Management of ovarian cysts in infants.

BACKGROUND: To discuss the experience of diagnosis and treatment of ovarian cyst in infants. MATERIALS AND METHODS: A retrospective review was conducted on 20 infants who suffered from ovarian cyst. RESULTS: There were no dysplasia ovarian was found in children which were preoperatively diagnosed simplex cyst. Within thirteen children preoperatively detected mixed cystic-solid lesion, six cases ovarian cysts disappeared and two cases underwent poor blood supply in the following time. CONCLUSION: Adverse effects for ovarian cyst in infants can be prevented by agressive surgical intervention. Harmful effects of ovarian cyst can be prevented by positive surgical intervention despite the diagnostic difficulties in children with clinical symptoms of this condition.

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. 7.5 x 10(10) AAV2-bFGF viral particles were injected to the dorsum of each of the 29 rats; these rats were divided into 3 groups according to the timing of flap elevation. At the time of surgery, 1 week, and 2 weeks after surgery, flaps of 3 x 7 cm were raised. One week after surgery, flap viability was measured. Vascularization and immunohistochemical staining of the bFGF were evaluated of histologic sections. Flap viability was significantly improved by the AAV2-bFGF gene therapy at the time of surgery, and the flaps with the greatest survival area were found in the rats injected with AAV2-bFGF, 2 weeks before surgery. However, flap viability was significantly decreased by the gene therapy 1 week before surgery. Histologically, vascularity was increased in the groups with AAV2-bFGF injection and immunohistochemical staining showed greatly enhanced bFGF expression by gene transfer. The novel approach of AAV2-bFGF gene therapy shows encouraging manifestations in improving survival of flaps when the flaps are prefabricated during or 2 weeks before surgery.

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. In addition, we developed rAd5 encoding the HIV-1(IIIB) envelope (Env) gene for the induction of Env-specific cellular immunity. RESULTS: The virus titers of the constructed rAd5 were similar to that of the parental Ad5 vector. Furthermore, high-dose immunization of rAd5 induced Env-specific CD8(+) cells and high levels of anti-ELDKWA antibodies. Moreover, an in vitro HIV-1 neutralization assay indicated that ELDKWA-specific mAbs derived from rAd5-immunized mice neutralized a wide range of HIV-1 strains. CONCLUSIONS: The present study outlines the development of an Ad5-based HIV-1 vaccine targeting the hypervariable regions of Ad5. The constructed rAd5 induced an HIV-1-specific cellular immune response and neutralizing antibodies against various strains of HIV-1 simultaneously.

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 purpose of the present study was to transfer the basic fibroblast growth factor gene through the adeno-associated viral-2 vector to injured digital flexor tendons and to investigate its effects on the healing strength of the tendon and on adhesion formation in a clinically relevant injury model. METHODS: One hundred and twenty-eight long toes from sixty-four white leghorn chickens were used. The flexor digitorum profundus tendons were cut completely in the digital sheath area and were repaired with the modified Kessler method. In Group 1, a total of 2 x 10(9) particles of adeno-associated viral vector harboring the basic fibroblast growth factor gene were injected into both ends of the cut tendon. In Group 2, the same amount of adeno-associated viral vector carrying the luciferase gene was injected. In Group 3 (the non-injection control group), the tendons were sutured without any injection. At the end of two, four, eight, and twelve weeks, the toes were harvested and the tendons were tested for determination of the load-to-failure strength. At the end of eight and twelve weeks, the energy required to flex the toes was tested. The morphology regarding healing status and adhesions around the tendon were evaluated at two, four, eight, and twelve weeks. RESULTS: The ultimate strength of repaired tendons that had been treated with adeno-associated viral vector-basic fibroblast growth factor was significantly greater than that of tendons that had been treated with the sham vector or simple repair both during the early healing period (two weeks, p < 0.01; four weeks, p < 0.01) and a later period (eight weeks, p < 0.05). At four weeks, the strength of tendons that had been treated with adeno-associated viral vector-basic fibroblast growth factor (8.9 +/- 1.9 N) was significantly greater than that of tendons that had been treated with sham vector (6.1 +/- 1.0 N) (p < 0.01) or simple suture (5.7 +/- 1.1 N) (p < 0.001). Statistically, the grading of adhesions was the same among all three groups at four and eight weeks, but at twelve weeks it was significantly less severe for tendons that had been treated with adeno-associated viral vector-basic fibroblast growth factor than for those that had been treated with simple suture (p < 0.05). The energy that was required to flex the toes after treatment with adeno-associated viral vector-basic fibroblast growth factor was not increased at eight or twelve weeks compared with that in the controls. CONCLUSIONS: The present study demonstrates that basic fibroblast growth factor gene transfer to digital flexor tendons by means of adeno-associated viral vector-2 significantly increases healing strength during the critical tendon healing period but does not increase adhesion formation.

