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PubMed2002

A minimal genome simian foamy virus type 1 vector system with efficient gene transfer.

Foamy viruses have several inherent features for the opportunity to develop efficient and versatile vectors for gene therapy. We have constructed a series of vectors and helper plasmids based on simian foamy virus type 1 (SFV-1) to establish the minimum vector genome required for efficient gene transduction. To characterize the efficiency of gene transduction by these vectors, the green fluorescent protein (GFP) coding sequence is linked to the human cytomegalovirus immediate gene promoter. Several deletion analyses of SFV-1 vectors revealed that the minimum genome with efficient GFP transduction contained the 5' untranslated region extending to the first 637 nucleotides of the gag gene, a 596 nucleotides of pol sequence from position 3137-3733, the 3' pol region at position 5200-5693, the 3' end polypurine tract, and the 3' LTR. An additional 1131 nucleotides can be removed from the 3' end LTR without affecting the efficiency of vector transduction. SFV-1 vector can therefore accommodate a minimum 8930 base-size heterologous DNA fragment. Furthermore, the efficiency of SFV-1 vector transduction was analyzed using different packaging plasmids. GFP transduction with packaging plasmid that contained the 5' R-U5 region of the LTR was compared with helper plasmids that had deletions in this region except for 22 nucleotides (positions 21-41), the first 61, 77, or 140 nucleotides of the R of the LTR. Transduction efficiencies were significantly reduced with the deletion mutations implicating that for optimum SFV-1 vector productions a packaging construct that includes the 5' R-U5 is required.

5' Untranslated Regions↗
PubMed1976

[Arthropodan vectors of human parasites: their pathology and defence reactions (author's transl)].

Many infectious diseases of man are transmitted by arthropods. It is not known whether the carriers were primarily susceptible or insusceptible as the cycle first came into existence. Similarly so little is understood, why some arthropods are suitable vectors and other close relatives are not suitable. Only the hypothesis that the agents are original parasites and not at all commensals or symbionts can reasonably explain the evolution of the cycles. It is shown by means of numerous examples how the vector can be damaged by the parasite. Some organs of the vector might become unfunctional, the reproduction rate might be lowered and the vector itself may die. On the other hand the vector has at its disposal defence mechanisms which are according to our present point of view limited to the midgut barrier and the hemolymph reactions. Both components of the system, the vector and the parasite together are capable of change under the influence of the other. For example through the high mortality of the susceptible part of a vector population the resistant and the tolerant individuals will be selected. On the contrary harmful parasites cannot transmit their genetic information when their virulence is so high that the vector will suffer death. Due to this the parasites succumb to a selection pressure and only the careful treatment of the vector is rewarding. Besides this rough pattern of actions and reactions exists also the possibility of developing finer adjustments, with the "molecular mimikry" as a well known example.

Adaptation, Biological↗
PubMed1998

Defective RNA packaging is responsible for low transduction efficiency of CAEV-based vectors.

Replication defective retroviral vectors are regularly used for transfer and expression of exogenous genes into dividing cells and in animals. Since lentiviruses are able to infect terminally differentiated and non-dividing cells, their use to produce replication defective vectors may overcome this limitation. We developed two replication-defective lentiviral vectors based on the genome of Caprine Arthritis Encephalitis Virus (CAEV). The first vector (pBNL2) carries the neo and lacZ marker genes. Neo gene is expressed from a genomic RNA and lacZ gene from a subgenomic RNA. The second vector (pCSHL) carries a single fusion gene encoding both phleomycin resistance and beta-galactosidase activity. Replication-competent CAEV was used as helper virus to provide the viral proteins for transcomplementation of these vectors. Our data demonstrated that the genomes of both vectors were packaged into CAEV virions and transduced into goat synovial membrane cells following infection. However, the vector titers remained 3 to 4 logs lower than those of CAEV. Further analysis showed a lack of accumulation of unspliced pBNL2 RNA into the cytoplasm of producer cells resulting in the packaging of pBNL2 sub-genomic RNA only. In contrast, RNA produced from pCSHL vector was correctly transported to the cytoplasm and more efficiently packaged than the pBNL2 sub-genomic RNA as revealed by slot-blot and quantitative RT/PCR analyses. However this higher packaging efficiency of pCSHL genome did not result in a higher transduction efficiency of lacZ gene.

