A block in release of progeny virus and a high particle-to-infectious unit ratio contribute to poor growth of enteric adenovirus types 40 and 41 in cell culture.

The fastidious enteric adenovirus (FEAd) types 40 (Ad40) and 41 (Ad41) are found in stool specimens of infants and young children in association with gastroenteritis. Although they can be isolated routinely from clinical specimens by using 293 cells, they are propagated with variable success in cell lines which support the replication of other adenovirus serotypes. HeLa cells are generally considered to be nonpermissive for the replication of FEAds, but in this study, Ad40 and Ad41 grew to comparable titers in individual 293 and HeLa cells. However, virus was not efficiently released from infected HeLa cells and thus did not undergo multiple cycles of infection in HeLa cell cultures. The block in virus release was not overcome in KB18 cells which, like 293 cells, constitutively express proteins encoded by the E1B region of a subgroup C adenovirus (in this case Ad2). Moreover, it was apparent from these studies that Ad40 and Ad41 have particle-to-infectious unit ratios several orders of magnitude greater than that for Ad5, even in 293 cells which express the E1A and E1B proteins of Ad5 and are considered to be permissive for replication of the FEAds. Neither the block in release of progeny virus nor the high particle-to-infectious unit ratio is explained solely by the defect in expression of the E1B 55K protein identified by Mautner et al. (V. Mautner, N. MacKay, and V. Steinthorsdottir, Virology 171:619-622, 1989; V. Mautner, N. MacKay, and K. Morris, Virology 179:129-138, 1990).     

重组p16腺病毒的构建及其对人白血病细胞的抑制作用

为了探讨腺病毒载体用于基因治疗的可行性及野生型 p1 6基因的抗肿瘤特性 ,构建了复制缺陷型重组 p1 6腺病毒 .首先将 p1 6全长 c DNA插入穿梭质粒 p Ad CMV产生重组质粒 p Ad-CMV- p1 6,然后通过脂质体介导与 p JM1 7共转染 2 93细胞 ,经同源重组产生 E1区缺失的重组腺病毒空斑 .用纯化后的腺病毒感染人白血病细胞株 HL- 60后 ,PCR及 Western blot分析显示在感染细胞中有外源性 p1 6 c DNA存在和 p1 6蛋白表达 ;被感染的 HL- 60细胞的生长受到明显抑制 ,而未感染细胞及对照腺病毒感染的细胞没有受到抑制 .结果表明 ,腺病毒作为一种新型基因转移载体 ,可有效地介导肿瘤抑制基因 p1 6的表达 ,在肿瘤基因治疗方面具有很大的应用前景 .     

Conditional replication of a recombinant adenovirus studied using neomycin as a selective marker

An E1B-defective adenovirus, named r2/Ad carrying the neo expression cassette, was constructed by homologous recombination. The construction, selection (using neomycin as a selective marker), and propagation of the recombinant virus was performed in human embryonic kidney 293 cells (HEK 293). An in vitro study demonstrated that this recombinant virus has the ability to replicate in and lyse some p53-deficient human tumor cells such as human glioma tumor cells (U251) and human bladder cells (EJ), but not in some cells with functional p53, such as human adenocarcinoma cells (A549) and human fibroblast cells (MRC-5). Also, based on the cytopathic effect (CPE), it was demonstrated, under identical conditions, that the U251 cells were more sensitive to r2/Ad replication than the EJ cells. In this paper, we report that r2/Ad could be very useful in studying the in vitro selective replication of E1B-defective adenovirus and has great potential in cancer gene therapy.     

Monoclonal antibodies against simian virus 40 tumor antigens: analysis of antigenic binding sites, using adenovirus type 2-simian virus 40 hybrid viruses.

The antigenic binding sites of two monoclonal antibodies are located in the COOH-terminal region (clone 412) and probably in an internal region (clone 7) of simian virus 40 large T antigen. A third monoclonal antibody (clone 122), which has been shown to bind nonviral T antigen, does not react with HeLa cells infected with nondefective adenovirus type 2 (Ad2)-simian virus 40 hybrid viruses Ad2+ND1, Ad2+ND2, or Ad2+ND4.     

