Replication-defective Viral Vectors Research Articles

Oncolytic Viruses: An Inventory of Shedding Data from Clinical Trials and Elements for the Environmental Risk Assessment.

Attenuated and/or genetically modified oncolytic viruses (OV) gain increasing interest as a promising approach for cancer therapy. Beside the assessment of subject safety, quality and efficacy aspects of medicinal products for human use, genetically modified viruses are also governed by EU regulatory frameworks requiring an environmental risk assessment (ERA). An important element to be assessed as part of the ERA is the incidence of exposure to OV of individuals, other than the trial subjects, and the environment. The evidence-based evaluation of shedding data is considered to be decisive in that context, as it may impact the OV capacity to be transmitted. This is particularly true for OV still able to (conditionally) replicate as opposed to replication-defective viral vectors commonly used in gene therapy or vaccination. To our knowledge, this article presents the most extensive and up-to-date review of shedding data reported with OV employed in clinics. Besides the identification of a topical need for improving the collection of shedding data, this article aims at providing an aid to the design of an appropriate shedding study, thereby relying on and further complementing principles described in existing guidelines issued by European and international institutions.

Protection of SARS-CoV-2 trial vaccines in human is a functnion of the viral genomes

Abstract Objectives Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has devastated mankind with complaints that many vaccines developed against the disease could not offer protection and guarantee safety, hence many vaccinated individuals either came down with the infection and died or transmitted the infection. Therefore permanent immunization may become difficult due to interplay between the viral and human genomes. Methods In view of this, literatures were searched on the trial vaccines against SARS-CoV-2.Concentration log10 (copies/ml) of the virus, log10 viral copies, populations of human vaccinated, protection indices of the vaccines, population of Tcells, lethal concentration 1 (LC1) of the virus, doses of the trial vaccines and vaccine regimens of the SARS-CoV-2 obtained from membrane ribonucleic acid (MRNA), replication defective viral vector (RDVV), inactivated pathogen vaccine (IPV), and protein subunit vaccine (PSV),virus like particle (VLP) and deoxyribonucleic acid vaccine (DNAV) were used with an intent to assessing the pathogenicity and virulence of the vaccines. Key findings Findings have shown that virion of 3.3 x10 8-9 could kill human over a period of ≥20 days, and 10 9-11virions have killed three in every 100 humans. However, viral load of detection (3.22 x 10 3), positivity threshold (3.3 x 10 3) and index patient value (6.6 x 10 6) respectively have been established. The protection index is between 11-99%. Conclusion The viral load of coronavirus found in the affected patients was relatively high and could be fatal. However DNAV based vaccine (2 mg) administered twice 4 weeks apart provided the best protection index that lasted for 40 – 60.

Immunogenicity and Safety Outcome of Homologous and Heterologous Prime-Boost of Inactivated Vaccine and Replication-Defective Viral Vectors Vaccine Against SARS-CoV-2 Among Hemodialysis Patients

Immunogenicity and Safety Outcome of Homologous and Heterologous Prime-Boost of Inactivated Vaccine and Replication-Defective Viral Vectors Vaccine Against SARS-CoV-2 Among Hemodialysis Patients

IDO1, FAT10, IFI6, and GILT Are Involved in the Antiretroviral Activity of γ-Interferon and IDO1 Restricts Retrovirus Infection by Autophagy Enhancement.

Gamma-interferon (γ-IFN) significantly inhibits infection by replication-defective viral vectors derived from the human immunodeficiency virus type 1 (HIV-1) or murine leukemia virus (MLV) but the underlying mechanism remains unclear. Previously we reported that knockdown of γ-IFN-inducible lysosomal thiolreductase (GILT) abrogates the antiviral activity of γ-IFN in TE671 cells but not in HeLa cells, suggesting that other γ-IFN-inducible host factors are involved in its antiviral activity in HeLa cells. We identified cellular factors, the expression of which are induced by γ-IFN in HeLa cells, using a microarray, and analyzed the effects of 11 γ-IFN-induced factors on retroviral vector infection. Our results showed that the exogenous expression of FAT10, IFI6, or IDO1 significantly inhibits both HIV-1- and MLV-based vector infections. The antiviral activity of γ-IFN was decreased in HeLa cells, in which the function of IDO1, IFI6, FAT10, and GILT were simultaneously inhibited. IDO1 is an enzyme that metabolizes an essential amino acid, tryptophan. However, IDO1 did not restrict retroviral vector infection in Atg3-silencing HeLa cells, in which autophagy did not occur. This study found that IDO1, IFI6, FAT10, and GILT are involved in the antiviral activity of γ-IFN, and IDO1 inhibits retroviral infection by inducing autophagy.