Animals↗
PubMed1994

Vector-parasite interactions for vaccine development.

The ingestion of blood by arthropod vectors of disease can be exploited in order to either kill the vector or render it incapable of disease transmission. This paper examines some approaches to identifying target molecules of vector origin, against which immunisation could result in blocking parasite transmission. Manipulation of the blood meal of vectors through such techniques as membrane feeding can help identify true target sites for attack, but just as useful, can identify structures or molecules that play no significant role in parasite development. Examples, mostly derived from the interactions between the malaria parasite, Plasmodium, and the mosquito midgut, illustrate the real need to understand the multiple aspects of vector-parasite interactions before they can be exploited for control purposes. The approaches outlined are however applicable directly to any vector-borne disease. Careful examination of the parasite life cycle in the vector, and comparisons with other parasites, vectors, non-vector insects and analogous vertebrate systems (the latter being often relatively well advanced) can result in the identification of specific and definable interactions which can then be further developed for vaccine purposes.

Animals↗
PubMed1994

A new vector for the high level expression of chimeric antibodies in myeloma cells.

We previously reported the expression of a mouse/human chimeric anti-ganglioside GD3 antibody, KM871 (IgG1,kappa) in mouse myeloma SP2/0 cells under the control of the ecotropic Moloney virus long terminal repeat by the co-transfection of chimeric heavy (H) and light (L) chain vectors (Shitara et al. (1993) Cancer Immunol. Immunother.). To establish an efficient and high level expression system for the chimeric antibody, we did comparative study on vector systems and host cells. An improved expression vector, named 'a tandem vector, pChi641HLGM4' was constructed, in which both of the chimeric H and L chain gene transcription units and a dihydrofolate reductase (dhfr) gene transcription unit were inserted. When two kinds of mouse myeloma cell lines, SP2/0 and P3U1, were used as host cells, frequency of the incidence of antibody-producing transfectants was markedly increased by the use of the tandem vector compared with the use of the mixture of each chimeric H vector and L chain vector. To select out appropriate host cells, transfection frequency and antibody production level were compared among SP2/0, P3U1 and rat myeloma YB2/0 cells by transfection of the tandem vector. YB2/0 cell was shown to have the highest potential in both the transfection frequency and the antibody production. Introduction of the tandem vector into YB2/0 cells and the subsequent amplification with 50-200 nM methotrexate gave rise to several clones that stably secreted 70-100 micrograms/10(6) cells per 24 h of the chimeric antibody. This productivity of the antibody is one of the highest levels which have been achieved by other investigators using transfected myeloma cells. Using this system it took only 2-3 months to establish the transfectant clones which stably produced the chimeric antibody.

Animals↗
PubMed1988

Examination of vectors with two dominant, selectable genes for DNA repair and mutation studies in mammalian cells.