Tropism of human adenovirus type 5-based vectors in swine and their ability to protect against transmissible gastroenteritis coronavirus.

The infection of epithelia] swine testicle and intestinal porcine epithelial (IPEC-1) cell lines by adenovirus type 5 (Ad5) has been studied in vitro by using an Ad5-luciferase recombinant containing the firefly luciferase gene as a reporter. Porcine cell lines supported Ad5 replication, showing virus titers, kinetics of virus production, and luciferase expression levels similar to those obtained in human 293 cells, which constitutively express the 5'-end 11% of the Ad5 genome. The tropism of Ad5-based vectors in swine and its ability to induce an efficient immune response against heterologous antigens expressed by foreign genes inserted in these vectors has been determined. Ad5 vectors replicate and express heterologous antigens in porcine lungs and mediastinal and mesenteric lymph nodes. Significant levels of heterologous antigen expression were also demonstrated in the small intestine (jejunum and ileum), but Ad5 replication in this organ was very poor, suggesting that Ad vectors undergo an abortive replication in the porcine small intestine. The tissues infected by Ad5 were dependent on the inoculation route. The oronasal route appeared to be best for inoculation of bronchus-associated lymphoid tissue infection, while the intraperitoneal route was best for gut-associated lymphoid tissue infection. Epithelial cells of bronchioles, macrophages, type II pneumocytes, and follicular dendritic cells were identified as targets for Ad5, while epithelial cells of the intestine were not infected by Ad5. Viruses with a deletion from 79.5 to 84.8 map units in the E3 region, with or without heterologous inserted genes, replicated to lower levels in porcine tissues than did wild-type Ad5. It was also shown that an Ad5 recombinant expressing the four antigenic sites (A, B, C, and D) of transmissible gastroenteritis coronavirus (TGEV) spike protein induced in swine immune responses which neutralized TGEV infectivity. In addition, porcine serum from Ad-TGEV-immune animals provide passive protection when mixed with fully virulent TGEV and orally administered to highly susceptible newborn piglets. These results taken together indicate that swine may be a good animal model for human Ad5 lung infection to aid in the evaluation of candidate adenovirus vaccines and that Ad5 may be suitable as a recombinant viral vaccine or for other applications in swine.     

Dependence of tumor-forming capacities of cells transformed by recombinants between adenovirus types 5 and 12 on expression of early region 1.

Recombinants between an adenovirus type 5 (Ad5) deletion mutant and the Ad12 DNA fragment containing early region 1 (E1) were isolated from cells cotransfected with the EcoRI-C fragment of Ad12 DNA and Ad5 dl312 (deletion in E1A) DNA (rcA) and from cells cotransfected with the SalI-C fragment of Ad12 DNA and Ad5 dl312 DNA (rcB). No recombinant was isolated from cells cotransfected with Ad5 dl313 (deletion in E1B) DNA and restriction fragments of Ad12 DNA. Both rcA and rcB are defective and able to replicate in human embryo kidney (HEK) and KB cells with complementation by dl312. Both rcA and rcB formed Ad12 T antigen g, but not T antigen f, in infected HEK and KB cells. In rcA- and rcB-infected cells, Ad5 E1B and Ad12 E1A genes are transcribed. Heteroduplex and size analyses of rcA-1 or rcB-1 DNA fragments hybridized with Ad12 DNA revealed that rcA-1 DNA has a deletion between 5 and 15 map units with an insertion of a portion of Ad12 DNA (10%) and that rcB-1 DNA has a deletion between 70 and 80 map units with an insertion of a portion of Ad12 DNA (10%). The transformed cell lines, RCAY and RCBY, were established after infection of rat 3Y1 cells with rcA and rcB, respectively. Both Ad5 and Ad12 DNA sequences are contained in these cells. In RCAY cells, Ad12 T antigen g is detected, but Ad12 T antigen f is not. In RCBY cells, both Ad12 T antigen g and f are detected. Only the Ad12 E1A gene is transcribed in RCAY cells, whereas Ad5 E1B, Ad12 E1A, and Ad12 E1B genes are transcribed in RCBY cells. In soft-agar cultures, RCBY cells form large colonies, whereas RCAY cells form only tiny colonies. RCBY cells form tumors as efficiently as 12WY cells in transplanted rats. RCAY cells formed tumors inefficiently. Ad5-transformed 5WY cells do not form tumors. These observations indicate that the efficient tumor formation by RCBY cells is dependent on the expression of the Ad12 E1A and E1B genes, whereas the inefficient tumor formation by RCAY cells is due to the expression of only the Ad12 E1A gene.     

