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Although human adenovirus serotype 5 (Ad5) is widely used as a vaccine vector for infectious diseases due to its high transduction efficiency, pre-existing immunity to Ad5 in many people reduces vaccine efficacy. To address this limitation, simian Ad vectors, such as ChAdOx1 and ChAdOx2, have been explored as alternative vaccine platforms. ChAdOx2 is based on simian Ad25 (SAd25), but the fundamental characteristics of gene transduction by SAd25-based vectors have not been fully elucidated. This study aimed to characterize the gene transduction efficiency, tissue distribution, and immunogenicity of an SAd25-based vector in comparison with those of the Ad5 vector following various routes of administration. Compared with intravenous administration of the Ad5 vector, intravenous administration of the SAd25 vector showed distinct biodistribution patterns, including reduced liver accumulation and predominant expression in the lung. Transduction by the SAd25 vector was not inhibited by human serum, whereas transduction by the Ad5 vector was inhibited, indicating that the SAd25 vector, but not the Ad5 vector, can evade pre-existing Ad immunity. Although intramuscular administration of the SAd25 vector induced lower transgene product-specific antibody production than intramuscular administration of the Ad5 vector, gene expression and Ad genome distribution mediated by the SAd25 vector, but not the Ad5 vector, were localized only to the muscle at the administration site. Intranasal administration of the SAd25 vector induced an antigen-specific antibody response in serum more rapidly than intranasal administration of the Ad5 vector. The SAd25 vector induced antigen-specific antibody production in bronchoalveolar lavage fluid (BALF) that was comparable to that induced by the Ad5 vector. These findings provide essential insights into the biological characteristics of the SAd25 vector, supporting its potential as a safe and effective vaccine vector.
The most promising new techniques for the study of in vivo mammalian mutagenesis make use of transgenic mice carrying a recoverable vector. Mutation systems in mammals can be based on the selection of altered phenotypes among cells sampled from the whole animal, but they are then limited to the very few cell types in which the marker gene is expressed. Such systems require both in vivo and in vitro cell proliferation for expression and verification of the mutations. To avoid these complications, the study of mutations in most tissues must be based on the detection of genetic alterations in a vector that is independent of the phenotype of the mammalian cell. The vector is only a small portion of the mammalian genome, and many of the procedures for recovering the vector are inhibited by the host DNA. For this reason, partial purification is necessary. The purification is made possible by using vectors which are not cut by restriction enzymes that cut the host DNA to pieces of an average size considerably smaller than the vector. The efficiency for measuring mutation frequencies depends on the number of vectors which can be recovered from a certain amount of DNA and is affected by the number of vectors per mammalian genome and the transfection efficiency of the partially-purified vector. In order to avoid selection against or for the spontaneous or induced mutations, the transfection efficiency of the vector from the transformed DNA and of the pure vector DNA should be of the same order of magnitude. Differences in the response to mutagens between the mammalian genome and the procaryotic vector may be expected due to the lack of unique mammalian topographical features in the vectors. Any mutation induction which depends preferentially on these unique features of the mammalian genome may not be detected in a shuttle vector system unless the vector has been engineered or specifically designed to include such topographical characters. The shortcoming of short-term tests that use mutagenicity for predicting human carcinogenicity is usually lack of correlation between mutagenesis in the short-term tests and the corresponding results in carcinogenesis bioassays in mammals. One factor which could contribute to the lack of correlation between the short-term test systems and the bioassays is that we are comparing mutations in totally different genes in different organisms. By using the phi X174 shuttle system, one of the variables may be eliminated.(ABSTRACT TRUNCATED AT 400 WORDS)
Three-dimensional vectorcardiography was used to characterize changes in the T-wave vector loop during exposure to cold pressor stress. Data were collected from 8 subjects during baseline, cold pressor, and recovery periods. Maximum vector length, polar angle of the longest vector, azimuth angle of the longest vector, sum of all vectors, polar angle of the vector sum, azimuth angle of the vector sum, surface area of the loop, and maximum distance between adjacent vectors were computed from the T-wave vector loop. The amplitude of the one-dimensional T-wave (TWA) was also computed. Interbeat interval and TWA decreased during hand immersion and increased during recovery. Of the vectorcardiographic measures, only maximum vector length and vector sum showed statistically significant change; both decreased during the task and returned to baseline during recovery. TWA accounted for 46% of the task related variance compared to 53% and 47% respectively, for vector sum and maximum vector length. Changes in TWA probably reflect a true change in cardiac electrical potential rather than a change in T-wave vector loop shape or orientation.