A series of vectors with two dominant selectable genes was constructed for repair and mutation studies following transfer into mammalian cells. The recombinant genes (SV-gpt and HSVtk-neo) were placed in different relative orientations and positions in the vectors. These variables were shown to affect transformation frequency of cells by the vectors especially where one of the genes had a relatively weak expression, modelled by truncating the promoter of the HSVtk-neo gene. The use of two-gene vectors to assess DNA repair was investigated by cutting the SV-gpt gene with a restriction endonuclease and monitoring correct rejoining by selecting for gene activity after transfer into various cell types. In such experiments, selection was first applied for the undamaged HSVtk-neo gene to eliminate transfer artefacts, followed by counterselection for the activity of the damaged SV-gpt gene. The measured frequency of correct rejoining of the damaged gene was found to vary both with the vector construct and with the recipient cell species (Chinese hamster V79 or human transformed fibroblasts). Despite this variation, correct rejoining was found to be consistently lower in radiosensitive (ataxia telangiectasia) human cells than in wild-type human cells, irrespective of the vector construct. In these experiments, some of the transformed cell colonies showed 'sectoring' on exposure to the counterselection, suggesting a slow determination of the fate of transferred DNA. For mutation studies a V79 cell clone carrying a single copy of one of these two-gene vectors was identified and shown to be stably integrated. Mutations of the SV-gpt gene in these cells were isolated while maintaining selection for the HSVtk-neo gene, to attempt to limit mutational loss of the total integrated sequence and provide at least one identifiable junction for analysis of deletion events. Spontaneous and X-ray-induced mutants were identified with a variety of genetic changes, as shown by Southern analysis, from presumed point mutations to deletions and rearrangements of the vector sequence. Rescue of integrated two-gene vector sequences from transformed cells, by recloning in E. coli, was shown to be feasible; thus alterations in transferred DNA can be analysed in detail.

Animals↗
PubMed1992

Identification of a generalised packaging sequence for D-type retroviruses and generation of a D-type retroviral vector.

In order to construct vectors based upon D-type, rather than C-type, retroviruses, we have identified a 624-bp fragment of Mason-Pfizer monkey virus (MPMV) which constitutes a packaging sequence for at least two D-type retroviruses. When this fragment was included in an extensively deleted D-type vector genome, the D-type viruses MPMV and SRV-5, but not the C-type viruses MLV-A or MLV-E, rescued the vector RNA from HeLa cells. The recombinant virus stocks have the host range of the rescuing D-type virus as shown by expression of an internal (SV40-puromycin) cassette replacing the retroviral structural genes. The recombinant MPMV was specifically neutralized by anti-MPMV serum and receptor interference was demonstrated when it was plated on cells productively infected with wild type MPMV. When the putative D-type packaging sequence was removed from the vector genome, even though the other sequence elements required for efficient reverse transcription remained, the vector was no longer rescued from HeLa cells. These results complement the recent demonstration of broad specificity of rescue of a C-type vector (carrying only the packaging sequence of Mo-MLV) by several different C-type, but not D-type, viruses. Replacement of the D-type packaging sequence by most of the extended packaging sequence of Mo-MLV prevented the otherwise D-type vector from being rescued by D-type viruses and did not allow it to be rescued by C-type viruses. This was probably because of the incompatibility of the D-type vector sequences with the C-type retroviral proteins involved in viral reverse transcription and integration. Hence, we have localized a packaging sequence that is recognized by D-type, but not by C-type, retroviruses and have constructed a D-type vector which may be useful in gene transfer experiments.

Betaretrovirus↗
PubMed1991

A system, using neural cell lines, to characterize HSV-1 vectors containing genes which affect neuronal physiology, or neuronal promoters.

Among the potential uses of defective herpes simplex virus (HSV-1) vectors are to study neuronal physiology, neuronal gene regulation, and to perform gene therapy of neuronal diseases. The prototype HSV-1 vector, pHSVlac, stably expresses Escherichia coli beta-galactosidase from the HSV-1 immediate early (IE) 4/5 promoter in cultured rat peripheral and CNS neurons, and in neurons in the adult rat brain. The LacZ gene and the IE 4/5 promoter in pHSVlac can be replaced with genes which affect neuronal physiology or cellular promoters, respectively. A system is required to characterize these HSV-1 vectors; cultured neurons, a mixture of different kinds of neurons and glia, cannot be used. In contrast, neural cell lines represent a homogenous population of neural cells available in virtually unlimited quantities. A system, using neural cell lines, to characterize HSV-1 vectors carrying other genes or promoters is now reported: First, 4 assays are described to detect HSV-1 vector DNA, RNA transcribed from the vector, and to quantitate beta-galactosidase expression. Second, 8 cell lines derived from rodents, primates, and humans were infected with pHSVlac virus and shown to express beta-galactosidase. The cell lines tested included adrenergic and cholinergic mouse neuroblastoma cells, rat pheochromocytoma cells, rodent pituicytes, and human neuroblastoma cells. Infection of these cell lines should prove useful for characterizing HSV-1 vectors with molecular and biochemical assays. Third, differentiated rat pheochromocytoma and mouse neuroblastoma cells, which resemble neurons, were infected with pHSVlac virus and shown to stably express beta-galactosidase. Infection of these cells should be useful for determining the effect of various HSV-1 vectors on neuronal physiology. Thus, HSV-1 vectors containing various genes or promoters can be characterized using the system described in this study.

