Replication-deficient Vector Research Articles

Accuracy estimation of foamy virus genome copying

BackgroundFoamy viruses (FVs) are the most genetically stable viruses of the retrovirus family. This is in contrast to the in vitro error rate found for recombinant FV reverse transcriptase (RT). To investigate the accuracy of FV genome copying in vivo we analyzed the occurrence of mutations in HEK 293T cell culture after a single round of reverse transcription using a replication-deficient vector system. Furthermore, the frequency of FV recombination by template switching (TS) and the cross-packaging ability of different FV strains were analyzed.ResultsWe initially sequenced 90,000 nucleotides and detected 39 mutations, corresponding to an in vivo error rate of approximately 4 × 10-4 per site per replication cycle. Surprisingly, all mutations were transitions from G to A, suggesting that APOBEC3 activity is the driving force for the majority of mutations detected in our experimental system. In line with this, we detected a late but significant APOBEC3G and 3F mRNA by quantitative PCR in the cells. We then analyzed 170,000 additional nucleotides from experiments in which we co-transfected the APOBEC3-interfering foamy viral bet gene and observed a significant 50% drop in G to A mutations, indicating that APOBEC activity indeed contributes substantially to the foamy viral replication error rate in vivo. However, even in the presence of Bet, 35 out of 37 substitutions were G to A, suggesting that residual APOBEC activity accounted for most of the observed mutations. If we subtract these APOBEC-like mutations from the total number of mutations, we calculate a maximal intrinsic in vivo error rate of 1.1 × 10-5 per site per replication. In addition to the point mutations, we detected one 49 bp deletion within the analyzed 260000 nucleotides.Analysis of the recombination frequency of FV vector genomes revealed a 27% probability for a template switching (TS) event within a 1 kilobase (kb) region. This corresponds to a 98% probability that FVs undergo at least one additional TS event per replication cycle. We also show that a given FV particle is able to cross-transfer a heterologous FV genome, although at reduced efficiency than the homologous vector.ConclusionOur results indicate that the copying of the FV genome is more accurate than previously thought. On the other hand recombination among FV genomes appears to be a frequent event.

Exploring the Chondrocytic Potential of Cranial Neural Crest Cells

Osteoarthritis is a degenerative joint disease where the joint cartilage becomes damaged. Despite the ability of cranial neural crest cells (CNCC) to form cartilage in the embryo, this population of cells has been overlooked when designing cell‐based therapies for osteoarthritis and associated diseases We have begun to explore the potential of CNCC in developing tissues. A LacZ‐encoded replication‐deficient retrovial vector was used to tag explants of CNCC excised from stage 10 chick embryos before injecting into the joint regions of developing limbs of stage 18‐28 embryos. Tagged chondrocytes were subsequently identified in the developing cartilage, particularly in the region of the developing joints. The results appear to demonstrate that cranial neural crest derived cells (CNCDC) retain their ability to give rise to cartilage when implanted into the developing limb and are restricted to particular regions of the limb. The chondrocytic potential of CNCC is now being refined to investigate the integrative properties of these cells in developing and regenerating cartilage. Funded by Brighton & Sussex Medical School.

Neural stem cells target intracranial glioma to deliver an oncolytic adenovirus in vivo

Adenoviral oncolytic virotherapy represents an attractive treatment modality for central nervous system (CNS) neoplasms. However, successful application of virotherapy in clinical trials has been hampered by inadequate distribution of oncolytic vectors. Neural stem cells (NSCs) have been shown as suitable vehicles for gene delivery because they track tumor foci. In this study, we evaluated the capability of NSCs to deliver a conditionally replicating adenovirus (CRAd) to glioma. We examined NSC specificity with respect to viral transduction, migration and capacity to deliver a CRAd to tumor cells. Fluorescence-activated cell sorter (FACS) analysis of NSC shows that these cells express a variety of surface receptors that make them amenable to entry by recombinant adenoviruses. Luciferase assays with replication-deficient vectors possessing a variety of transductional modifications targeted to these receptors confirm these results. Real-time PCR analysis of the replication profiles of different CRAds in NSCs and a representative glioma cell line, U87MG, identified the CRAd-Survivin (S)-pk7 virus as optimal vector for further delivery studies. Using in vitro and in vivo migration studies, we show that NSCs infected with CRAd-S-pk7 virus migrate and preferentially deliver CRAd to U87MG glioma. These results suggest that NSCs mediate an enhanced intratumoral distribution of an oncolytic vector in malignant glioma when compared with virus injection alone.