The spread of insect-borne animal virus diseases is influenced by a number of factors. Hosts migrate, move or are conveyed over long distances: vectors are carried on the wind for varying distances in search of hosts and breeding sites; weather and climate affect hosts and vectors through temperature, moisture and wind. As parasites of host and vector, viruses are carried by animals, birds and insects, and their spread can be correlated with the migration of hosts and the carriage of vectors on winds associated with the movements of the Intertropical Convergence Zone (ITCZ) and warm winds to the north and south of the limits of the ITCZ. The virus is often transmitted from a local cycle to a migratory cycle and back again.Examples of insect-borne virus diseases and their spread are analysed. Japanese, Murray Valley, Western equine, Eastern equine and St Louis encephalitis represent viruses transmitted by mosquito-bird or pig cycles.THE AREAS EXPERIENCING INFECTION WITH THESE VIRUSES CAN BE DIVIDED INTO A NUMBER OF ZONES: A, B, C, D, E and F. In zone A there is a continuous cycle of virus in host and vector throughout the year; in zone B, there is an upsurge in the cycle during the wet season, but the cycle continues during the dry season; there is movement of infected vectors between and within zones A and B on the ITCZ and the virus is introduced to zone C by infected vectors on warm winds; persistence may occur in zone C if conditions are right. In zone D, virus is introduced each year by infected vectors on warm winds and the arrival of the virus coincides with the presence of susceptible nestling birds and susceptible piglets. The disappearance of virus occurs at the time when migrating mosquitoes and birds are returning to warmer climates. The virus is introduced to zone E only on occasions every 5-10 years when conditions are suitable. Infected hosts introduced to zone F do not lead to circulation of virus, since the climate is unsuitable for vectors. Zones A, B and C correspond to endemic and zones D and E to epidemic conditions.Similar zones can be recognized for African horse sickness, bluetongue, Ibaraki disease and bovine ephemeral fever - examples of diseases transmitted in a midge-mammal cycle. In zones A and B viruses are transported by infected midges carried on the wind in association with the movement of ITCZ and undergo cycles in young animals. In these zones and in zone C there is a continual movement of midges on the warm wind between one area and another, colonizing new sites or reinforcing populations of midges already present. Virus is introduced at times into fringe areas (zones D and E) and, as there is little resistance in the host, gives rise to clinical signs of disease. In some areas there is persistence during adverse conditions; in others, the virus is carried back to the endemic zones by infected midges or vectors.Examples of viruses maintained in a mosquito/biting fly-mammal cycle are Venezuelan equine encephalitis and vesicular stomatitis. These viruses enter a migratory cycle from a local cycle and the vectors in the migratory cycle are carried over long distances on the wind. Further examples of virus spread by movement of vectors include West Nile, Rift Valley fever, yellow fever, epizootic haemorrhagic disease of deer and Akabane viruses.In devising means of control it is essential to decide the relationship of host, vector and virus and the nature of the zone in which the area to be controlled lies. Because of the continual risk of reintroduction of infected vectors, it is preferable to protect the host by dipping, spraying or by vaccination rather than attempting to eliminate the local population of insects.
