Application of AAV2-mediated bFGF gene therapy on survival of ischemic flaps: effects of timing of gene transfer.

Necrosis of surgically transferred flaps is a major problem in reconstructive surgery. We investigated efficacy of a new vector system-adeno-associated viral 2 (AAV2)-mediated bFGF gene transfer to enhance survival of the ischemic flap. Thirty-eight Sprague-Dawley rats were divided into 3 gene therapy groups and 1 nontreated control of 9 or 10 each.

Designed recombinant adenovirus type 5 vector induced envelope-specific CD8(+) cytotoxic T lymphocytes and cross-reactive neutralizing antibodies against human immunodeficiency virus type 1.

Background: A monoclonal antibody (mAb) 2F5 binds to the membrane-proximal external region (MPER) of the transmembrane subunit gp41 of human immunodeficiency virus type 1 (HIV-1) is known to broadly neutralize HIV-1 strains. The Adenovirus type 5 vector (Ad5) has been widely applied for HIV-1 vaccine, and hexon hypervariable region 5 (HVR5) is exposed on viral surface and easily target host immune responses against Ad5.

Methods: We constructed a recombinant adenovirus type 5 vector (rAd5) with a 2F5-binding epitope (ELDKWA) of MPER on Ad5-HVR5.

Adeno-associated virus-2-mediated bFGF gene transfer to digital flexor tendons significantly increases healing strength. an in vivo study.

Background: Treatment of the disrupted intrasynovial flexor tendon is troublesome and can be complicated by the rupture of weak repairs and the formation of adhesions. The central issue underlying the unsatisfactory outcomes is the lack of sufficient healing capacity, which prohibits aggressive postoperative tendon motion. Transfer of genes that are critical to healing by means of an efficient vector system offers a promising way of strengthening the repair.

The Atg5 Atg12 conjugate associates with innate antiviral immune responses.

Autophagy is an essential process for physiological homeostasis, but its role in viral infection is only beginning to be elucidated. We show here that the Atg5-Atg12 conjugate, a key regulator of the autophagic process, plays an important role in innate antiviral immune responses. Atg5-deficient mouse embryonic fibroblasts (MEFs) were resistant to vesicular stomatitis virus replication, which was largely due to hyperproduction of type I interferons in response to immunostimulatory RNA (isRNA), such as virus-derived, double-stranded, or 5'-phosphorylated RNA.

Chimeric adenovirus type 5/35 vector encoding SIV gag and HIV env genes affords protective immunity against the simian/human immunodeficiency virus in monkeys.

Replication-defective adenovirus type 5 (Ad5) vector-based vaccines are widely known to induce strong immunity against immunodeficiency viruses. To exploit this immunogenicity while overcoming the potential problem of preexisting immunity against human adenoviruses type 5, we developed a recombinant chimeric adenovirus type 5 with type 35 fiber vector (rAd5/35). We initially produced a simian immunodeficiency virus (SIV) gag DNA plasmid (rDNA-Gag), a human immunodeficiency virus type 1 (HIV-1) 89.

Blocking of the TLR5 activation domain hampers protective potential of flagellin DNA vaccine.

Flagellin is a key component of the flagella of many pathogens, including Pseudomonas aeruginosa. Flagellin is an attractive vaccine candidate because it is readily produced and manipulated as a recombinant protein and has intrinsic adjuvant activity mediated through TLR5. Although DNA vaccines encoding native Pseudomonas B-type (FliC) or A-type (FlaA) flagellin are strongly immunogenic, the resultant Ab response interferes with the interaction of homologous flagellin with TLR5.

Chimeric adenovirus 5/35 vector containing the clade C HIV gag gene induces a cross-reactive immune response against HIV.

Most of the recent HIV studies have focused on the clade B virus subtype. However, it is estimated that half the HIV patients in developing countries are infected with virus belonging to clade C. Therefore, a vaccine against HIV clade C is urgently required.

Tendon healing in vitro: adeno-associated virus-2 effectively transduces intrasynovial tenocytes with persistent expression of the transgene, but other serotypes do not.

Background: Transfer of exogenous growth factor genes to injured tendons offers a promising method for strengthening tendon repairs. Adeno-associated virus vectors have advantages of being both nonpathogenic and nontoxic. The authors explored the efficiency of transduction of intrasynovial tenocytes with different serotypes of adeno-associated virus (AAV) and the persistency of its expression of a growth factor transgene.

Isolation and identification of adenovirus from conjunctival scrapings over a two-year period (between 2001 and 2003) in Yokohama, Japan.