A helper virus-free packaging system for recombinant adeno-associated virus vectors

Adeno-associated virus (AAV) is a human parvovirus that is currently receiving widespread attention for its potential use as a gene therapy vector. Construction of the recombinant AAV vector (rAAV) involves replacing most of the viral genome with a transgene of interest and then packaging this recombinant genome into an infectious virion. Most current protocols for generating rAAV entail the co-transfection of a vector plasmid and a packaging plasmid that expresses the viral replication and structural genes onto adenovirus (Ad) infected cells growing in culture. Limitations of this procedure include (1) contamination of rAAV with the Ad helper virus, (2) low yields of rAAV and (3) production of replication-competent AAV. In this report we describe new helper plasmids (pSH3 and pSH5) that eliminate the Ad co-infection requirement. The helper plasmids express the AAV rep and cap genes and the Ad E2A, VAI and E4 genes. When the helper plasmids are co-transfected onto human 293 cells with a vector plasmid in the absence of Ad infection, the rAAV vector yield is up to 80-fold greater than those obtained with the pAAV/Ad packaging plasmid. Moreover, replication competent AAV in the rAAV preparations is less than 0.00125%. The major advantages of this system are (1) the absence of infectious adenovirus and (2) the use of only two plasmids, which enhances transfection efficiencies and hence vector production. We believe that this two-plasmid transfection system will allow for more widespread use of the AAV vector system because of its simplicity and high yields. This system will be especially useful for preclinical analyses of multiple rAAV vectors.     

Oncogenicity by adenovirus is not determined by the transforming region only.

We have constructed a nondefective recombinant virus between the nononcogenic adenovirus 5 (Ad5) and the highly oncogenic Ad12. The recombinant genome consists essentially of Ad5 sequences, with the exception of the transforming early region 1 (E1) which is derived from Ad12. HeLa cells infected with the recombinant virus were shown to contain the Ad12-specific E1 proteins of 41 kilodaltons (E1a) and 19 and 54 kilodaltons (both encoded by E1b). The recombinant virus replicated efficiently in human embryonic kidney cells and HeLa cells, showing that the transforming regions of Ad5 and Ad12 had similar functions in productive infection. After the recombinant virus was injected into newborn hamsters, no tumors were produced during an observation period of 200 days. Thus, despite the fact that all products required for oncogenic transformation in vitro were derived from the highly oncogenic Ad12, the recombinant virus did not produce tumors in vivo. These data show that tumor induction by adenovirus virions is not determined only by the gene products of the transforming region.     

Immunological Cross-Reactivity Between Simian Virus 40 Large T Antigen and D2 Hybrid T Antigen

A specific antiserum was raised in rabbits against D2 hybrid T antigen that had been purified from HeLa cells infected with the adenovirus/simian virus 40 hybrid, Ad2(+)D2. The specificity of this serum was compared with that of a conventional hamster antiserum against simian virus 40-induced tumors by immunoprecipitation and by a new radioimmune assay that can detect nanogram quantities of D2 hybrid T antigen.     

Defective and nondefective adenovirus vectors for expressing foreign genes in vitro and in vivo.