An avian cell line designed for production of highly attenuated viruses

Several viral vaccines, including highly promising vectors such as modified vaccinia Ankara (MVA), are produced on chicken embryo fibroblasts. Dependence on primary cells complicates production especially in large vaccination programs. With primary cells it is also not possible to create packaging lines for replication-deficient vectors that are adapted to proliferation in an avian host. To obviate requirement for primary cells permanent lines from specific tissues of muscovy duck were derived (AGE1.CR, CS, and CA) and further modified: we demonstrate that stable expression of the structural gene pIX from human adenovirus increases titers for unrelated poxvirus in the avian cells. This augmentation appears to be mediated via induction of heat shock and thus provides a novel cellular substrate that may allow further attenuation of vaccine strains.

Gene Therapy for Bladder Overactivity and Nociception with Herpes Simplex Virus Vectors Expressing Preproenkephalin

Interstitial cystitis/painful bladder syndrome (IC/PBS) is a major challenge to treat. We studied the effect of targeted and localized expression of enkephalin in afferent nerves that innervate the bladder by gene transfer using replication-defective herpes simplex virus (HSV) vectors in a rat model of bladder hyperactivity and pain. Replication-deficient HSV vectors encoding preproenkephalin, which is a precursor for Met- and Leu-enkephalin, or control vector encoding the lacZ reporter gene, were injected into the bladder wall of female rats. After viral vector injection, quantitative polymerase chain reaction showed high preproenkephalin transgene levels in bladder and dorsal root ganglia innervating the bladder in enkephalin vector-treated animals. Functionally, enkephalin vector-treated animals showed reductions in bladder hyperactivity and nociceptive behavior induced by intravesical application of capsaicin; however, vector-mediated expression of enkephalin did not alter normal voiding. This antinociceptive effect of enkephalin gene therapy was antagonized by naloxone hydrochloride administration. Together, our results with HSV vectors encoding preproenkephalin demonstrated physiological improvement in visceral pain induced by bladder irritation. Thus, gene therapy may represent a potentially useful treatment modality for bladder hypersensitive disorders such as IC/PBS.

A Natural Human Retrovirus Efficiently Complements Vectors Based on Murine Leukemia Virus

BackgroundMurine Leukemia Virus (MLV) is a rodent gammaretrovirus that serves as the backbone for common gene delivery tools designed for experimental and therapeutic applications. Recently, an infectious gammaretrovirus designated XMRV has been identified in prostate cancer patients. The similarity between the MLV and XMRV genomes suggests a possibility that the two viruses may interact when present in the same cell.Methodology/Principal FindingsWe tested the ability of XMRV to complement replication-deficient MLV vectors upon co-infection of cultured human cells. We observed that XMRV can facilitate the spread of these vectors from infected to uninfected cells. This functional complementation occurred without any gross rearrangements in the vector structure, and the co-infected cells produced as many as 104 infectious vector particles per milliliter of culture medium.Conclusions/SignificanceThe possibility of encountering a helper virus when delivering MLV-based vectors to human cells in vitro and in vivo needs to be considered to ensure the safety of such procedures.

Atraumatic Oral Spray Immunization with Replication-Deficient Viral Vector Vaccines

The development of needle-free vaccines is one of the recently defined "grand challenges in global health" (H. Varmus, R. Klausner, R. Klausner, R. Zerhouni, T. Acharya, A. S. Daar, and P. A. Singer, Science 302:398-399, 2003). To explore whether a natural pathway to the inductive site of the mucosa-associated lymphatic tissue could be exploited for atraumatic immunization purposes, replication-deficient viral vector vaccines were sprayed directly onto the tonsils of rhesus macaques. Tonsillar immunization with viral vector vaccines encoding simian immunodeficiency virus (SIV) antigens induced cellular and humoral immune responses. Viral RNA levels after a stringent SIV challenge were reduced, providing a level of protection similar to that observed after systemic immunization with the same vaccines. Thus, atraumatic oral spray immunization with replication-deficient vectors can overcome the epithelial barrier, deliver the vaccine antigen to the mucosa-associated lymphatic tissue, and avoid induction of tolerance, providing a novel approach to circumvent acceptability problems of syringe and needle vaccines for children and in developing countries.

