176), and the median survival time from the diagnosis date of XDR-TB was 51 months (range 0-127, 95%CI 32.53-69.47). Cure and treatment completion were classified as favourable outcome and treatment failure, death during treatment and default as poor outcome. Previous TB treatment with second-line drugs (aOR 2.76, 95%CI 1.02-7.44) and cavitary disease (aOR 3.01, 95%CI 1.12-8.08) were independent risk factors for poor outcome. Use of linezolid (aOR 0.10, 95%CI 0.01-0.69) and surgical resection (aOR 0.18, 95%CI 0.04-0.78) were associated with favourable outcome. CONCLUSION: There was high mortality in non-HIV-infected patients with XDR-TB at a TB referral hospital in South Korea. Adjunctive surgical treatment and linezolid improved the outcome for selected patients with XDR-TB.
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Antituberculosos/uso terapéutico , Tuberculosis Extensivamente Resistente a Drogas/mortalidad , Neumonectomía/métodos , Adulto , Tuberculosis Extensivamente Resistente a Drogas/terapia , Femenino , Estudios de Seguimiento , Infecciones por VIH , Hospitales Especializados/estadística & datos numéricos , Humanos , Corea (Geográfico)/epidemiología , Masculino , Estudios Retrospectivos , Tasa de Supervivencia/tendencias , Resultado del TratamientoRESUMEN
Lentiviral-based vectors hold great promise as gene delivery vehicles for the treatment of a wide variety of diseases. We have previously reported the development of a nonprimate lentiviral vector system based on the equine infectious anaemia virus (EIAV), which is able to efficiently transduce dividing and nondividing cells both in vitro and in vivo. Here, we report on the application of EIAV vectors for the systemic delivery of an antibody fusion protein designed for the treatment of cancer. The therapeutic potential of a single chain antibody against the tumour-associated antigen, 5T4, fused to immune enhancer moieties has been demonstrated in vitro and here we evaluate the genetic delivery of a 5T4 scFv fused to B7.1 (scFvB7) using an EIAV vector. The kinetics and concentration of protein produced following both intravenous (i.v.) and intramuscular (i.m.) administration was determined in immune competent adult mice. In addition, the immune response to the EIAV vector and the transgene were determined. Here, we show that a single injection of EIAV expressing scFv-B7 can give rise to concentrations of protein in the range of 1-5 microg/ml that persist in the sera for more than 50 days. After a second injection, concentrations of scFv-B7.1 rose as high as 20 microg/ml and levels greater than 2 microg/ml were present in the sera of all mice injected i.v. after 210 days despite the detection of antibodies against both the transgene and viral envelope for the duration of this study. These results demonstrate the potential of EIAV as a gene therapy vector for long-term production of therapeutic recombinant proteins.
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Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Virus de la Anemia Infecciosa Equina/genética , Neoplasias/terapia , Transducción Genética/métodos , Animales , Anticuerpos/sangre , Antígeno B7-1/genética , Línea Celular Tumoral , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Citometría de Flujo , Expresión Génica , Proteínas Fluorescentes Verdes/genética , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/inmunología , Ligando Coestimulador de Linfocitos T Inducibles , Inyecciones Intramusculares , Inyecciones Intravenosas , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos BALB C , Neoplasias/inmunología , Neoplasias/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Proteínas Recombinantes de Fusión/sangre , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Factores de Tiempo , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
This unit describes the maintenance of cell lines used with vaccinia virus, both in monolayer cultures and in suspension. The suspended cell culture is then used in the preparation of vaccinia virus stocks. The preparation of chick embryo fibroblasts (CEF) is also presented for use in the production of the highly attenuated and host range-restricted modified vaccinia virus Ankara (MVA) strain of vaccinia virus. Additionally, support protocols are presented for the titration of standard and MVA vaccinia virus stocks.
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Virus Vaccinia/crecimiento & desarrollo , Animales , Técnicas de Cultivo de Célula , Línea Celular , Células Cultivadas , Embrión de Pollo , Fibroblastos , Células HeLa , Humanos , Ensayo de Placa ViralRESUMEN
This unit first describes how to infect cells with vaccinia virus and then transfect them with a plasmid-transfer vector to generate a recombinant virus. Methods are also presented for purifying vaccinia virus and for isolating viral DNA, which can be used during transfection. Also presented are selection and screening methods used to isolate recombinant viruses and a method for the amplification of recombinant viruses. Finally, a method for live immunostaining that has been used primarily for detection of recombinant modified vaccinia virus Ankara (MVA) is presented.