We have constructed recombinant adenoviruses (Ad), with functional or defective E1a genes, which harbor either the hepatitis B (HB) virus s gene encoding the HB surface antigen, as well as the pre-S2 epitopes, or the bacterial gene encoding chloramphenicol acetyltransferase (CAT) under control of the Ad major late promoter (MLP). The recombinant viruses defective for E1a (Ad.MLP.S2 and Ad.CAT), which can be efficiently propagated only on 293 cells that complement this defect, and the nondefective (Ad.MLP.S2.E1A) recombinant were used to infect a wide spectrum of cells of different origin. The yields of HBs and CAT proteins obtained with these different recombinant viruses demonstrate no real advantage to using nondefective vectors, whatever the cell type infected. The injection into chimpanzees of Ad.MLP.S2 does not elicit the production of antibodies, but can immunologically prime the animals, resulting in a partial protection against HBV challenge.     

Recombinant Interferon-γ Lentivirus Co-Infection Inhibits Adenovirus Replication Ex Vivo

Recombinant interferon-γ (IFNγ) production in cultured lentivirus (LV) was explored for inhibition of target virus in cells co-infected with adenovirus type 5 (Ad5). The ability of three different promoters of CMV, EF1α and Ubiquitin initiating the enhanced green fluorescence protein (GFP) activities within lentiviruses was systematically assessed in various cell lines, which showed that certain cell lines selected the most favorable promoter driving a high level of transgenic expression. Recombinant IFNγ lentivirus carrying CMV promoter (LV-CMV-IFNγ) was generated to co-infect 293A cells with a viral surrogate of recombinant GFP Ad5 in parallel with LV-CMV-GFP control. The best morphologic conditions were observed from the two lentiviruses co-infected cells, while single adenovirus infected cells underwent clear pathologic changes. Viral load of adenoviruses from LV-CMV-IFNγ or LV-CMV-GFP co-infected cell cultures was significantly lower than that from adenovirus alone infected cells (P = 0.005–0.041), and the reduction of adenoviral load in the co-infected cells was 86% and 61%, respectively. Ad5 viral load from LV-CMV-IFNγ co-infected cells was significantly lower than that from LV-CMV-GFP co-infection (P = 0.032), which suggested that IFNγ rather than GFP could further enhance the inhibition of Ad5 replication in the recombinant lentivirus co-infected cells. The results suggest that LV-CMV-IFNγ co-infection could significantly inhibit the target virus replication and might be a potential approach for alternative therapy of severe viral diseases.     

Common features of polyomavirus mutants selected on PCC4 embryonal carcinoma cells.

The genomic rearrangements of six polyomavirus mutants selected on PCC4 embryonal carcinoma cells have been compared and their common characteristics pointed out. All mutants show a duplication which includes at least the adenovirus type 5 (Ad5) E1A-like enhancer core sequence plus a deletion of variable size and location. The presence of the second enhancer core sequence, the SV40-like enhancer, is not required for expression of the PyEC PCC4 phenotype. Two of these mutants are also able to express polyomavirus T antigen on F9 and LT1 cells. Multiadaptation seems to require the duplication of the Ad5 E1A-like core sequence, the maintenance of the SV40-like core sequence and a local change in DNA stability.     

Hybrid of Monkey and Human Adenoviruses

Following joint replication of monkey SA7 adenovirus (C8 strain) and human adenovirus type 2 in green monkey kidney tissue culture, a virus possessing the properties of a hybrid was obtained. It was designated Ad2C8. Ad2C8 preparations contained two types of viral particles: human adenovirus type 2, and hybrid particles. The hybrid virions multiplied in green monkey kidney cells in the presence of human adenovirus types 1, 2, and 3, but not 3 and 7, and acquired the capsid of the helper adenovirus. The hybrid can serve as a helper for human adenoviruses. It can apparently induce T antigen of the C8 virus but, in contrast to the latter, does not induce tumors in hamsters.