Modified Vaccinia Virus Ankara Induces Toll-Like Receptor-Independent Type I Interferon Responses

Modified vaccinia virus Ankara (MVA) is a highly attenuated vaccinia virus strain undergoing clinical evaluation as a replication-deficient vaccine vector against various infections and tumor diseases. To analyze the basis of its high immunogenicity, we investigated the mechanism of how MVA induces type I interferon (IFN) responses. MVA stimulation of bone marrow-derived dendritic cells (DC) showed that plasmacytoid DC were main alpha IFN (IFN-alpha) producers that were triggered independently of productive infection, viral replication, or intermediate and late viral gene expression. Increased IFN-alpha levels were induced upon treatment with mildly UV-irradiated MVA, suggesting that a virus-encoded immune modulator(s) interfered with the host cytokine response. Mice devoid of Toll-like receptor 9 (TLR9), the receptor for double-stranded DNA, mounted normal IFN-alpha responses upon MVA treatment. Furthermore, mice devoid of the adaptors of TLR signaling MyD88 and TRIF and mice deficient in protein kinase R (PKR) showed IFN-alpha responses that were only slightly reduced compared to those of wild-type mice. MVA-induced IFN-alpha responses were critically dependent on autocrine/paracrine triggering of the IFN-alpha/beta receptor and were independent of IFN-beta, thus involving "one-half" of a positive-feedback loop. In conclusion, MVA-mediated type I IFN secretion was primarily triggered by non-TLR molecules, was independent of virus propagation, and critically involved IFN feedback stimulation. These data provide the basis to further improve MVA as a vaccine vector.

Mucosal Vaccine Vectors: Replication-Competent Versus Replication-Deficient Poxviruses

The mucosal surfaces of the respiratory, gastrointestinal, and genitourinary tract are entry points for a variety of pathogens and they serve as the first line of defense against infection. To prevent transmission of mucosal pathogens, it is often necessary to target the vaccine to the mucosal surface. Viral vectors, such as poxviruses expressing gene inserts, capable of overcoming the formidable array of host defenses at the mucosal surfaces, are an attractive vaccination strategy for mucosal immunization against infectious diseases. Replication-competent vectors, such as vaccinia, are highly effective, but have a number of safety concerns that may limit their widespread use in humans. In contrast, replication-deficient vectors, such as modified vaccinia ankara (MVA), ALVAC, and NYVAC, may be safer vectors than live vectors, but these vectors may not be as effective at inducing immune responses. Co-delivery of viral vectors with genetic adjuvants (cytokines) or deletion of certain immunomodulatory genomic sequences from these viral vectors may increase vaccine efficiency and are promising strategies for a new generation of mucosal vaccines.

Replication-deficient mutant Herpes Simplex Virus-1 targets professional antigen presenting cells and induces efficient CD4 + T helper responses

Both neutralizing antibodies and cytotoxic T-cells are necessary to control a viral infection. However, vigorous T helper responses are essential for their elicitation and maintenance. Here we show that a recombinant replication-deficient Herpes Simplex Virus (HSV)-1 vector encoding the Human Immunodeficiency Virus (HIV)-1 matrix protein p17 (T0-p17) was capable of infecting professional antigen presenting cells (APCs) in vitro and in vivo. The injection of T0-p17 in the mouse dermis generated a strong p17-specific CD4 + T helper response preceding both p17-specific humoral and effector T cell responses. Moreover, we show that T0-p17 infection did not interfere with the endogenous processing of the transgene encoded antigen, since infected APCs were able to evoke a strong recall response in vitro. Our results demonstrate that replication-deficient HSV vectors can be appealing candidates for the development of vaccines able to trigger T helper responses.