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Virus Vaccinia/genética , Animales , Línea Celular , Células Cultivadas , Embrión de Pollo , Concanavalina A , ADN Viral/genética , ADN Viral/aislamiento & purificación , Vectores Genéticos , Humanos , Recombinación Genética , Transfección , Virus Vaccinia/aislamiento & purificaciónRESUMEN
This unit describes the maintenance of cell lines used with vaccinia virus, both in monolayer cultures and in suspension. The suspended cell culture is then used in the preparation of vaccinia virus stocks. The preparation of chick embryo fibroblasts (CEF) is also presented for use in the production of the highly attenuated and host range-restricted modified vaccinia virus Ankara (MVA) strain of vaccinia virus. Additionally, support protocols are presented for the titration of standard and MVA vaccinia virus stocks.
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Células Cultivadas/virología , Virus Vaccinia/crecimiento & desarrollo , Animales , Técnicas de Cultivo de Célula/métodos , Embrión de Pollo , Fibroblastos/citología , Fibroblastos/virologíaRESUMEN
This unit first describes how to infect cells with vaccinia virus and then transfect them with a plasmid-transfer vector to generate a recombinant virus. Methods are also presented for purifying vaccinia virus and for isolating viral DNA, which can be used during transfection. Also presented are selection and screening methods used to isolate recombinant viruses and a method for the amplification of recombinant viruses. Finally, a method for live immunostaining that has been used primarily for detection of recombinant modified vaccinia virus Ankara (MVA) is presented.
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Recombinación Genética/genética , Virus Vaccinia/genética , Animales , ADN Viral/genética , Vectores Genéticos/genética , Humanos , Modelos Genéticos , Virus Vaccinia/crecimiento & desarrollo , Replicación ViralRESUMEN
The oncofoetal antigen 5T4 is a 72 kDa glycoprotein expressed at the cell surface. It is defined by a monoclonal antibody, mAb5T4, that recognises a conformational extracellular epitope in the molecule. Overexpression of 5T4 antigen by tumours of several types has been linked with disease progression and poor clinical outcome. Its restricted expression in non-malignant tissue makes 5T4 antigen a suitable target for the development of antibody directed therapies. The use of murine monoclonal antibodies for targeted therapy allows the tumour specific delivery of therapeutic agents. However, their use has several drawbacks, including a strong human anti-mouse immune (HAMA) response and limited tumour penetration due to the size of the molecules. The use of antibody fragments leads to improved targeting, pharmacokinetics and a reduced HAMA. A single chain antibody (scFv) comprising the variable regions of the mAb5T4 heavy and light chains has been expressed in Escherichia coli. The addition of a eukaryotic leader sequence allowed production in mammalian cells. The two 5T4 single chain antibodies, scFv5T4WT19 and LscFv5T4, described the same pattern of 5T4 antigen expression as mAb5T4 in normal human placenta and by FACS. Construction of a 5T4 extracellular domain-IgGFc fusion protein and its expression in COS-7 cells allowed the relative affinities of the antibodies to be compared by ELISA and measured in real time using a biosensor based assay. MAb5T4 has a high affinity, K(D)=1.8x10(-11) M, as did both single chain antibodies, scFv5T4WT19 K(D)=2.3x10(-9) M and LscFv5T4 K(D)=7.9x10(-10) M. The small size of this 5T4 specific scFv should allow construction of fusion proteins with a range of biological response modifiers to be prepared whilst retaining the improved pharmacokinetic properties of scFvs.
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Anticuerpos Monoclonales/inmunología , Fragmentos de Inmunoglobulinas/aislamiento & purificación , Región Variable de Inmunoglobulina/aislamiento & purificación , Glicoproteínas de Membrana/inmunología , Secuencia de Aminoácidos , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/genética , Antígenos de Neoplasias/inmunología , Secuencia de Bases , Clonación Molecular , Ensayo de Inmunoadsorción Enzimática , Femenino , Terapia Genética , Humanos , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/inmunología , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Inmunohistoquímica , Glicoproteínas de Membrana/análisis , Datos de Secuencia Molecular , Mutación , Placenta/inmunología , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Neoplasias Gástricas/inmunología , Resonancia por Plasmón de SuperficieRESUMEN
Many human and mouse tumor antigens are normal, nonmutated tissue differentiation antigens. Consequently, immunization with these "self" antigens could induce autoimmunity. When we tried to induce immune responses to five mouse melanocyte differentiation antigens, gp100, MART-1, tyrosinase, and tyrosinase-related proteins (TRP) 1 and TRP-2, we observed striking depigmentation and melanocyte destruction only in the skin of mice inoculated with a vaccinia virus encoding mouse TRP-1. These mice rejected a lethal challenge of B16 melanoma, indicating the immune response against TRP-1 could destroy both normal and malignant melanocytes. Cytotoxic T lymphocytes specific for TRP-1 could not be detected in depigmented mice, but high titers of IgG anti-TRP-1 antibodies were present. Experiments with knockout mice revealed an absolute dependence on major histocompatibility complex class II, but not major histocompatibility complex class I, for the induction of both vitiligo and tumor protection. Together, these results suggest that the deliberate induction of self-reactivity using a recombinant viral vector can lead to tumor destruction, and that in this model, CD4(+) T lymphocytes are an integral part of this process. Vaccine strategies targeting tissue differentiation antigens may be valuable in cancers arising from nonessential cells and organs such as melanocytes, prostate, testis, breast, and ovary.
