Clones y vectores infectivos derivados de coronavirus y sus aplicaciones

Referencia OEPM: P9902673.-- Fecha de solicitud: 03/12/1999.-- Titular: Consejo Superior de Investigaciones Científicas (CSIC).Clones y vectores infectivos derivados de coronavirus y sus aplicaciones. El clon infectivo derivado de un coronavirus comprende un cDNA que codifica el gRMA de un coronavirus clonado bajo una secuencia promotora de la transcripción. El vector viral recombinante comprende un clon infectivo modificado para contener un ácido nucleico heterólogo insertado en dicho clon infectivo bajo condiciones que permiten la expresión de dicho ácido nucleico heterólogo. Los clones y vectores infectivos son útiles tanto en investigación básica como aplicada, en el desarrollo de sistemas de expresión eficientes de productos de interés (proteínas, enzimas, anticuerpos, etc.), vectores vacunales y terapia génica.Peer reviewe

Transgenic mice secreting coronavirus neutralizing antibodies into the milk

Ten lines of transgenic mice secreting transmissible gastroenteritis coronavirus (TGEV) neutralizing recombinant monoclonal antibodies (rMAbs) into the milk were generated. The rMAb light- and heavy-chain genes were assembled by fusing the genes encoding the variable modules of the murine MAb 6A. C3, which binds an interspecies conserved coronavirus epitope essential for virus infectivity, and a constant module from a porcine myeloma with the immunoglobulin A (IgA) isotype. The chimeric antibody led to dimer formation in the presence of J chain. The neutralization specific activity of the recombinant antibody produced in transiently or stably transformed cells was 50-fold higher than that of a monomeric rMAb with the IgG1 isotype and an identical binding site. This rMAb had titers of up to 104 by radioimmunoassay (RIA) and neutralized virus infectivity up to 104- fold. Of 23 transgenic mice, 17 integrated both light and heavy chains, and at least 10 of them transmitted both genes to the progeny, leading to 100% of animals secreting functional TGEV neutralizing antibody during lactation. Selected mice produced milk with TGEV-specific antibody titers higher than 106 as determined by A, neutralized virus infectivity by 10-fold, and produced up to 6 mg of antibody per ml. Antibody expression levels were transgene copy number independent and integration site dependent. Comicroinjection of the genomic β-lactoglobulin gene with rMAb light and heavy-chain genes led to the generation of transgenic mice carrying the three transgenes. The highest antibody titers were produced by transgenic mice that had integrated the antibody and β-lactoglobulin genes, although the number of transgenic animals generated does not allow a definitive conclusion on the enhancing effect of β-lactoglobulin cointegration. This approach may lead to the generation of transgenic animals providing lactogenic immunity to their progeny against enteric pathogens.This work has been supported by grants from the Consejo Superior de Investigaciones Científicas, the Comisión Interministerial de Ciencia y Tecnología (CICYT), The Instituto Nacional de Investigación y Tecnología Agraria y Alimentación project SC-GAN94-119, La Consejería de Educación y Cultura de la Comunidad de Madrid, and Laboratorios Fort Dodge from Spain and the European Communities (Projects Science and Biotech). I.S., J.C., and J.M.S.-M. received fellowships from the Consejo Superior de Investigaciones Científicas, the Department of Education, University and Research of the Gobierno Vasco, and the Colegio Oficial de Veterinarios de la Comunidad de Madrid (Spain), respectively. C.B.A.W. and A.J.C. are supported by the BBSRC

Targeted recombination demonstrates that the spike gene of transmissible gastroenteritis coronavirus is a determinant of its enteric tropism and virulence