SARS-CoV-2 Vaccines in Kidney Transplant Recipients: Will They Be Safe and Effective and How Will We Know?

SARS-CoV-2 Vaccines in Kidney Transplant Recipients: Will They Be Safe and Effective and How Will We Know?

Viral and other therapies for recurrent glioblastoma: is a 24-month durable response unusual?

A phase I trial of an engineered poliovirus for the treatment of recurrent glioblastoma (GBM) has attracted attention due to 8 survivors reaching the 24-month and 5 reaching the 36-month survival landmarks.1 Genetically engineered viruses (oncolytic viruses) have been in trials for GBM for almost two decades.2 These replication-competent (tumor-selective, oncolytic, replication-conditional) viruses or replication-defective viral vectors (gene therapy) deliver cytotoxic payloads to tumors, leading to immunogenic death and intratumoral inflammatory responses. This transforms the tumor microenvironment from immunologically naïve ("cold") to inflamed ("hot"), increasing immune cell recognition of tumor antigens and the durable responses observed in virotherapy.3,4 Several current and past virotherapy trials have reported a "tail" of apparent responders at the 24-month landmark. Other modalities have also reported a "tail" of seemingly long-term survivors. These trials seem to show that these responder "tails" characterize a defined subset of GBM patients.

Expression and role of VLA-1 in resident memory CD8 T cell responses to respiratory mucosal viral-vectored immunization against tuberculosis

Lung resident memory T cells (TRM) characterized by selective expression of mucosal integrins VLA-1 (α1β1) and CD103 (αEβ7) are generated following primary respiratory viral infections. Despite recent progress, the generation of lung TRM and the role of mucosal integrins following viral vector respiratory mucosal immunization still remains poorly understood. Here by using a replication-defective viral vector tuberculosis vaccine, we show that lung Ag-specific CD8 T cells express both VLA-1 and CD103 following respiratory mucosal immunization. However, VLA-1 and CD103 are acquired in differential tissue sites with the former acquired during T cell priming in the draining lymph nodes and the latter acquired after T cells entered the lung. Once in the lung, Ag-specific CD8 T cells continue to express VLA-1 at high levels through the effector/expansion, contraction, and memory phases of T cell responses. Using a functional VLA-1 blocking mAb, we show that VLA-1 is not required for trafficking of these cells to the lung, but it negatively regulates them in the contraction phase. Furthermore, VLA-1 plays a negligible role in the maintenance of these cells in the lung. Our study provides new information on vaccine-inducible lung TRM and shall help develop effective viral vector respiratory mucosal tuberculosis vaccination strategies.

Regulable Transgene Expression in Dorsal Root Ganglia of a Replication-Defective Herpes Simplex Virus Type 1 Vector by Means of Sciatic Nerve Injection.