Recently, we described a retrovirus vector system with which to study formation of cDNA genes (R. Dornburg and H. M. Temin, Mol. Cell. Biol. 6:2328-2334, 1988; Mol. Cell. Biol. 8:64-72, 1990; J. Virol. 64:886-889, 1990). For these studies, retrovirus vectors were constructed in which the U3 region of the 3' long terminal repeat (LTR) was deleted. After one round of retrovirus replication, such vectors formed a provirus with two U3-minus LTRs. However, the insertion of some additional sequences into such vectors promoted vector rearrangements with an efficiency greater than 95%. Such rearranged vectors behaved like vectors with two wild-type LTRs. Proviruses derived from such vectors were investigated by Southern blot analysis, polymerase chain reaction, and DNA sequencing. We found that the U3 region was reconstituted, resulting in vectors with LTRs like wild-type virus. The sequences that reconstituted the U3 region of the vector LTR were derived from LTR sequences present in the helper cell. Since no retroviral protein coding sequences were detected in infected target cells, recombination of vector sequences with coencapsidated helper cell sequences during reverse transcription seems very unlikely. Thus, it appears that the recombination (or gene conversion) events leading to a vector with reconstituted LTRs occurred at the DNA level. The high frequency of this recombination (or gene conversion) was dependent on internal vector sequences.
Recombinant retroviral vectors producing multicistronic mRNAs were constructed. Picornavirus putative internal ribosome entry sites (IRES) were used to confer cap-independent translation of an internal cistron. Internal cistrons were engineered by ligation of various lengths of the IRES of encephalomyocarditis (EMC) virus or polio virus to the E. coli chloramphenicol acetyltransferase (CAT) gene. The IRES/CAT fusions were introduced into retroviral vectors 3' to the translation stop codon of the neomycin phosphotransferase (NEO) gene, and the molecular constructs transfected into retroviral vector packaging lines. Retroviral vector producer cells efficiently express the internal CAT gene product only when the full length IRES is used. Both the EMC/CAT and polio/CAT retroviral vectors produced high titer vector supernatant capable of productive transduction of target cells. To test the generality of this gene transfer system, a retroviral vector containing an IRES fusion to the human adenosine deaminase (ADA) gene was constructed. Producer cell supernatant was used to transduce NIH/3T3 cells, and transduced cells were shown to express NEO, and ADA. Novel three-gene-containing retroviral vectors were constructed by introducing the EMC/ADA fusion into either an existing internal-promoter-containing vector, or a polio/CAT bicistronic vector. Producer cell clones of the three-gene vectors synthesize all three gene products, were of high titer, and could productively transduce NIH/3T3 cells. By utilizing cap-independent translation units, IRES vectors can produce polycistronic mRNAs which enhance the ability of retroviral-mediated gene transfer to engineer cells to produce multiple foreign proteins.
Expression of chloramphenicol acetyltransferase (cat) by suitable vectors in chloroplasts of cultured tobacco cells, delivered by high-velocity microprojectiles, is reported here. Several chloroplast expression vectors containing bacterial cat genes, placed under the control of either psbA promoter region from pea (pHD series) or rbcL promoter region from maize (pAC series) have been used in this study. In addition, chloroplast expression vectors containing replicon fragments from pea, tobacco, or maize chloroplast DNA have also been tested for efficiency and duration of cat expression in chloroplasts of tobacco cells. Cultured NT1 tobacco cells collected on filter papers were bombarded with tungsten particles coated with pUC118 (negative control), 35S-CAT (nuclear expression vector), pHD312 (repliconless chloroplast expression vector), and pHD407, pACp18, and pACp19 (chloroplast expression vectors with replicon). Sonic extracts of cells bombarded with pUC118 showed no detectable cat activity in the autoradiograms. Nuclear expression of cat reached two-thirds of the maximal 48 hr after bombardment and the maximal at 72 hr. Cells bombarded with chloroplast expression vectors showed a low level of expression until 48 hr of incubation. A dramatic increase in the expression of cat was observed 24 hr after the addition of fresh medium to cultured cells in samples bombarded with pHD407; the repliconless vector pHD312 showed about 50% of this maximal activity. The expression of nuclear cat and the repliconless chloroplast vector decreased after 72 hr, but a high level of chloroplast cat expression was maintained in cells bombarded with pHD407. Organelle-specific expression of cat in appropriate compartments was checked by introducing various plasmid constructions into tobacco protoplasts by electroporation. Although the nuclear expression vector 35S-CAT showed expression of cat, no activity was observed with any chloroplast vectors.