Animals↗
PubMedDate not supplied

The use of integrating DNA vectors to analyse the molecular defects in ionising radiation-sensitive mutants of mammalian cells including ataxia telangiectasia.

Integrating DNA vectors, encoding selectable recombinant genes, were used to assess rejoining and recombination in wild-type mammalian cells and their ionising radiation-sensitive mutants. To provide a simple model of an important radiation-induced lesion - the DNA double-strand break - the vectors were cut with restriction endonucleases at specific single sites. If these breaks were made in the coding sequence of a selectable gene, the fidelity of the rejoin/recombination process could be measured by survival of vector-transformed cells in selective medium. Rejoining was assessed using vectors without internal homologies, while recombination was measured using pairs of fragments or deletion vectors carrying homologous regions. Initial experiments were made with vectors carrying a single selectable gene but, to overcome potential artefacts, 2-gene vectors were then constructed where one gene acts as a linked marker and (unbroken) control for the other (broken) gene. Available data are reviewed to show that, compared to their respective wild-type counterparts: (1) an ataxia telangiectasia (A-T) cell line and the hamster irs1 mutant show a consistent reduction in the fidelity of rejoining double-strand breaks (while the hamster mutants irs2, irs3, xrs series, and EM9 show wild-type fidelity); (2) the hamster EM9 mutant shows a reduction in ability to recombine homologous vector fragments (while the A-T line and probably the xrs mutants show show wild-type abilities); and (3) the xrs mutants show a reduction in overall transformation frequency with vector DNA, whether broken or not, while the other mutants tested show approximately wild-type frequencies. A critical account of the techniques and data is given, together with speculations on the molecular nature of the processes which are defective in these mutants, leading to radiosensitivity.

Ataxia Telangiectasia↗
PubMed1988

Bunyavirus-vector interactions.

Recent advances in the genetics and molecular biology of bunyaviruses have been applied to understanding bunyavirus-vector interactions. Such approaches have revealed which virus gene and gene products are important in establishing infections in vectors and in transmission of viruses. However, much more information is required to understand the molecular mechanisms of persistent infections of vectors which are lifelong but apparently exert no untoward effect. In fact, it seems remarkable that LAC viral antigen can be detected in almost every cell in an ovarian follicle, yet no untoward effect on fecundity and no teratology is seen. Similarly the lifelong infection of the vector would seem to provide ample opportunity for bunyavirus evolution by genetic drift and, under the appropriate circumstances, by segment reassortment. The potential for bunyavirus evolution by segment reassortment in vectors certainly exists. For example the Group C viruses in a small forest in Brazil seem to constitute a gene pool, with the 6 viruses related alternately by HI/NT and CF reactions, which assay respectively M RNA and S RNA gene products (Casals and Whitman, 1960; Shope and Causey, 1962). Direct evidence for naturally occurring reassortant bunyaviruses has also been obtained. Oligonucleotide fingerprint analyses of field isolates of LAC virus and members of the Patois serogroup of bunyaviruses have demonstrated that reassortment does occur in nature (El Said et al., 1979; Klimas et al., 1981; Ushijima et al., 1981). Determination of the genotypic frequencies of viruses selected by the biological interactions of viruses and vectors after dual infection and segment reassortment is an important issue. Should a virus result that efficiently interacts with alternate vector species, the virus could be expressed in different circumstances with serious epidemiologic consequences. Dual infection of vectors with different viruses is not unlikely, because many bunyaviruses are sympatric in nature. For example, the Ae. trivittatus-cottontail rabbit and the Ae. triseriatus-squirrel arbovirus cycles are sympatric in the ecotone between their respective grassland and forest ecosystems (LeDuc, 1979). Should a LaCrosse virus variant or reassortant evolve that was efficiently vectored by Ae. trivittatus mosquitoes, significantly more human infections with La Crosse virus would likely occur. Unlike Ae. triseriatus, Ae. trivittatus mosquitoes are not restricted to forested areas and consequently are more likely to encounter and to feed upon humans.(ABSTRACT TRUNCATED AT 400 WORDS)