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. Similar hyperresponse to isRNA was also observed in Atg7-deficient MEFs, in which Atg5-Atg12 conjugation is impaired. Overexpression of Atg5 or Atg12 resulted in Atg5-Atg12 conjugate formation and suppression of isRNA-mediated signaling. Molecular interaction studies indicated that the Atg5-Atg12 conjugate negatively regulates the type I IFN production pathway by direct association with the retinoic acid-inducible gene I (RIG-I) and IFN-beta promoter stimulator 1 (IPS-1) through the caspase recruitment domains (CARDs). Thus, in contrast to its role in promoting the bactericidal process, a component of the autophagic machinery appears to block innate antiviral immune responses, thereby contributing to RNA virus replication in host cells.

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. This reduces the ability of the host to clear homologous, but not heterologous, flagellin-expressing P. aeruginosa. To circumvent this problem, a DNA vaccine encoding a mutant FliC R90A flagellin was developed. The mutant Ag encoded by this vaccine was highly immunogenic, but its ability to interact with TLR5 was reduced by >100-fold. Vaccination with this flagellin mutant DNA vaccine induced cross-reactive Abs against both FliC and FlaA, but few Abs capable of interfering with TLR5 activation. The flagellin mutant DNA vaccine provided excellent protection against both FliC- and FlaA-expressing P. aeruginosa. These findings suggest that vaccines against flagellated pathogens should avoid inducing Abs against TLR5 and raise the possibility that flagellated bacteria evade host elimination by facilitating the production of Abs that reduce the host's ability to mount an innate immune response.

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) 89.6 env DNA plasmid (rDNA-Env) and a recombinant Ad5/35 vector encoding the SIV gag and HIV env gene (rAd5/35-Gag and rAd5/35-Env). Prime-boost vaccination with rDNA-Gag and -Env followed by high doses of rAd5/35-Gag and -Env elicited higher levels of cellular immune responses than did rDNAs or rAd5/35s alone. When challenged with a pathogenic simian human immunodeficiency virus (SHIV), animals receiving a prime-boost regimen or rAd5/35s alone maintained a higher number of CD4(+) T cells and remarkably suppressed plasma viral RNA loads. These findings suggest the clinical promise of an rAd5/35 vector-based vaccine.

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. In this study, we evaluate the immunogenicity and protective immunity of an adenovirus vector (Ad) in BALB/c mice and cynomolgus monkeys. We developed an HIV vaccine containing the HIV clade C gag gene using a replication-defective chimeric adenovirus type 5 (Ad5) vector incorporating Ad35 fiber (Ad5/35); this vector has exhibited low hepatotoxicity in animal models. We observed that immunization with the Ad5/35 vaccine generated heightened HIV-specific immune responses in both mice and monkeys. Furthermore, the Ad5/35 vector vaccine produced a cross-immunity against challenge with recombinant vaccinia viruses expressing HIV clade B gag. These results demonstrate that Ad5/35 vaccines expressing HIV clade C gag may be promising candidates for clinical trials.

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. METHODS: Tenocytes were obtained from cultures of rat intrasynovial tendons and distributed to 82 wells in eight culture plates and to 30 culture dishes. The tenocytes in the wells were treated with AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, and AAV8 vectors containing the lacZ gene, and plasmid vectors (pCMVbeta-lacZ). The tenocytes were stained with in situ beta-galactosidase 5 days later. The basic fibroblast growth factor (bFGF) gene was cloned to the AAV2 vector to construct the AAV2-bFGF vector, which transduced tenocytes in culture dishes. Expression of the transgene was measured over 3 weeks and analyzed statistically. RESULTS: AAV2 effectively delivered exogenous genes to proliferating intrasynovial tenocytes. In contrast, other tested adeno-associated viruses transduced tenocytes minimally or not at all. The efficiency of gene transfer by AAV2, indicated by the percentage of cells with positive beta-galactosidase staining, was significantly greater than that by a plasmid vector (p = 0.001). Expression of the bFGF gene in tenocytes transduced with the AAV2-bFGF was significantly higher than that in the control over the 3-week period (p < 0.01). CONCLUSIONS: Gene transfer to tenocytes by AAV2 is more efficient than that by a plasmid vector. However, other adeno-associated virus serotypes cannot effectively transduce tenocytes. The bFGF gene can be delivered to intrasynovial tenocytes by the AAV2 vector effectively, and the gene transfer significantly increases expression of bFGF gene over 3 weeks.