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Antígenos de Neoplasias/inmunología , Autoantígenos/inmunología , Vacunas contra el Cáncer , Citotoxicidad Inmunológica , Melanoma Experimental/inmunología , Glicoproteínas de Membrana , Oxidorreductasas , Proteínas/inmunología , Vitíligo/inmunología , Animales , Autoinmunidad , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , ADN Recombinante , Femenino , Vectores Genéticos , Humanos , Masculino , Melanocitos/inmunología , Melanoma Experimental/prevención & control , Ratones , Ratones Noqueados , Vacunación , Virus VacciniaRESUMEN
BACKGROUND: Construction of recombinant viruses that can serve as vaccines for the treatment of experimental murine tumors has recently been achieved. The cooperative effects of immune system modulators, including cytokines such as interleukin 12 (IL-12) and costimulatory molecules such as B7-1, may be necessary for activation of cytotoxic T lymphocytes. Thus, we have explored the feasibility and the efficacy of inclusion of these immunomodulatory molecules in recombinant virus vaccines in an experimental antitumor model in mice that uses Escherichia coli beta-galactosidase as a target antigen. METHODS: We developed a "cassette" system in which three loci of the vaccinia virus genome were used for homologous recombination. A variety of recombinant vaccinia viruses were constructed, including one virus, vB7/beta/IL-12, that contains the following five transgenes: murine B7-1, murine IL-12 subunit p35, murine IL-12 subunit p40, E. coli lacZ (encodes beta-galactosidase, the model antigen), and E. coli gpt (xanthine-guanine phosphoribosyltransferase, a selection gene). The effects of the recombinant viruses on lung metastases and survival were tested in animals that had been given an intravenous injection of beta-galactosidase-expressing murine colon carcinoma cells 3 days before they received the recombinant virus by intravenous inoculation. RESULTS: Expression of functional B7-1 and IL-12 by virally infected cells was demonstrated in vitro. Lung tumor nodules (i.e., metastases) were reduced in mice by more than 95% after treatment with the virus vB7/beta/IL-12; a further reduction in lung tumor nodules was observed when exogenous IL-12 was also given. Greatest survival of tumor-bearing mice was observed in those treated with viruses encoding beta-galactosidase and B7-1 plus exogenous IL-12. CONCLUSION: This study shows the feasibility of constructing vaccinia viruses that express tumor antigens and multiple immune cofactors to create unique immunologic microenvironments that can modulate immune responses to cancer.
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Antígenos de Neoplasias/genética , Antígeno B7-1/genética , Vacunas contra el Cáncer/genética , Interleucina-12/genética , Neoplasias Experimentales/terapia , Virus Vaccinia/genética , Animales , Antígeno B7-1/inmunología , Western Blotting , Vacunas contra el Cáncer/uso terapéutico , Escherichia coli/enzimología , Escherichia coli/genética , Estudios de Factibilidad , Femenino , Citometría de Flujo , Interleucina-12/inmunología , Operón Lac , Neoplasias Pulmonares/secundario , Neoplasias Pulmonares/terapia , Ratones , Ratones Endogámicos BALB C , Pentosiltransferasa/genética , Células Tumorales Cultivadas , Virus Vaccinia/inmunología , beta-Galactosidasa/genética , beta-Galactosidasa/inmunologíaRESUMEN
The severely attenuated and host range (HR) restricted modified vaccinia virus Ankara (MVA) was derived by >500 passages in chick embryo fibroblasts, during which multiple deletions and mutations occurred. To determine the basis of the HR defect, we prepared cosmids from the parental vaccinia virus Ankara genome and transfected them into nonpermissive monkey BS-C-1 cells that had been infected with MVA. Recombinant viruses that formed macroscopic plaques were detected after transfections with DNA segments that mapped near the left end of the viral genome. Plaque-forming viruses, generated by transfections with four individual cosmids and one pair of minimally overlapping cosmids, were purified, and their HRs were evaluated in BS-C-1 cells, rabbit RK-13 cells, and human HeLa, MRC-5, and A549 cells. The acquisition of the K1L and SPI-1 HR genes and the repair of large deletions were determined by polymerase chain reaction or pulse-field gel electrophoresis of NotI restriction enzyme digests of genomic DNA. The following results indicated the presence of previously unrecognized vaccinia virus HR genes: (1) the major mutations that restrict HR are within the left end of the MVA genome because the phenotypes of some recombinants approached that of the parental virus, (2) acquisition of the K1L gene correlated with the ability of recombinant viruses to propagate in RK-13 cells but did not enhance replication in human or monkey cell lines, (3) acquisition of the SPI-1 gene correlated with virus propagation in A549 cells but not with the extent of virus spread in monkey or other human cell lines, (4) there are at least two impaired HR genes because rescue occurred with nonoverlapping or minimally overlapping cosmids and recombinant viruses with intermediate HRs were isolated, and (5) at least one of the new HR genes did not map within any of the major deletions because the size of the left terminal NotI fragment was not appreciably altered in some recombinant viruses.