Aedes↗
PubMed1996

The potential of extrachromosomal replicating vectors for gene therapy.

Persistence of DNA vectors in target cells is advantageous in most applications of gene therapy. Particularly when target cells are undergoing proliferation, vector longevity will depend on either the integration of the vector into the chromosomes or the ability of the vector to replicate and be retained extrachromosomally. Vectors that efficiently integrate in a nonrandom fashion are currently unavailable, and those that can replicate extrachromosomally provide a major alternative strategy. Several classes of such vectors are under development, carrying mechanisms for prolonging DNA retention in mammalian nuclei that extend vector lifetime in non-proliferating cells as well. The vectors utilize either chromosomal or viral elements to mediate replication and retention, and have a large size capacity for insertion of genes of interest. I discuss the state of the art for these vectors, including the assets and limitations of their future use in gene therapy.

Animals↗
PubMed1988

A versatile class of positive-selection vectors based on the nonviability of palindrome-containing plasmids that allows cloning into long polylinkers.

Several families of positive-selection cloning vectors were constructed, based on the principle of palindrome nonviability first used by Hagan and Warren [Gene 19 (1982) 147-151]. Each vector, derived from either pBR322 or RSF1010 (a broad-host-range plasmid), contains a long inverted repeat (2 x 366 to 2 x 1008 bp) ending in a symmetrical polylinker. Plasmids with long palindromes are not viable in most strains of Escherichia coli and in at least one Gram-positive bacterium. These palindrome-containing vectors therefore transform such strains at a very low frequency unless a DNA fragment is cloned within the polylinker at the center of the palindrome. Transformation by plasmids lacking an insert is reduced by two to four orders of magnitude. Such vectors can be propagated in a palindrome-tolerant strain; however, long symmetrical deletions then occur within the palindrome. To suppress the resulting deletion derivatives, vectors have been constructed so that an extensive deletion would remove the selectable marker. Alternatively, the vectors can be propagated in any strain of E. coli so long as the palindrome is interrupted by a nonpalindromic DNA fragment. We also present several symmetrical polylinkers and drug-resistance cassettes within the vectors. These components can be interchanged to make new positive-selection vectors as needed, and the cassettes are useful in insertional mutagenesis as well. A general method is described to convert virtually any small or medium-sized plasmid into a positive-selection vector.

Base Sequence↗
PubMed1989

Construction and properties of an Epstein-Barr-virus-derived cDNA expression vector for human cells.

A cDNA expression vector containing the element oriP and the sequence encoding the Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) as well as the hygromycin B-resistance dominant marker gene has been constructed. Its characteristics have been compared to a similar vector lacking the EBV sequences. (a) The EBV+ vector is maintained as an episome with a copy number of approx. 50 per cell, whereas the number of the integrated EBV- copies is in general smaller than 10, when simian virus 40-transformed xeroderma pigmentosum fibroblasts (XP20S-SV) constitute the recipient cell line. (b) The presence of the EBV sequences in the vector resulted in a five- to ten-fold higher transfection efficiency with the Ca.phosphate precipitation technique. (c) cDNA inserts in the EBV+ vector are shown to be efficiently and properly expressed in the recipient cell. (d) If transfection is performed with a mixture of EBV+ vectors with different inserts, transfectants are shown to harbour different plasmids within one cell. (e) The ratio between these plasmids in one cell can be shifted in favour of a vector with a particular insert, when selection for this insert is performed. (f) Reconstruction experiments indicated that isolation of a low-abundance sequence from a mixture of vectors is at least 100-fold more efficient with the EBV+ system, than with the EBV- system. (g) Rescue of the episomal vector from transfected cells can be readily achieved.