Targeted and controllable gene delivery to neurons is essential to efforts to facilitate peripheral nerve regeneration. The authors investigated both the in vitro and in vivo expression profiles of a tetracycline-controlled, replication-defective, herpes simplex virus type 1-based vector. Mouse primary dorsal root ganglia cells were infected with QR9TO-LacZ in the absence or presence of tetracycline. LacZ gene expression was examined. It was also injected into sciatic nerves in CD-1 mice fed with and without tetracycline. LacZ expression in the upstream dorsal root ganglia was examined. Following inoculation with QR9TO-LacZ, approximately 40 percent of the cultured primary dorsal root ganglia cells exhibited strong LacZ activity in the presence of tetracycline at 48 and 72 hours, whereas little was detected in those in the absence of tetracycline. Quantitative analysis revealed that the β-galactosidase activity within cells exposed to tetracycline increased 181-fold at 48 hours (p < 0.001) and 47-fold at 72 hours after infection (p < 0.05) compared with those without tetracycline. However, this LacZ transgene activity in the presence of tetracycline tapered off to less than sevenfold over baseline 168 hours after infection (p < 0.05). Furthermore, successful uptake of this replication-defective viral vector was evident in upstream dorsal root ganglia after sciatic nerve injection in mice. In addition, its expression profile was similar to that in vitro, as strong β-galactosidase activity was evident only in mice fed with a doxycycline-containing diet, and it tapered off by 168 hours. The replication-defective herpes simplex virus type 1-based vector, which provides tightly regulated transgene expression in dorsal root ganglia by means of peripheral nerve injection, represents an appealing approach to improve peripheral nerve regeneration.

Cytomegalovirus Vaccines

An effective cytomegalovirus (CMV) vaccine could prevent the majority of birth defects caused by congenital CMV infections. Candidate vaccines in clinical evaluation include live attenuated, protein subunit, DNA, and viral-vectored approaches. Subunit approaches have focused on the CMV proteins pp65 and IE1 as important inducers of cytotoxic T cells and glycoprotein B (gB) as an important inducer of neutralizing antibodies. A vaccine comprised of recombinant gB protein with MF59 adjuvant reduced the incidence of primary infection by 50%. Recent revelations regarding CMV entry pathways into different cell types suggest a possible course for improvement. A 5-subunit pentameric complex is uniquely required for endothelial and epithelial cell entry. Sera from naturally infected subjects contain high-potency neutralizing activities specific for this complex, whereas the gB/MF59 vaccine fails to induce comparable neutralizing activities. A vaccine's ability to induce salivary antibodies that neutralize epithelial cell entry may be especially important for preventing oral transmission as the first cells infected are presumably epithelial cells of the oral mucosa. In addition, recent evidence suggests that antibodies can inhibit postentry CMV spread between endothelial and epithelial cells. Such activities may serve to limit viral replication in tissues or impair dissemination to the placenta and fetus. Thus, inclusion of epitopes derived from the pentameric complex may provide enhanced efficacy by inducing potent neutralizing/spread-inhibiting antibodies that target virus replication in a broad spectrum of cell types. Next-generation vaccine candidates in preclinical development incorporate peptides, subunits, or multisubunit complexes representing parts or all of the pentameric complex. Approaches include peptides, recombinant proteins, DNA, replication-defective viral vectors, genetically disabled CMV, and inactivated CMV virions. The diversity of novel strategies under development engenders optimism that a successful candidate will emerge.

Quantitative RT-PCR for titration of replication-defective recombinant Semliki Forest virus

Virus titration may constitute a drawback in the development and use of replication-defective viral vectors like Semliki Forest virus (SFV). The standardization and validation of a reverse transcription quantitative PCR (qRT-PCR) method for SFV titration is presented here. The qRT-PCR target is located within the nsp1 gene of the non-structural polyprotein SFV region (SFV RNA), which allows the strategy to be used for several different recombinant SFV constructs. Titer determinations were carried out by performing virus titration and infection assays with SFVs containing an RNA coding region for the rabies virus glycoprotein (RVGP) or green fluorescent protein (GFP). Results showed that the standardized qRT-PCR is applicable for different SFV constructs, and showed good reproducibility. To evaluate the correlation between the amount of functional SFV RNA in a virus lot and its infectivity in BHK-21 cell cultures, a temperature mediated titer decrease was performed and successfully quantitated by qRT-PCR. When used for cell infection at the same multiplicity of infection (MOI), the temperature treated SFV-RVGP samples induced the same levels of RVGP expression. Similarly, when different SFV-GFP lots with different virus titers, as accessed by qRT-PCR, were used for cell infection at the same MOI, the cultures showed comparable amounts of fluorescent cells. The data demonstrate a good correlation between the amount of virus used for infection, as measured by its SFV RNA, and the protein synthesis in the cells. In conclusion, the qRT-PCR method developed here is accurate and enables the titration of replication-defective SFV vectors, an essential aid for viral vector development as well as for establishment of production bioprocesses.