The titer of retroviral vectors can be increased by cocultivation of retrovirus packaging cells that produce a vector with packaging cells having a different host range. Multiple rounds of infection occur in such cultures, producing an amplification of vector copy number and titer. Production of a vector with a very high titer of over 10(10) CFU per ml of conditioned medium has been reported, although replication-competent helper virus was also present. Since helper-free virus is a requirement for many applications of retroviral vectors, we repeated this procedure with a modified vector and achieved a 2- to 10-fold amplification of vector titer in the absence of helper virus, up to 2 x 10(7) CFU/ml. We have also repeated these experiments with the same vector and methods described previously or have assayed virus from the high-titer vector-producing cell line reported previously and observed maximum titers of 10(8) CFU/ml, invariably accompanied by helper virus. Thus, while amplification of vector titer in the absence of helper virus is possible, some unexplained difference in the assays for virus titer must account for our inability to obtain the exceptionally high vector titers that were reported previously.
Gene targeting has been used to direct mutations into specific chromosomal loci in murine embryonic stem (ES) cells. The altered locus can be studied in vivo with chimeras and, if the mutated cells contribute to the germ line, in their offspring. Although homologous recombination is the basis for the widely used gene targeting techniques, to date, the mechanism of homologous recombination between a vector and the chromosomal target in mammalian cells is essentially unknown. Here we look at the nature of gene targeting in ES cells by comparing an insertion vector with replacement vectors that target hprt. We found that the insertion vector targeted up to ninefold more frequently than a replacement vector with the same length of homologous sequence. We also observed that the majority of clones targeted with replacement vectors did not recombine as predicted. Analysis of the recombinant structures showed that the external heterologous sequences were often incorporated into the target locus. This observation can be explained by either single reciprocal recombination (vector insertion) of a recircularized vector or double reciprocal recombination/gene conversion (gene replacement) of a vector concatemer. Thus, single reciprocal recombination of an insertion vector occurs 92-fold more frequently than double reciprocal recombination of a replacement vector with crossover junctions on both the long and short arms.
We used rep+ and rep- recombinant AAV-plasmid vectors containing the nonselectable marker chloramphenicol acetyltransferase (CAT) driven by the AAV p40 promoter, and having a selectable marker, neo, inserted in the plasmid genome, and driven by a herpesvirus thymidine kinase gene promoter. Each vector was transfected into human 293 cells or HeLa cells and the neo gene was used to select geneticin-resistant (genr) cells containing integrated vectors. The genr cells were then screened for expression of the unselected marker CAT. For 293 cells, most clones from the rep- vector gave high CAT expression whereas only 50% of those from the rep+ vector expressed CAT, generally at low level. For HeLa cells about 25% of the clones derived from either the rep+ or rep- vector expressed CAT, and several clones from the rep+ vector gave very high yields. We also analyzed integrated rep+ vectors by rescue after superinfection with adenovirus and by Southern blotting. The AAV-CAT genome could be rescued from 50% of HeLa cell clones but not from 293 cell clones. Lack of rescuability reflected rearrangement of the AAV genome termini or the rep gene. Western blotting showed low level constitutive expression of rep protein in one 293 cell clone and two HeLa cell clones. Thus, the AAV p40 promoter (as well as p5 and p19) can function in integrated vectors to express unselected markers which can subsequently be rescued. Expression and rescue depended upon several parameters including the cell type, the initial structure of the vector (rep+ or rep-) but not continued expression of rep, and possibly global effects of the surrounding chromatin.