Development of immunostimulatory virotherapy using non-transmissible Sendai virus-activated dendritic cells

Dendritic cell (DC)-based immunotherapy has been clinically evaluated, however, still requires modification to improve its outcomes. We previously demonstrated that DCs activated by replication competent recombinant Sendai virus (SeV) showed dramatic efficacy over that seen in use of current DC vaccine for immunotherapy against malignancies; however, application of replication-deficient vector is more relevant in clinical setting. We here show that F-gene-deleted non-transmissible Sendai virus (SeV/dF)-activated DCs (DCs/SeV/dF) has strong antitumor effects against murine SCCVII tumor, that was well-known as a less immunogenic cell line. SeV/dF shows high transfection efficiency to DCs and leads them to upregulate costimulatory molecules. Intratumoral injection of DCs/SeV/dF resulted in a marked and representative inhibition of the tumor, even when the tumors were well-vascularized. This is the first demonstration that non-transmissible SeV vector, SeV/dF, could be a DC-activator; DC/SeV/dF-based cancer immunotherapy may, therefore, warrant further investigation.

Armed Interference: Oncolytic Viruses Engineered to Carry Antitumor shRNAs

A diverse array of oncolytic viruses are being developed for the treatment of cancer.1, 2 These therapeutic agents are naturally and/or genetically targeted to replicate selectively in cancer cells. The resulting "oncolysis" is a novel mechanism of action (MOA) for cancer treatment and seems in many cases to be effective against apoptosis-resistant cells. In addition to this primary MOA, oncolytic viruses can demonstrate secondary MOAs such as induction of tumor-specific cytotoxic T lymphocytes,3 anti-angiogenic cytokines,4 and chemosensitization.5

Degradation of Fibrillar Collagen in a Human Melanoma Xenograft Improves the Efficacy of an Oncolytic Herpes Simplex Virus Vector

Oncolytic viral therapy provides a promising approach to treat certain human malignancies. These vectors improve on replication-deficient vectors by increasing the viral load within tumors through preferential viral replication within tumor cells. However, the inability to efficiently propagate throughout the entire tumor and infect cells distant from the injection site has limited the capacity of oncolytic viruses to achieve consistent therapeutic responses. Here we show that the spread of the oncolytic herpes simplex virus (HSV) vector MGH2 within the human melanoma Mu89 is limited by the fibrillar collagen in the extracellular matrix. This limitation seems to be size specific as nanoparticles of equivalent size to the virus distribute within tumors to the same extent whereas smaller particles distribute more widely. Due to limited viral penetration, tumor cells in inaccessible regions continue to grow, remaining out of the range of viral infection, and tumor eradication cannot be achieved. Matrix modification with bacterial collagenase coinjection results in a significant improvement in the initial range of viral distribution within the tumor. This results in an extended range of infected tumor cells and improved virus propagation, ultimately leading to enhanced therapeutic outcome. Thus, fibrillar collagen can be a formidable barrier to viral distribution and matrix-modifying treatments can significantly enhance the therapeutic response.

Feline foamy virus-mediated marker gene transfer: Identification of essential genetic elements and influence of truncated and chimeric proteins

Retroviral vectors derived from foamy or spumaretroviruses are considered promising tools for targeted gene delivery and vaccination purposes. In order to fully exploit this potential, we identified essential cis-acting sequences on the feline foamy virus (FFV) genome by constructing and analyzing a series of FFV-based replication-deficient vector genomes. Cis-acting sequences essentially required for marker gene transfer were found to be localized at two sites on the FFV genome: (i) in the 5′-untranslated region and close to the gag ATG and (ii) in the central part of the pol gene. The presence of two cis-acting sequences and their relative location on the FFV genome are similar but not identical to the functionally corresponding elements described for simian and primate foamy viruses.

Human Adenoviruses: Propagation, Purification, Quantification, and Storage

Detailed protocols are described for the propagation of adenoviruses (Ads) and adenovirus (Ad) vectors and their purification by CsCl equilibrium density gradient centrifugation. A discussion of monolayer and spinner cell culture techniques suitable, respectively, for small- and large-scale growth of adenoviruses is provided. Protocols for cloning into and growth of Ad replication-deficient vectors using a convenient commercially available system are described. Lastly, time-tested plaque titration protocols for the accurate and convenient measurement of the infectivity of adenoviruses and adenovirus vectors are provided in detail.