Evaluation of the immune response elicited by vaccination with viral vectors encoding FMDV capsid proteins and boosted with inactivated virus

The aim of the present study was to assess the effect of introducing a priming step with replication-defective viral vectors encoding the capsid proteins of FMDV, followed by a boost with killed virus vaccines, using a suitable BALB/c mice model. Additionally, the immune response to other combined vector immunization regimens was studied. For this purpose, we analyzed different prime-boost immunizations with recombinant adenovirus (Ad), herpesvirus amplicons (Hs) and/or killed virus (KV) vaccines. The highest antibody titers were found in the group that received two doses of adjuvanted KV (P<0.002). Antibody titers were higher in those groups receiving a mixed regimen of vectors, compared to immunization with either vector alone (P<0.0001). Priming with any of the viral vectors induced a shift of the cytokine balance toward a Th1 type immune response regardless of the delivery system used for boosting. The highest IgG1 titer was induced by two doses of adjuvanted KV (P=0.0002) and the highest IgG2a titer corresponded to the group primed with Ad and boosted with KV (P=0.01). Re-stimulation of all groups of mice with 0.5 μg of inactivated virus five months later resulted in a fast increase of antibody titers in all the groups tested. After virus stimulation, antibody titers in the groups that received KV alone or Ad prime-KV boost, were indistinguishable (P=0.800). Protection from challenge was similar (75%) in the groups of animals that received Ad prime-Hs boost or Ad prime-KV boost, or two doses of oil-adjuvanted KV. The data presented in this study suggest that sequential immunization with viral vectors-based vaccines combined with protein-based vaccines have the potential to enhance the quality of the immune response against FMDV.

Synthetic riboswitches for external regulation of genes transferred by replication-deficient and oncolytic adenoviruses

Therapeutic gene transfer by replication-defective viral vectors or, for cancer treatment, by replication-competent oncolytic viruses shows high promise for treatment of major diseases. To ensure safety, timing or dosing in patients, external control of therapeutic gene expression is desirable or even required. In this study, we explored the potential of artificial aptazymes, ligand-dependent self-cleaving ribozymes, as an innovative tool for regulation of therapeutic gene expression. Importantly, aptazymes act on RNA intrinsically, independent of regulatory protein–nucleic acid interactions and stoichiometry, are non-immunogenic and of small size. These are key advantages compared with the widely used inducible promoters, which were also reported to lose regulation at high copy numbers, e.g. after replication of oncolytic viruses. We characterized aptazymes in therapeutic gene transfer utilizing adenovectors (AdVs), adeno-associated vectors (AAVs) and oncolytic adenoviruses (OAds), which are all in advanced clinical testing. Our results show similar aptazyme-mediated regulation of gene expression by plasmids, AdVs, AAVs and OAds. Insertion into the 5′-, 3′- or both untranslated regions of several transgenes resulted in ligand-responsive gene expression. Notably, aptazyme regulation was retained during OAd replication and spread. In conclusion, our study demonstrates the fidelity of aptazymes in viral vectors and oncolytic viruses and highlights the potency of riboswitches for medical applications.

Evaluation of biolistic gene transfer methods in vivo using non-invasive bioluminescent imaging techniques