Targeted recombination within the S (spike) gene of transmissible gastroenteritis coronavirus (TGEV) was promoted by passage of helper respiratory virus isolates in cells transfected with a TGEV-derived defective minigenome carrying the S gene from an enteric isolate. The minigenome was efficiently replicated in trans and packaged by the helper virus, leading to the formation of true recombinant and pseudorecombinant viruses containing the S proteins of both enteric and respiratory TGEV strains in their envelopes. The recombinants acquired an enteric tropism, and their analysis showed that they were generated by homologous recombination that implied a double crossover in the S gene resulting in replacement of most of the respiratory, attenuated strain S gene (nucleotides 96 to 3700) by the S gene of the enteric, virulent isolate. The recombinant virus was virulent and rapidly evolved in swine testis cells by the introduction of point mutations and in-phase codon deletions in a domain of the S gene (nucleotides 217 to 665) previously implicated in the tropism of TGEV. The helper virus, with an original respiratory tropism, was also found in the enteric tract, probably because pseudorecombinant viruses carrying the spike proteins from the respiratory strain and the enteric virus in their envelopes were formed. These results demonstrated that a change in the tropism and virulence of TGEV can be engineered by sequence changes in the S gene.This work has been supported by grants from the Comisión Interministerial de Ciencia y Tecnología (CICYT), La Consejería de Educación y Cultura de la Comunidad de Madrid, Fort-Dodge Veterinaria from Spain, and the European Union (Projects FAIR and Biotech)

Biology

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Clinical manifestations of intermediate allele carriers in Huntington disease

Objective: There is controversy about the clinical consequences of intermediate alleles (IAs) in Huntington disease (HD). The main objective of this study was to establish the clinical manifestations of IA carriers for a prospective, international, European HD registry. Methods: We assessed a cohort of participants at risk with <36 CAG repeats of the huntingtin (HTT) gene. Outcome measures were the Unified Huntington's Disease Rating Scale (UHDRS) motor, cognitive, and behavior domains, Total Functional Capacity (TFC), and quality of life (Short Form-36 [SF-36]). This cohort was subdivided into IA carriers (27-35 CAG) and controls (<27 CAG) and younger vs older participants. IA carriers and controls were compared for sociodemographic, environmental, and outcome measures. We used regression analysis to estimate the association of age and CAG repeats on the UHDRS scores. Results: Of 12,190 participants, 657 (5.38%) with <36 CAG repeats were identified: 76 IA carriers (11.56%) and 581 controls (88.44%). After correcting for multiple comparisons, at baseline, we found no significant differences between IA carriers and controls for total UHDRS motor, SF-36, behavioral, cognitive, or TFC scores. However, older participants with IAs had higher chorea scores compared to controls (p 0.001). Linear regression analysis showed that aging was the most contributing factor to increased UHDRS motor scores (p 0.002). On the other hand, 1-year follow-up data analysis showed IA carriers had greater cognitive decline compared to controls (p 0.002). Conclusions: Although aging worsened the UHDRS scores independently of the genetic status, IAs might confer a late-onset abnormal motor and cognitive phenotype. These results might have important implications for genetic counseling. ClinicalTrials.gov identifier: NCT01590589

Clinical and genetic characteristics of late-onset Huntington's disease

Background: The frequency of late-onset Huntington's disease (&gt;59 years) is assumed to be low and the clinical course milder. However, previous literature on late-onset disease is scarce and inconclusive. Objective: Our aim is to study clinical characteristics of late-onset compared to common-onset HD patients in a large cohort of HD patients from the Registry database. Methods: Participants with late- and common-onset (30–50 years)were compared for first clinical symptoms, disease progression, CAG repeat size and family history. Participants with a missing CAG repeat size, a repeat size of ≤35 or a UHDRS motor score of ≤5 were excluded. Results: Of 6007 eligible participants, 687 had late-onset (11.4%) and 3216 (53.5%) common-onset HD. Late-onset (n = 577) had significantly more gait and balance problems as first symptom compared to common-onset (n = 2408) (P &lt;.001). Overall motor and cognitive performance (P &lt;.001) were worse, however only disease motor progression was slower (coefficient, −0.58; SE 0.16; P &lt;.001) compared to the common-onset group. Repeat size was significantly lower in the late-onset (n = 40.8; SD 1.6) compared to common-onset (n = 44.4; SD 2.8) (P &lt;.001). Fewer late-onset patients (n = 451) had a positive family history compared to common-onset (n = 2940) (P &lt;.001). Conclusions: Late-onset patients present more frequently with gait and balance problems as first symptom, and disease progression is not milder compared to common-onset HD patients apart from motor progression. The family history is likely to be negative, which might make diagnosing HD more difficult in this population. However, the balance and gait problems might be helpful in diagnosing HD in elderly patients