Over a 2-year period between 2001 and 2003, a total of 115 conjunctival scrapings were collected from patients with keratoconjuctivitis from several hospitals in Yokohama, Japan. Out of 115, 94 (82.4%) cases of adenoviruses were detected by polymerase chain reaction (PCR); 60 (52.

Tissue reactions of adenoviral, adeno-associated viral, and liposome-plasmid vectors in tendons and comparison with early-stage healing responses of injured flexor tendons.

Purpose: Delivery of growth factor genes that may substantially increase the healing rate of injured digital flexor tendons is a new application of gene therapy. Adenoviral, adeno-associated viral (AAV), and liposome-plasmid vectors have been used to deliver genes to tendons, but the tendon reactions to these vectors--particularly in contrast to the healing responses in the injured tendons--were unknown. This study was designed to compare the tissue reactions of the earlier-mentioned vectors in tendons with the healing responses of injured flexor tendons.

The degree of apoptosis as an immunostimulant for a DNA vaccine against HIV-1 infection.

To regulate the expression of the apoptotic gene, we constructed bicistronic DNA vaccines that encode for HIV env and caspase-3 mutant (casp 3m) that are expressed via the encephalomyocarditis virus internal ribosomal entry site (IRES) or cytomegalovirus (CMV) promoter-dependent translations. While IRES-casp 3m induced weak apoptosis and caused little reduction in antigen expression, CMV-casp 3m elicited strong apoptosis and led to a marked decrease in the antigen expression. Therefore, IRES-casp 3m augmented HIV-specific immune responses, and IRES-casp 3m induced significant protection against the vaccinia-HIV chimeric virus.

Induction of robust immune responses against human immunodeficiency virus is supported by the inherent tropism of adeno-associated virus type 5 for dendritic cells.

The ability of adeno-associated virus serotype 1 to 8 (AAV1 to AAV8) vectors expressing the human immunodeficiency virus type 1 (HIV-1) Env gp160 (AAV-HIV) to induce an immune response was evaluated in BALB/c mice. The AAV5 vector showed a higher tropism for both mouse and human dendritic cells (DCs) than did the AAV2 vector, whereas other AAV serotype vectors transduced DCs only poorly. AAV1, AAV5, AAV7, and AAV8 were more highly expressed in muscle cells than AAV2.

A fused gene of nucleoprotein (NP) and herpes simplex virus genes (VP22) induces highly protective immunity against different subtypes of influenza virus.

We evaluated the immunogenicity and protective activity of plasmid DNA vaccines encoding the influenza virus NP gene (pNP) alone or in combination with the herpes simplex virus type 1 protein 22 gene (pVP22). Optimal immune responses were observed in BALB/c mice immunized with the combination of pVP22 plus pNP, as assessed by enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot (ELISPOT) and intracellular cytokine staining (ICCS). These mice also showed maximal resistance following challenge with the A/PR/8/34 (H1N1) and A/Udron/72 (H3N2) strains of influenza virus.

Multivalent DNA vaccine protects mice against pulmonary infection caused by Pseudomonas aeruginosa.

For efficacious vaccine development against Pseudomonas aeruginosa (P. aeruginosa), the immunogenicity of multivalent DNA vaccine was evaluated. Three different plasmids each targeting a fusion of outer membrane proteins (OprF/OprI), a protein regulating type III secretion system (PcrV), or an appendage (PilA) were prepared and mice were immunized with single (monovalent) or a combination of these plasmids (multivalent) via intramuscular electroporation (imEPT) or gene gun.

Removal of empty capsids from type 1 adeno-associated virus vector stocks by anion-exchange chromatography potentiates transgene expression.

Production of recombinant adeno-associated virus (rAAV) results in substantial quantities of empty capsids or virus-like particles (VLPs), virus protein shells without the vector genome. The contaminating VLPs would interfere with transduction by competing for cell-surface receptors and, when administered in vivo, contribute to antigen load, which may elicit a stronger immune response. Density-gradient ultracentrifugation provides a means to separate VLPs from rAAV particles, but is not feasible for large-scale preparations of vectors.

Robust HIV-specific immune responses were induced by DNA vaccine prime followed by attenuated recombinant vaccinia virus (LC16m8 strain) boost.

Recombinant vaccinia virus-based vaccine combined with DNA vaccine has produced a protective immune response against HIV infection in non-human primates. In this study, we explored the immunogenicity of a recombinant vaccinia virus (LC16m8 strain), which has been used in children without severe side effects. The vaccinia virus expressing an HIV(89.