BackgroundGene therapy continues to hold great potential for treating many different types of disease and dysfunction. Safe and efficient techniques for gene transfer and expression in vivo are needed to enable gene therapeutic strategies to be effective in patients. Currently, the most commonly used methods employ replication-defective viral vectors for gene transfer, while physical gene transfer methods such as biolistic-mediated ("gene-gun") delivery to target tissues have not been as extensively explored. In the present study, we evaluated the efficacy of biolistic gene transfer techniques in vivo using non-invasive bioluminescent imaging (BLI) methods.ResultsPlasmid DNA carrying the firefly luciferase (LUC) reporter gene under the control of the human Cytomegalovirus (CMV) promoter/enhancer was transfected into mouse skin and liver using biolistic methods. The plasmids were coupled to gold microspheres (1 μm diameter) using different DNA Loading Ratios (DLRs), and "shot" into target tissues using a helium-driven gene gun. The optimal DLR was found to be in the range of 4-10. Bioluminescence was measured using an In Vivo Imaging System (IVIS-50) at various time-points following transfer. Biolistic gene transfer to mouse skin produced peak reporter gene expression one day after transfer. Expression remained detectable through four days, but declined to undetectable levels by six days following gene transfer. Maximum depth of tissue penetration following biolistic transfer to abdominal skin was 200-300 μm. Similarly, biolistic gene transfer to mouse liver in vivo also produced peak early expression followed by a decline over time. In contrast to skin, however, liver expression of the reporter gene was relatively stable 4-8 days post-biolistic gene transfer, and remained detectable for nearly two weeks.ConclusionsThe use of bioluminescence imaging techniques enabled efficient evaluation of reporter gene expression in vivo. Our results demonstrate that different tissues show different expression kinetics following gene transfer of the same reporter plasmid to different mouse tissues in vivo. We evaluated superficial (skin) and abdominal organ (liver) targets, and found that reporter gene expression peaked within the first two days post-transfer in each case, but declined most rapidly in the skin (3-4 days) compared to liver (10-14 days). This information is essential for designing effective gene therapy strategies in different target tissues.

Loss of the Brm-Type SWI/SNF Chromatin Remodeling Complex Is a Strong Barrier to the Tat-Independent Transcriptional Elongation of Human Immunodeficiency Virus Type 1 Transcripts

To elucidate the epigenetic regulation of Tat-independent human immunodeficiency virus (HIV) transcription following proviral integration, we constructed an HIV type 1 (HIV-1)-based replication-defective viral vector that expresses a reporter green fluorescent protein (GFP) product from its intact long terminal repeat (LTR). We transduced this construct into human tumor cell lines that were either deficient in or competent for the Brm-type SWI/SNF complex. One day after transduction, single cells that expressed GFP were sorted, and the GFP expression profiles originating from each of these clones were analyzed. Unlike clones of the SWI/SNF-competent cell line, which exhibited clear unimodal expression patterns in all cases, many clones originating from Brm-deficient cell lines either showed a broad-range distribution of GFP expression or were fully silenced. The resorting of GFP-negative populations of these isolated clones showed that GFP silencing is either reversible or irreversible depending upon the proviral integration sites. We further observed that even in these silenced clones, proviral gene transcription initiates to accumulate short transcripts of around 60 bases in length, but no elongation occurs. We found that this termination is caused by tightly closed nucleosome-1 (nuc-1) at the 5' LTR. Also, nuc-1 is remodeled by exogenous Brm in some integrants. From these results, we propose that Brm is required for the occasional transcriptional elongation of the HIV-1 provirus in the absence of Tat. Since the Brm-type SWI/SNF complex is expressed at marginal levels in resting CD4+ T cells and is drastically induced upon CD4+ T-cell activation, we speculate that it plays crucial roles in the early Tat-independent phase of HIV transcription in affected patients.

Recombinant Viral Vaccines Expressing Merozoite Surface Protein-1 Induce Antibody- and T Cell-Mediated Multistage Protection against Malaria

SummaryProtecting against both liver and blood stages of infection is a long-sought goal of malaria vaccine design. Recently, we described the use of replication-defective viral vaccine vectors expressing the malaria antigen merozoite surface protein-1 (MSP-1) as an antimalarial vaccine strategy that elicits potent and protective antibody responses against blood-stage parasites. Here, we show that vaccine-induced MSP-1-specific CD4+ T cells provide essential help for protective B cell responses, and CD8+ T cells mediate significant antiparasitic activity against liver-stage parasites. Enhanced survival is subsequently seen in immunized mice following challenge with sporozoites, which mimics the natural route of infection more closely than when using infected red blood cells. This effect is evident both in the presence and absence of protective antibodies and is associated with decreased parasite burden in the liver followed by enhanced induction of the cytokine IFN-γ in the serum. Multistage immunity against malaria can thus be achieved by using viral vectors recombinant for MSP-1.