Antigens, Viral↗
PubMed1991

A family of Corynebacterium glutamicum/Escherichia coli shuttle vectors for cloning, controlled gene expression, and promoter probing.

A new family of vectors including cloning vectors (pEK0; pEC5), an expression vector (pEKEx1), and promoter probe vectors (pEKpllacZ; pEKplCm), has been constructed. All these shuttle vectors are based on the replication origins of the corynebacterial pBL1 and the Escherichia coli ColE1 plasmids, and thus are able to replicate in Corynebacterium glutamicum and E. coli. Plasmids pEK0 and pEC5 carry multiple restriction sites useful for gene cloning and the kanamycin- or chloramphenicol-resistance-encoding gene from Tn903 or from Tn9, respectively. In C. glutamicum, both vectors are compatible with vectors containing the corynebacterial pHM1519 replicon. Based on plasmid pEK0, the expression vector pEKEx1 was developed to allow for isopropyl-beta-D-thiogalactopyranoside-inducible expression of inserted genes in C. glutamicum and E. coli. Also based on pEK0, the promoter probe vectors pEKpllacZ and pEKplCm were constructed to carry the promoterless lacZ or cat reporter genes downstream from useful cloning sites, for assaying the transcriptional activity of cloned fragments.

Cloning, Molecular↗
PubMed1995

Integrative vectors for heterologous gene expression in Streptomyces spp.

Integrative expression vectors for heterologous expression of the genes in Streptomyces were developed. The vectors are comprised of a strong constitutive promoter, PE, a synthetic ribosome-binding site, ATG start codon, multiple cloning site, transcription terminator and hygromycin-resistance-encoding gene. The vectors also contain a ColE1 replicon for propagation in Escherichia coli and a wide-host-range Streptomyces integration element, the mini-circle, to direct the insertion of the vectors into the Streptomyces genome at the mini-circle attachment site. HyR transformants are stable in the absence of drug selection. Conjugative derivatives were also constructed by incorporating oriT, the origin of transfer of the IncP plasmid RK2, into these vectors, and conjugal transfer was demonstrated from an appropriate E. coli donor to Steptomyces lividans (Sl). Derivatives of these vectors potentially useful for gene disruption, as well as complementation, are also described. Replicative forms of the constructed mini-circle-based vectors in Sl, that co-exist with the integrated copy of the vector, were also present without any apparent instability problems. The utility of the vectors was demonstrated by expression of the gene encoding 31-O-methyltransferase, which is involved in methylation at position 31 of the immunosuppressive drug FK506, in Sl.

Base Sequence↗
PubMed2005

Enhanced long-term expression from helper virus-free HSV-1 vectors packaged in the presence of deletions in genes that modulate the function of VP16, U L 46 and U L 47.