In Vivo Administration of Replication-Deficient Mutant HSV-1 Targets Professional APCs and Induces Efficient CD4+ T Helper Responses

Both neutralizing antibodies and cytotoxic T cells are necessary to control a viral infection. However, vigorous T helper responses are essential for their elicitation and maintenance. These findings have critical implications in the design of vaccination strategies aimed at triggering and sustaining antigen specific CD4+ in addition to CD8+ effector immune responses. Here we show that a recombinant replication-deficient HSV-1 vector encoding the HIV-1 matrix protein p17 (T0-p17) is capable to infect professional APCs in vitro and in vivo without interfering with the endogenous MHC class II processing of the transgene encoded antigen. Moreover, we show that injection of T0-p17 in the mouse dermis generates a strong p17specific CD4+ T helper response preceding both cytotoxic and humoral responses. Importantly, T0-p17 infected peritoneal macrophages were capable to trigger a longlasting expansion of p17-specific CD4+ T cells in vitro. Because of their capability to infect professional APCs without interfering with their biological functions, replication-deficient HSV vectors are appealing candidates for the development of vaccines able to trigger strong T helper responses. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005

Liver-Directed Gamma Interferon Gene Delivery in Chronic Hepatitis C

Gamma interferon (IFN-gamma) has been shown to inhibit replication of subgenomic and genomic hepatitis C virus (HCV) RNAs in vitro and to noncytolytically suppress hepatitis B virus (HBV) replication in vivo. IFN-gamma is also known for its immunomodulatory effects and as a marker of a successful cellular immune response to HCV. Therapeutic expression of IFN-gamma in the liver may therefore facilitate resolution of chronic hepatitis C, an infection that is rarely resolved spontaneously. To analyze immunomodulatory and antiviral effects of liver-specific IFN-gamma expression in vivo, we intravenously injected two persistently HCV-infected chimpanzees twice with a recombinant, replication-deficient HBV vector and subsequently with a recombinant adenoviral vector. These vectors expressed human IFN-gamma under control of HBV- and liver-specific promoters, respectively. Gene transfer resulted in a transient increase of intrahepatic IFN-gamma mRNA, without increase in serum alanine aminotransferase levels. Ex vivo analysis of peripheral blood lymphocytes demonstrated enhanced CD16 expression on T cells and upregulation of the liver-homing marker CXCR3. Moreover, an increased frequency of HCV-specific T cells was detected ex vivo in the peripheral blood and in vitro in liver biopsy-derived, antigen-nonspecifically expanded T-cell lines. None of these immunologic effects were observed in the third chimpanzee injected with an HBV control vector. Despite these immunologic effects of the experimental vector, however, IFN-gamma gene transfer did not result in a significant and long-lasting decrease of HCV titers. In conclusion, liver-directed IFN-gamma gene delivery resulted in HCV-specific and nonspecific activation of cellular immune responses but did not result in effective control of HCV replication.

AdEasy-based cloning system to generate tropism expanded replicating adenoviruses expressing transgenes late in the viral life cycle

Replicating adenoviral vectors (RAds) hold great promise for the treatment of cancer. Significant therapeutic effects of these vectors do not only rely on tumor targeting but also on efficient release of viral progeny from host cells. Cytotoxic genes expressed late in the adenoviral life cycle can significantly enhance viral release and spreading. Therefore, an adenoviral cloning system that allows easy integration of established tumor targeting techniques together with late expression of transgenes can be a valuable tool for the development of RAds. We expanded the features of the widely used AdEasy adenoviral cloning system toward the production of tropism modified replicating adenoviral vectors that express transgenes late in the viral life cycle. Three vectors (pIRES, pFIBER and pAdEasy-Sce) that facilitate easy manipulation of the adenoviral fiber region were established. Unique BstBI and I-Sce-1 restriction sites facilitate the introduction of retargeting peptides in the fiber HI-loop and of genes of interest in the fiber transcription unit. We validated the system by constructing an E1-positive adenovirus with an RGD motif in the fiber HI-loop and green fluorescent protein (GFP) expressed from the fiber transcription unit (AdDelta24Fiber-rgd-GFP). Additionally, assessment of E1-negative replication-deficient vectors confirmed strict dependence upon E1 expression for the expression of transgenes inserted into the fiber transcription unit. This flexible cloning system allows for straightforward construction of tropism expanded replicating adenoviral vectors that express transgenes late in the adenoviral life cycle.

