Attenuated poxvirus expressing three immunodominant CMV antigens as a vaccine strategy for CMV infection.

BACKGROUND: Human cytomegalovirus (CMV) infection is an important risk factor in the post-transplant (Tx) recovery phase for both hematopoietic stem cell Tx (HSCT) and solid organ Tx (SOT) recipients. CMV infection may be prevented or controlled by simultaneously inducing both CMV-specific neutralizing antibody (nAb) and cellular immunity. Soluble (s) UL55 (surface glycoprotein), UL83 (tegument protein) and UL123/e4 (nuclear protein) are immunodominant in eliciting both CMV nAb and cellular immunity. An attenuated poxvirus, modified vaccinia Ankara (MVA) was selected to develop this vaccine strategy in Tx recipients, because of its clinical safety record, large foreign gene capacity, and capability to activate strong humoral and cellular immune responses against recombinant antigens. OBJECTIVES: A subunit vaccine that targets multiple CMV antigens will be used to gain maximal coverage and protective function against CMV infection. rMVA simultaneously expressing sUL55, UL83 and UL123/e4 will be generated, and humoral and cellular immunity it elicits will be characterized, after murine immunization and in vitro to amplify clinical recall responses. STUDY DESIGN: rMVA will be constructed in two steps using UL123/e4-pLW22 followed by sUL55-UL83-pLW51 transfer plasmids. Western blots will be used to characterize expression levels of each antigen. Primary immunity will be evaluated in mouse models, while recall responses to the virally expressed CMV antigens will be assessed in human peripheral blood. RESULTS: We generated CMV-MVA via homologous recombination, and demonstrated high expression levels of sUL55, UL83 and UL123/e4 by Western blot. CMV-MVA immunization potently induced both humoral and cellular immunity to sUL55, UL83 and UL123 after murine immunization, and cellular immunity to UL83 and UL123 by in vitro amplification of T cell recall responses in human PBMC. CONCLUSIONS: rMVA promotes high level expression of three immunodominant CMV antigens, which is reflected in results of immunization studies in which high titers of UL55-specific antibodies and CD4+ T-help are detected, as well as high levels of UL83-specific and moderate levels of UL123-specific CD8+ CTL.     

Genotyping hepatitis C virus by heteroduplex mobility analysis using temperature gradient capillary electrophoresis

The genotype of the infecting hepatitis C virus (HCV) helps determine the patient's prognosis and the duration of treatment. Heteroduplex mobility analysis (HMA) is a rapid, inexpensive method for genotyping of HCV that does not require sequencing. We developed an HMA that uses temperature gradient capillary electrophoresis (TGCE) to differentiate HCV genotypes. A 56-bp region of the HCV 5' untranslated region (UTR) that was conserved within a genotype yet whose sequence differed between genotypes was amplified for HMA-TGCE analysis. HCV amplicons of types 1, 2a, 2b, 3a, 4, and 6a were hybridized in pairs and analyzed by TGCE. Amplicons hybridized to the same subtype yielded one homoduplex peak, while hybridization of different subtypes resulted in heteroduplexes and generated multiple TGCE peaks. Heteroduplexes contain thermodynamically unstable nucleotide mismatches that reduced their TGCE mobilities compared to those of homoduplexes. Three HCV subtypes (subtypes 1a, 3a, and 4) generated unique peak patterns when they were combined with each genotype analyzed and were chosen as the reference genotypes. A blinded study with 200 HCV-infected samples was 97% accurate compared to genotyping by 5' UTR sequence analysis. The majority of discordant results were unexpected sequence variants; however, five of nine sequence variants were correctly genotyped. The assay also detected and correctly genotyped mixed HCV infections. Compared to conventional HMA, TGCE improves the resolution, with better separation of heteroduplexes and homoduplexes. All common HCV genotypes can be detected and differentiated by this HMA-TGCE assay.     

Fiber optic probe for polarized reflectance spectroscopy in vivo: design and performance

We present the design and construction of a fiber optic probe for elastic light scattering spectroscopy in vivo with polarized excitation and polarization sensitive detection. The performance of the fiber probe is evaluated using a suspension of polystyrene spheres placed atop a diffusely scattering substrate, and it demonstrates that the size-dependent characteristics of the scatterers can be extracted in the presence of a highly diffusely scattering background using a linear combination of forward and backward Mie scattering components of the scatterers. Subsequently, Mie theory calculations are performed over a broad range of diagnostically relevant parameters of nuclei-mean diameter, size distribution, and relative refractive index-to understand how the polarized reflectance measurements with the fiber probe can be used to extract morphological information about epithelial tissue. Finally, the feasibility of in vivo measurements with the fiber optic based polarization sensitive light scattering spectroscopy is demonstrated.     