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. These results suggest that the appropriate level of apoptosis is important for DNA vaccine development.

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 (52.1%) by cell culture isolation; and 16 (14.0%) by enzyme-linked immunosorbent assay (ELISA). The serotypes were determined by PCR- restriction fragment length polymorphism analysis (PCR-RFLP) and by the neutralization test (NT). PCR-RFLP was performed using a combination of endonucleases such as HhaI, AluI, and HaeIII. Of the 94 PCR-positive samples, the serotypes of 91 (96.8%) were identified by PCR-RFLP analysis (adenovirus 3: 50%, 4: 11%, and 8: 32%). Out of the 115 samples, 60 samples were identified by the neutralization (adenovirus 3, 4, 7, and 8). When both PCR-RFLP and the neutralization techniques were used, 53.2%, 11.7%, 1.1%, and 34% of the samples were identified as adenovirus 3, 4, 7, and 8, respectively. In contrast to the results of a nationwide surveillance report, adenovirus 3 was found as a major cause of keratoconjunctivitis in the Yokohama area. The nationwide surveillance report did not reflect accurately the epidemiological situation in the local area. In order to obtain surveillance data that would be useful for the prevention of an adenovirus conjunctivitis epidemic, it seems that local epidemiology is more important than that nationwide surveillance.

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. METHODS: Forty-two flexor digitorum profundus tendons of 6 New Zealand white rabbits were used. Eighteen tendons were divided into 3 groups of 6 each and injected with different vectors: adenoviral vector, AAV2-luciferase vector, or pCMV-beta vector with liposome. Another 12 tendons were cut and repaired. At 3, 7, and 14 days, the tendons were harvested and stained with hematoxylin and eosin. Normal flexor tendons were harvested as controls. RESULTS: The tissue reactions of the liposome-plasmid vector in tendons were the most prominent among the 3 vectors tested. The adenoviral vector elicited a moderate degree of tissue reaction. The AAV2 vector caused remarkable reactions in epitenon but almost no reactions in endotenon. Early-stage tissue reactions were more robust in the injured tendons. Compared with early-stage inflammatory and healing responses, the reactions elicited by these vectors were less severe. CONCLUSIONS: The 3 gene delivery systems tested elicit less severe tissue reactions in flexor tendons compared with early-stage inflammatory changes in injured tendons. Adenoviral and AAV vectors elicit less severe tissue reactions than liposome-plasmid vectors. The AAV2 vector appears to cause almost no reaction in endotenon. In terms of tissue reactions, the adenoviral and AAV2 vectors, in particular AAV2, are suitable gene delivery systems for future gene transfer to the tendon in vivo.

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. An immunogenicity study of AAV serotypes indicates that AAV1, AAV5, AAV7, and AAV8 vectors expressing the Env gp160 gene induced higher HIV-specific humoral and cell-mediated immune responses than the AAV2 vector did, with the AAV5 vector producing the best responses. Furthermore, mice injected with DCs that had been transduced ex vivo with an AAV5 vector expressing the gp160 gene elicited higher HIV-specific cell-mediated immune responses than did DCs transduced with AAV1 and AAV2 vectors. We also found that AAV vectors produced by HEK293 cells and insect cells elicit similar levels of antigen-specific immune responses. These results demonstrate that the immunogenicity of AAV vectors depends on their tropism for both antigen-presenting cells (such as DCs) and non-antigen-presenting cells (such as muscular cells) and that AAV5 is a better vector than other AAV serotypes. These results may aid in the development of AAV-based vaccine and gene therapy.