Towards an effective genital herpes vaccine: past lessons and future prospects.

In 2007, the most effective means to acquire life-long immunity to genital herpes is to engage in romantic activity with a partner who is infected with herpes simplex virus 2 (HSV-2). Three out of four people who acquire HSV-2 in this manner are blissfully unaware of the molecular hitchhikers that they will carry for life, hidden away in neurons of their peripheral nervous system. Such persons derive a huge benefit from these molecular hitchhikers: life-long immunity from the disease of genital herpes. The natural approach to acquiring HSV-2 has a serious downside: 2 to 5% of HSV-2 infected people endure considerable pain and distress, as they may experience recurrent outbreaks of genital herpes every three to twelve months for the rest of their lives. About 1.5 billion people worldwide are infected with HSV-2, and ~50 million suffer from recurrent outbreaks of genital herpes. It is absurd to suggest that unprotected sex is the best means to vaccinate against HSV-2 and genital herpes. Yet, the sad reality in 2007 is that the medical community has about as much power to prevent genital herpes as doctors did who practiced in ancient Rome. The development of acyclovir in the late 1970s was a major breakthrough. It has provided a tool by which doctors and patients can restrict the severity and frequency of genital herpes outbreaks. However, a preventative vaccine that confers life-long protection against genital herpes continues to elude us. Tens of thousands of studies have been published on herpes simplex virus 1 (HSV-1) and HSV-2 in the past fifty years. These studies provide a wealth of information about the epidemiology, clinical presentation, immunology, molecular biology, and animal biology of HSV-1. Given that HSV-1 and HSV-2 share a nearly identical set of 75 genes, we possess an amazingly detailed knowledge about the biology of the infectious agents that cause recurrent herpes. So, how is it possible that progress in treating genital herpes has remained at a standstill for 30 years? I have grappled with this question for the past decade, and summarize my conclusions herein. My personal view is that a dichotomy in logic exists between 1. the approaches being most seriously considered to prevent genital herpes, and 2. the biology of herpes simplex virus infections. As the most thoroughly studied herpes vaccine candidate, the glycoprotein D subunit vaccine merits specific attention. However, I believe that the larger questions are: Why has an effective herpes vaccine eluded us for so long? What approaches are most likely to lead to an effective herpes vaccine? HSV-2 subunit vaccines: what have we learned? If one thinks of vaccine development in purely molecular terms, then an ideal HSV-2 vaccine would be a protein subunit of the virus that “jump starts” the adaptive immune response to HSV-2. Thus, HSV-2 vaccine development has focused on how to deliver immunodominant epitopes of HSV-2 to the immune system (1, 6, 25, 32, 38). Glycoprotein D is an immunodominant protein of HSV-1 and HSV-2. Clinical trials have been performed to determine if HSV-2 glycoprotein D can be used to protect people from HSV-2 infection and genital herpes. On both counts, the glycoprotein D subunit vaccine is marginally effective (29–31). Likewise, glycoprotein 120 subunit vaccines fail to protect people against human immunodeficiency virus infection and AIDS (5, 33, 39). Other attempts to produce HSV-2 vaccines have included killed, inactivated HSV-2 vaccines (27, 28), and replication-defective viral vectors (3, 7, 9, 20). In each case, I have reservations about the likelihood of success of the approach. Before proceeding, I wish to make clear my respect for the scientists who performed the studies on the HSV-2 glycoprotein D subunit vaccine. These individuals blazed a trail that simply did not exist ten years earlier. The glycoprotein D subunit may not be critical to the final formulation of a genital herpes vaccine, but what we have learned is invaluable. These studies have lifted a veil from the eyes of the scientific community, and have served as a catalyst that has scientists today asking the question, “What is necessary to attain an effective genital herpes vaccine?” There is no bigger success in science than to change (for the better) how your peers look at the natural world.