Protection against P. aeruginosa with an adenovirus vector containing an OprF epitope in the capsid

Pseudomonas aeruginosa is an important opportunistic pathogen that can cause chronic and often life-threatening infections of the respiratory tract, particularly in individuals with cystic fibrosis (CF). Because infections with P. aeruginosa remain the major cause of the high morbidity and mortality of CF, a vaccine against P. aeruginosa would be very useful for preventing this disorder. The outer membrane protein F (OprF) of P. aeruginosa is a promising vaccine candidate and various B cell epitopes within OprF have been identified. Given that adenovirus (Ad) vectors have strong immunogenic potential and can function as adjuvants for genetic vaccines, the present study evaluates the immunogenic and protective properties of a novel replication-deficient Ad vector in which the Ad hexon protein was modified to include a 14-amino acid epitope of P. aeruginosa OprF (Epi8) in loop 1 of the hypervariable region 5 of the hexon (AdZ.Epi8). Immunization of C57BL/6 mice with AdZ.Epi8 resulted in detectable serum anti-P. aeruginosa and anti-OprF humoral responses. These responses were haplotype dependent, with higher serum anti-OprF titers in CBA mice than in BALB/c or C57BL/6 mice. AdZ.Epi8 induced Epi8-specific IFN-gamma-positive CD4 and CD8 T cell responses and resulted in protection against a lethal pulmonary challenge with agar-encapsulated P. aeruginosa. Importantly, repeated administration of AdZ.Epi8 resulted in boosting of the anti-OprF humoral and anti-Epi8 cellular response, whereas no boosting effect was present in the response against the transgene beta-galactosidase. These observations suggest that Ad vectors expressing pathogen epitopes in their capsid will protect against an extracellular pathogen and will allow boosting of the epitope-specific humoral response with repeated administration, a strategy that should prove useful in developing Ad vectors as vaccines where humoral immunity will be protective.

Induction of human histo-blood group A antigen expression in mouse cells by gene therapy using lentiviral vectors harbouring human ABH-related glycosyltransferase genes

We recently discovered that ABO incompatibility, which evokes a rapid humoral immune response in adult heart transplantation, is not a barrier in infant heart transplantation, and that infant recipients of ABO-incompatible hearts develop specific B-cell tolerance to donor A/B antigens. An animal model of ABO-incompatible heart transplantation would allow detailed investigation of the mechanism(s) of acceptance of ABO-incompatible grafts, using experimental methods that would not be possible in humans. To determine the feasibility of such a model, the human α-1,2-fucosyltransferase (H-transferase; for H antigen expression) gene was cloned into a lentiviral vector, and the human α-1,3-N-acetylgalactosaminyltransferase (A-transferase; for A antigen expression) gene was cloned into a bicistronic lentiviral vector also containing the green fluorescent protein (GFP) gene; these replication-deficient vectors were denoted H-trs and A-GFP, respectively. Synthesis of the human histo-blood group A antigen in humans is dependent on expression of these two glycosyltransferases. HeLa cells, a human cell line known to be of blood group O origin, expressed cell surface A antigen as measured by cellular ELISA when transfected with A-GFP alone, and the level of A antigen expression was enhanced by transfection with H-trs in addition to A-GFP. Cell surface H antigen expression was observed on both mouse fibroblast and mouse endothelial cells only when infected with H-trs lentiviral particles. Expression of A antigen was dependent on infection with both H-trs and A-GFP lentiviral particles, approaching levels on human group A cells. These collective results indicate that expression of human histo-blood group A antigen at the cell surface can be induced in mouse cells by infection with H-trs and A-GFP, and that such A antigen expression is dependent on H-trs expression.