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Virus Vaccinia/genética , Virus Vaccinia/fisiología , Vacunas Virales , Replicación Viral , Animales , Embrión de Pollo , Cósmidos , ADN Viral/química , Electroforesis en Gel de Campo Pulsado , Marcadores Genéticos , Haplorrinos , Células HeLa , Humanos , Reacción en Cadena de la Polimerasa , Conejos , Mapeo Restrictivo , Virus Vaccinia/metabolismoRESUMEN
The utility of modified vaccinia virus Ankara (MVA) as a vector for eliciting AIDS virus-specific cytotoxic T lymphocytes (CTL) was explored in the simian immunodeficiency virus (SIV)/rhesus monkey model. After two intramuscular immunizations with recombinant MVA-SIVSM gag pol, the monkeys developed a Gag epitope-specific CTL response readily detected in peripheral blood lymphocytes by using a functional killing assay. Moreover, those immunizations also elicited a population of CD8+ T lymphocytes in the peripheral blood that bound a specific major histocompatibility complex class I/peptide tetramer. These Gag epitope-specific CD8+ T lymphocytes also were demonstrated by using both functional and tetramer-binding assays in lymph nodes of the immunized monkeys. These observations suggest that MVA may prove a useful vector for an HIV-1 vaccine. They also suggest that tetramer staining may be a useful technology for monitoring CTL generation in vaccine trials in nonhuman primates and in humans.
Asunto(s)
Proteínas de Fusión gag-pol/inmunología , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/inmunología , Linfocitos T Citotóxicos/inmunología , Virus Vaccinia/genética , Virus Vaccinia/inmunología , Vacunas contra el SIDA/aislamiento & purificación , Animales , Secuencia de Bases , Cartilla de ADN/genética , Epítopos , Proteínas de Fusión gag-pol/genética , Vectores Genéticos , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Inmunización , Macaca mulatta , Conformación Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunologíaRESUMEN
Many tumor-associated antigens are nonmutated, poorly immunogenic tissue differentiation antigens. Their weak immunogenicity may be due to "self"-tolerance. To induce autoreactive T cells, we studied immune responses to gp100/pmel 17, an antigen naturally expressed by both normal melanocytes and melanoma cells. Although a recombinant vaccinia virus (rVV) encoding the mouse homologue of gp100 was nonimmunogenic, immunization of normal C57BL/6 mice with the rVV encoding the human gp100 elicited a specific CD8(+) T cell response. These lymphocytes were cross-reactive with mgp100 in vitro and treated established B16 melanoma upon adoptive transfer. To understand the mechanism of the greater immunogenicity of the human version of gp100, we characterized a 9-amino acid (AA) epitope, restricted by H-2Db, that was recognized by the T cells. The ability to induce specific T cells with human but not mouse gp100 resulted from differences within the major histocompatibility complex (MHC) class I-restricted epitope and not from differences elsewhere in the molecule, as was evidenced by experiments in which mice were immunized with rVV containing minigenes encoding these epitopes. Although the human (hgp10025-33) and mouse (mgp10025-33) epitopes were homologous, differences in the three NH2-terminal AAs resulted in a 2-log increase in the ability of the human peptide to stabilize "empty" Db on RMA-S cells and a 3-log increase in its ability to trigger interferon gamma release by T cells. Thus, the fortuitous existence of a peptide homologue with significantly greater avidity for MHC class I resulted in the generation of self-reactive T cells. High-affinity, altered peptide ligands might be useful in the rational design of recombinant and synthetic vaccines that target tissue differentiation antigens expressed by tumors.