Herpes simplex virus (HSV-1) gene expression is hypothesized to shut off recombinant gene expression from HSV-1 vectors, but in a helper virus-free HSV-1 vector system, a number of promoters support only short-term expression. Thus paradoxically, recombinant gene expression remains short-term in the absence of almost all (approximately 99%) of the HSV-1 genome. To resolve this paradox, we hypothesize that specific HSV-1 proteins that affect the virion can shut off recombinant gene expression. In an earlier study, we examined the effects on recombinant gene expression of five different proteins that affect the HSV-1 virion. We found that vectors packaged in the presence of mutated vhs or U S 11 exhibited minimal changes in gene expression, vectors packaged in the presence of a mutated U S 3 supported improved gene transfer (numbers of cells at 4 days), and vectors packaged in the presence of mutated U L 13 or VP16 supported improved long-term expression. The capability of the VP16 transcriptional complex to reduce gene expression deserves additional study because VP16 is a powerful enhancer that interacts with a number of cellular and viral proteins. In particular, U L 46 and U L 47 are known to modulate the effects of VP16 on immediate early promoters. In this study, we examined expression from a HSV-1 vector that contains a neuronal-specific promoter and was packaged in the presence of deletions in U L 46, or U L 47, or both U L 46 and U L 47. In the rat striatum, each of these vector stocks supported both improved gene transfer (numbers of cells at 4 days) and improved long-term expression (2 months). Vectors packaged in the presence of a deletion in both U L 46 and U L 47 supported larger improvements in gene expression compared to vectors packaged in the presence of deletions in either gene alone. The implications of these results for strategies to improve long-term expression are discussed.

Animals↗
PubMed2005

HIV-based vectors and angiogenesis following rabbit hindlimb ischemia.

BACKGROUND: Numerous medical and surgical options exist for the treatment of vessel ischemia, which some patients fail or cannot tolerate. These investigations were designed to determine the effects of lentiviral-delivered vascular endothelial-derived growth factor (VEGF) and angiopoietin-2 (Ang-2) on collateralization in a rabbit model of hindlimb ischemia. MATERIALS AND METHODS: Self-inactivating human immunodeficiency virus (HIV)-based vectors were constructed encoding VEGF or Ang-2, co-transfected with vesicular stomatitis virus glycoprotein (VSV G) into 293T cells, and vector supernatants (1 x 10(8) IU/ml after concentration) were harvested. New Zealand white rabbits had ligation of either the right or left external iliac artery and excision of the ipsilateral femoral artery. Ten days later, empty, VEGF, or VEGF+Ang-2 vector supernatant was injected intramuscularly (IM) into the ipsilateral thigh. Ankle systolic blood pressure (SBP) ratios were recorded and venous blood samples collected on postoperative days (POD) 10, 25, and 40. On POD 40, run-off angiography was performed to measure vessel collateralization. Capillary density was determined by thin sectioning of muscle. RESULTS: A significant increase was noted in SBP in the VEGF-treated animals over time. Capillary density was not elevated despite significantly increased large vessel collateralization in rabbits receiving VEGF, which was counteracted by Ang-2. Antibodies against vector components were detected in exposed serum. CONCLUSIONS: Arterial collateralization and SBP increased significantly following VEGF vector administration, which was reversed by the Ang-2 vector. Development of antibody against VSV G can limit repeated injections of vector. Future experiments will involve the addition of other pro-angiogenic factors, repeated vector administration, and alternative routes of vector delivery.

Angiopoietin-2↗
PubMed2006

Viral vectors for malaria vaccine development.

A workshop on viral vectors for malaria vaccine development, organized by the PATH Malaria Vaccine Initiative, was held in Bethesda, MD on October 20, 2005. Recent advancements in viral-vectored malaria vaccine development and emerging vector technologies were presented and discussed. Classic viral vectors such as poxvirus, adenovirus and alphavirus vectors have been successfully used to deliver malaria antigens. Some of the vaccine candidates have demonstrated their potential in inducing malaria-specific immunity in animal models and human trials. In addition, emerging viral-vector technologies, such as measles virus (MV), vesicular stomatitis virus (VSV) and yellow fever (YF) virus, may also be useful for malaria vaccine development. Studies in animal models suggest that each viral vector is unique in its ability to induce humoral and/or cellular immune responses. Those studies have also revealed that optimization of Plasmodium genes for mammalian expression is an important aspect of vaccine design. Codon-optimization, surface-trafficking, de-glycosylation and removal of toxic domains can lead to improved immunogenicity. Understanding the vector's ability to induce an immune response and the expression of malaria antigens in mammalian cells will be critical in designing the next generation of viral-vectored malaria vaccines.

Adenoviridae↗