Scalable generation of high-titer recombinant adeno-associated virus type 5 in insect cells.

We established a method for production of recombinant adeno-associated virus type 5 (rAAV5) in insect cells by use of baculovirus expression vectors. One baculovirus harbors a transgene between the inverted terminal repeat sequences of type 5, and the second expresses Rep78 and Rep52. Interestingly, the replacement of type 5 Rep52 with type 1 Rep52 generated four times more rAAV5 particles.

Induction of positive cellular and humoral immune responses by a prime-boost vaccine encoded with simian immunodeficiency virus gag/pol.

It is believed likely that immune responses are responsible for controlling viral load and infection. In this study, when macaques were primed with plasmid DNA encoding SIV gag and pol genes (SIVgag/pol DNA) and then boosted with replication-deficient vaccinia virus DIs recombinant expressing the same genes (rDIsSIVgag/pol), this prime-boost regimen generated higher levels of Gag-specific CD4+ and CD8+ T cell responses than did either SIVgag/pol DNA or rDIsSIVgag/pol alone. When the macaques were i.

A modified HIV challenge assay in mice by using luciferase-expressing vaccinia virus.

In this study, we developed a simple and sensitive assay in mice for a challenge experiment by using a recombinant vaccinia virus dual-expressing antigen (HIV Env gp160) and firefly luciferase. This assay can detect the vaccine effect at real-time in vivo by using a small amount of mouse serum. The luciferase activity in mouse serum was in agreement with the viral titer in the ovary.

Repair of articular cartilage defect by intraarticular administration of basic fibroblast growth factor gene, using adeno-associated virus vector.

The objective of this study was to establish the potency of adeno-associated virus (AAV) as a viral vector to transport the basic fibroblast growth factor (bFGF) gene into synovial tissue, and to evaluate the consequent repair of articular cartilage defects. In the in vitro study, LacZ- and bFGF-encoding genes were transduced into rabbit synoviocytes by recombinant adeno-associated virus (AAV) vector, and the cells were cultured for 2 weeks. The percentage of successfully transduced LacZ-positive cells was assessed by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining, and the concentration of bFGF in the culture supernatant was confirmed by bFGF-specific enzyme-linked immunosorbent assay.

Hyperproduction of IFN-gamma by CpG oligodeoxynucleotide-induced exacerbation of atopic dermatitis-like skin lesion in some NC/Nga mice.

Under conventional conditions, NC/Nga mice spontaneously develop an atopic dermatitis (AD)-like skin lesion accompanied by immunoglobulin E (IgE) hyperproduction and the expression of T helper 2 (Th2) cytokines. CpG DNA activates a strong interferon-gamma (IFN-gamma)-dominated T helper 1 (Th1) response, while inhibiting Th2-dependent allergies. In this study, we examined whether CpG oligodeoxynucleotide (ODN) could prevent the development of the skin lesions in NC/Nga mice.

Tendon healing in vitro: bFGF gene transfer to tenocytes by adeno-associated viral vectors promotes expression of collagen genes.

Purpose: Adeno-associated virus-mediated gene transfer is promising in the delivery of genes to tendons because this vector stimulates few adverse tissue reactions. Basic fibroblast growth factor (bFGF) promotes collagen production in healing tendons. We transferred the exogenous bFGF gene to proliferating tenocytes by adeno-associated viral (AAV) vectors and investigated its effects on the expression of the collagen genes in an in vitro tenocyte model.

Cross-reactive protection against influenza A virus by a topically applied DNA vaccine encoding M gene with adjuvant.

The skin is rich with immunocompetent cells and therefore immunization through the skin is an attractive alternative to the invasive vaccination methods currently used. In this study the backs of mice were gently shaved, hydrated, and painted with a DNA vaccine encoding influenza M protein with adjuvant. The immunized mice were then challenged with two mouse-adapted strains of the influenza virus A: A/PR/8/34 (H1N1) and A/Udorn/72 (H3N2).

The F(ab)'2 fragment of an Abeta-specific monoclonal antibody reduces Abeta deposits in the brain.

This work examines whether administering the F(ab' )2 fragment of an IgG1 monoclonal antibody (mAb) targeting the N-terminal 1-13 amino acids of the beta-amyloid peptide (Abeta mAb) reduces amyloid deposition in Alzheimer's disease (AD). The F(ab')2 fragment was injected intraperitoneally or intracranially into Tg2576 mice, a murine model of human AD. Both routes of administration significantly reduced Abeta plaque formation in the brain, as determined immunohistochemically and by monitoring levels of Abeta1-40 and Abeta1-42 peptide.