A phagemid vector library for cloning DNA with four nucleotide 5' or 3' overhangs has been constructed. This library is based on the pT7T3 vector (Pharmacia) which is a modification of the phagemid pTZ18U vector. We have chosen pT7T3 as the parent vector because it can be used for Sanger's dideoxy sequencing and for the generation of RNA probes with either the T7 or T3 promoter. Each member of the cloning vector series pBM has recognition sites for both of the restriction enzymes BspM1 and BstX1 in addition to the basic multiple cloning sites. BspM1 recognizes the sequence 5'...ACCTGC NNNN/NNNN...3' whereas BstX1 recognizes the sequence 5'...CCAN NNNN/NTGG...3'. Thus these two sites can be overlapped, so that only 256 vectors (instead of 512 vectors) need be constructed to cover all the theoretical possible combinations of sites which give complementary cohesive ends for cloning DNA with four nucleotide 5' or 3' overhangs. This vector library can be used for amplification cloning of DNA in a tandem array by choosing appropriate vectors which have nonpalindromic sequences. We have obtained approximately 200 members of the 256 possible clones and have organized the vectors using a MacIntosh HyperCard program for easy retrieval.
We report yeast/Escherichia coli shuttle vectors suitable for fusing yeast promoter and coding sequences to the lacZ gene of E. coli. The vectors contain a region of multiple unique restriction sites including EcoRI, KpnI, SmaI, BamHI, XbaI, SalI, PstI, SphI and HindIII. The region with the unique cloning sites has been introduced in both orientations with respect to lacZ and occurs proximal to the eighth codon of the gene. All the restriction sites have been phased to three different reading frames. Two series of vectors have been constructed. The first series (YEp) has two origins of replication (ori), i.e., of the yeast 2 mu circle and of the ColE1 plasmid of E. coli, and can therefore replicate autonomously in both organisms. These shuttle vectors also have the ApR gene of E. coli and either the yeast LEU2 or URA3 genes to allow for selection of both E. coli and yeast transformants. The second series of vectors (YIp) are identical in all respects to the YEp vectors except that they lack the 2 mu ori. The YIp vectors can be used to integrate lacZ fusions into yeast chromosomal DNA. None of the vectors express beta-galactosidase (beta Gal) in yeast or E. coli in the absence of inserted yeast promoter sequences. The 5'-nontranslated sequences and parts of the coding sequences of various yeast genes have been cloned into representative lacZ fusion vectors. In-frame gene fusions can be detected by beta Gal activity when either yeast or E. coli clones are plated on media containing XGal indicator. Quantitative determinations of promoter activity were made by colorimetric assay of beta Gal activity in whole cells. Fusion of the yeast CYC1 gene to lacZ in one of the vectors allowed detection of regulated expression of this gene when cells were grown under conditions of catabolite repression or derepression.
Bacterial beta-glucuronidase (gus) and neomycin phosphotransferase (neo) genes were introduced into coat protein replacement vectors based on DNA A of tomato golden mosaic virus (TGMV). Recombinant gus and neo vectors up to 1.1 kbp larger than DNA A were shown to replicate stably in transgenic plants containing partial dimers (master copies) of the vectors integrated into their chromosomal DNA in the absence of DNA B. Beta-glucuronidase and neomycin phosphotransferase activities in independently transformed plants were proportional to the copy number of the double-stranded forms of the vector. Deletion analysis has shown that an essential part of the TGMV coat protein promoter, including a TATA box, lies within 76 nt upstream of the initiation codon of the gene. An increase in expression of a neo gene was obtained by replacing this 76 nt sequence by an 800 nt sequence containing a cauliflower mosaic virus 35S RNA promoter with no effect on the ability of the vector to replicate or on its stability in transgenic plants. Systemic infection of plants by agroinoculation with TGMV vectors larger than DNA A in the presence of DNA B resulted in deletions in the vector DNA in some, but not all, plants. Possible reasons for vector instability in systemically infected plants, and vector stability in transgenic plants containing master copies of the vector, are discussed.