Immunoprecipitation of virus-specific immediate-early and early polypeptides from cells lytically infected with human cytomegalovirus strain AD 169

By immunoprecipitation of human cytomegalovirus-infected cell-specific polypeptides (ICPs) with a variety of human cytomegalovirus-positive sera and analysis by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, we can identify at least 20 ICSP bands from lytic infections by 6 hr postinfection (pi). Three of these polypeptide bands (78K, 73K, and 68K) may represent more than one species of polypeptide. Four polypeptide bands (78K, 77K, 73K, and 31K) can be identified as immediate-early based on their synthesis in the presence of actinomycin-D after removal from a protein synthesis block mediated by cycloheximide (CH). An immediate-early 78K polypeptide and an early 49K polypeptide are synthesized only transiently during the first 4 hr pi. Most immediate-early polypeptide synthesis is enhanced after removal of a 5 hr CH block. Taken together, these results identify many previously undetected immediate-early and early ICSPs and suggest that several regulatory events are occurring during the early phase of the lytic cycle.     

Altered chromatin landscape in circulating T follicular helper and regulatory cells following grass pollen subcutaneous and sublingual immunotherapy

1. Download : Download high-res image (285KB)
2. Download : Download full-size image

     

A mouse model of spinal and bulbar muscular atrophy

Spinal and bulbar muscular atrophy (SBMA) is an adult-onset motor neuron disease, caused by the expansion of a trinucleotide repeat (TNR) in exon 1 of the androgen receptor (AR) gene. This disorder is characterized by degeneration of motor and sensory neurons, proximal muscular atrophy, and endocrine abnormalities, such as gynecomastia and reduced fertility. We describe the development of a transgenic model of SBMA expressing a full-length human AR (hAR) cDNA carrying 65 (AR(65)) or 120 CAG repeats (AR(120)), with widespread expression driven by the cytomegalovirus promoter. Mice carrying the AR(120) transgene displayed behavioral and motor dysfunction, while mice carrying 65 CAG repeats showed a mild phenotype. Progressive muscle weakness and atrophy was observed in AR(120) mice and was associated with the loss of alpha-motor neurons in the spinal cord. There was no evidence of neurodegeneration in other brain structures. Motor dysfunction was observed in both male and female animals, showing that in SBMA the polyglutamine repeat expansion causes a dominant gain-of-function mutation in the AR. The male mice displayed a progressive reduction in sperm production consistent with testis defects reported in human patients. These mice represent the first model to reproduce the key features of SBMA, making them a useful resource for characterizing disease progression, and for testing therapeutic strategies for both polyglutamine and motor neuron diseases.     

Discrimination of human pathogenic subspecies of Francisella tularensis by using restriction fragment length polymorphism

We describe the use of two insertion sequence elements (ISFtu1 and ISFtu2) in Francisella tularensis to type strains by restriction fragment length polymorphism (RFLP). The RFLP profiles of 17 epidemiologically unrelated isolates were determined and compared. Our results showed that RFLP profiles can be used to assign F. tularensis strains into five main groups corresponding to strains of F. tularensis subsp. tularensis, F. tularensis strain ATCC 6223, strains of F. tularensis subsp. holarctica, strains of F. tularensis subsp. holarctica from Japan, and F. tularensis subsp. mediaasiatica. The results confirm the genetic identities of these subspecies and also support the suggestion that strains of F. tularensis subsp. holarctica from Japan should be considered members of a separate biovar. These findings should support future studies to determine the genetic differences between strains of F. tularensis at the whole-genome level.     

Cloning and expression of the major secreted cathepsin B-like protein from juvenile Fasciola hepatica and analysis of immunogenicity following liver fluke infection

The functions of the cathepsin B-like proteases in liver flukes are unknown and analysis has been hindered by a lack of protein for study, since the protein is produced in small amounts by juvenile flukes. To circumvent this, we isolated and characterized a cDNA encoding the major secreted cathepsin B from Fasciola hepatica. The predicted preproprotein is 339 amino acids in length, with the mature protease predicted to be 254 amino acids long, and shows significant similarity to parasite and mammalian cathepsin B. Only one of the two conserved histidine residues required for cathepsin B exopeptidase activity is predicted to be present. Recombinant preproprotein was produced in yeast, and it was shown that the recombinant proprotein can undergo a degree of self-processing in vitro to the mature form, which is active against gelatin and synthetic peptide substrates. The recombinant protein is antigenic in vaccinated rats, and antibodies to the protein are detected early after infection of rats and sheep with F. hepatica. The kinetics of the response to cathepsin B and cathepsin L after infection of sheep and rats confirm the temporal expression of these proteins during the life cycle of the parasite.     

Development of neural processes mediating rhyme judgments: Phonological and orthographic interactions

Development of neural processes underlying integration of phonological and orthographic information were assessed by measuring event-related brain potentials (ERPs), judgment accuracies and reaction times (RTs) in 20 children (9-10 years) and 20 adults performing visual rhyme judgments. Half the trials were phonologically and orthographically congruent across the prime-target pairs (e.g., thrown-own, cake-own), and half were incongruent (e.g., gown-own, cone-own). For both children and adults, behavioral performance was most affected when different phonological representations had to be encoded from similar orthographic representations (e.g., gown-own), and the amplitudes of the N350 reflected effects of both rhyme and phonological/orthographic congruency. Latencies of the N350 were shorter over the left hemisphere only in adults, and phonological/orthographic incongruence produced greater delays in children's processing. Therefore, it appears that neural systems mediating rhyming judgments develop early; however, adults exhibit increased efficiency for left-hemisphere processing and are less affected by interference from incongruent phonological and orthographic codes.