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. The susceptibility of immunized mice to virus infection was significantly increased following depletion of either CD4+ or CD8+ T cells. These results indicate that a plasmid DNA vaccine encoding pVP22 plus NP induces a high level of cross-protective immunity against influenza virus subtypes.

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. Immunization with multivalent DNA vaccine via imEPT induced the most potent protection against lethal pneumonia. Although the serum levels of IgG binding to whole bacteria cells were comparable between groups, the strongest immune protection was associated with the serum levels of Th1-dominated multivalent IgG, the bronchoalveolar levels of macrophage inflammatory protein 2 (MIP-2) and IFN-gamma, and the number of neutrophils and macrophages in the bronchoalveolar lavage following intranasal challenge. These results implied the possible clinical application of multivalent DNA vaccine against P. aeruginosa.

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. Since the compositions of the VLP and vector differ by the single-stranded DNA genome, we hypothesized that the isoelectric point of the vector may differ from that of the VLP. In an attempt to separate type 1 rAAV particles from VLPs by ion-exchange chromatography, we tested a number of buffer systems and found that trimethylammonium sulfate, or [(CH3)4N]2SO4, effectively separated rAAV1 particles from VLPs. The rAAV1-GFP chromatographically separated from VLPs induced stronger GFP expression in HEK293 cells than rAAV1-GFP contaminated with VLPs. The transduction of mouse muscles with rAAV1-SEAP (secreted form of alkaline phosphatase) isolated from VLPs also showed higher serum SEAP levels than rAAV1-SEAP with VLPs. These results suggest that chromatographic separation of rAAV1 from empty capsids increased the efficacy of rAAV1.

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(89.6)env gene (vLC-Env) alone or combined with a DNA vaccine expressing the HIV(89.6)env gene (pCAG-Env) was characterized in BALB/c mice. Vaccination of vLC-Env induced much higher HIV-specific humoral and cell-mediated immune responses than that of pCAG-Env. Priming with pCAG-Env further enhanced vLC-Env induced immune responses, especially cell-mediated immune response. Moreover, efficient expression of Env protein was achieved following infection of bone marrow dendritic cells by vLC-Env in vitro. Administration of vLC-Env-infected dendritic cells to mice generated a high cell-mediated immune response. These results demonstrate that priming with pCAG-Env and boosting with vLC-Env represents a logical candidate for vaccination against HIV infection.

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. This assay would be applicable as a challenge model for infectious diseases.

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.v. challenged with pathogenic simian/HIV, the prime-boost group maintained high CD4+ T cell counts and reduced plasma viral loads up to 30 wk after viral challenge, whereas the rDIsSIVgag/pol group showed only a partial attenuation of the viral infection, and the group immunized with SIVgag/pol DNA alone showed none at all. The protection levels were better correlated with the levels of virus-specific T cell responses than the levels of neutralization Ab responses. These results demonstrate that a vaccine regimen that primes with DNA and then boosts with a replication-defective vaccinia virus DIs generates anti-SIV immunity, suggesting that it will be a promising vaccine regimen for HIV-1 vaccine development.

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. We replaced the N-terminal portion of type 5 VP1 with the equivalent portion of type 2 to generate infectious AAV5 particles. The rAAV5 with the modified VP1 required alpha2-3 sialic acid for transduction, as revealed by a competition experiment with an analog of alpha2-3 sialic acid. rAAV5-GFP/Neo with a 4.4-kb vector genome produced in HEK293 cells or Sf9 cells transduced COS cells with similar efficiencies. Surprisingly, Sf9-produced humanized Renilla green fluorescent protein (hGFP) vector with a 2.4-kb vector genome induced stronger GFP expression than the 293-produced one. Transduction of murine skeletal muscles with Sf9-generated rAAV5 with a 3.4-kb vector genome carrying a human secreted alkaline phosphatase (SEAP) expression cassette induced levels of SEAP more than 30 times higher than those for 293-produced vector 1 week after injection. Analysis of virion DNA revealed that in addition to a 2.4- or 3.4-kb single-stranded vector genome, Sf9-rAAV5 had more-abundant forms of approximately 4.7 kb, which appeared to correspond to the monomer duplex form of hGFP vector or truncated monomer duplex SEAP vector DNA. These results indicated that rAAV5 can be generated in insect cells, although the difference in incorporated virion DNA may induce different expression patterns of the transgene.