High-efficiency gene transfer into cultured embryonic motoneurons using recombinant lentiviruses

Primary neurons are a common tool for investigating gene function for survival and morphological and functional differentiation. Gene transfer techniques play an important role in this context. However, the efficacy of conventional gene transfer techniques, in particular for primary motoneurons is low so that it is not possible to distinguish whether the observed effects are representative for all neurons or only for the small subpopulation that expresses the transfected cDNA. In order to develop techniques that allow high gene transfer rates, we have optimized lentiviral-based gene transfer for cultured motoneurons by using a replication-defective viral vector system. These techniques result in transduction efficacies higher than 50%, as judged by EGFP expression under the control of SFFV or CMV promoters. Under the same conditions, survival and morphology of the cultured motoneurons was not altered, at least not when virus titers did not exceed a multiplicity of infection of 100. Under the same cell culture conditions, electroporation resulted in less than 5% transfected motoneurons and reduced survival. Therefore we consider this lentivirus-based gene transfer protocol as a suitable tool to study the effects of gene transfer on motoneuron survival, differentiation and function.

Characterization of Human Immunodeficiency Virus Gag-Specific Gamma Interferon-Expressing Cells following Protective Mucosal Immunization with Alphavirus Replicon Particles

A safe, replication-defective viral vector that can induce mucosal and systemic immune responses and confer protection against many infectious pathogens, such as human immunodeficiency virus type 1 (HIV-1), may be an ideal vaccine platform. Accordingly, we have generated and tested alphavirus replicon particles encoding HIV-1 Gag from Sindbis virus (SIN-Gag) and Venezuelan equine encephalitis virus (VEE-Gag), as well as chimeras between the two (VEE/SIN-Gag). Following intramuscular (i.m.), intranasal (i.n.), or intravaginal (IVAG) immunization with VEE/SIN-Gag and an IVAG challenge with vaccinia virus encoding HIV Gag (VV-Gag), a larger number of Gag-specific CD8+ intracellular gamma interferon-expressing cells (iIFNEC) were detected in iliac lymph nodes (ILN), which drain the vaginal/uterine mucosa (VUM), than were observed after immunizations with SIN-Gag. Moreover, a single i.n. or IVAG immunization with VEE/SIN-Gag induced a larger number of cells expressing HIV Gag in ILN, and immunizations with VEE/SIN-Gag through any route induced better protective responses than immunizations with SIN-Gag. In VUM, a larger percentage of iIFNEC expressed alpha4beta7 or alpha(Ebeta)7 integrin than expressed CD62L integrin. However, in spleens (SP), a larger percentage of iIFNEC expressed alpha4beta7 or CD62L than expressed alpha(Ebeta)7. Moreover, a larger percentage of iIFNEC expressed the chemokine receptor CCR5 in VUM and ILN than in SP. These results demonstrate a better induction of cellular and protective responses following immunizations with VEE/SIN-Gag than that following immunizations with SIN-Gag and also indicate a differential expression of homing and chemokine receptors on iIFNEC in mucosal effector and inductive sites versus systemic lymphoid tissues.

Insertional oncogenesis in gene therapy: how much of a risk?

Insertional mutagenesis is an unavoidable consequence of the transposition of genetic material. Whether it involves an integrating virus, a transposable element, a replicationdefective viral vector or plasmid DNA, the ectopic chromosomal integration of DNA is a mutagenic event that may disrupt chromatin or gene structure, thereby altering gene transcription, regulation and/or coding sequences. The gravest concern associated with such mutations is the risk of cell transformation. The acquisition of the malignant phenotype may follow tumor suppressor gene inactivation, typically a recessive event, or oncogene trans-activation, which is likely to exert a dominant effect. The propensity of transposable elements to induce experimental tumors in animals has long been exploited to study the genetic basis of cancer. The earliest analyses of the therapeutic potential of gene transfer have thus recognized the risk of insertional oncogenesis as inherent to the use of integrating vectors. Yet, there have been extremely few reports documenting insertional oncogenesis in rodent, dog and non-human primate gene transfer studies. Based on these experimental data, gene therapists and their patients have justifiably assumed that the risk of insertional oncogenesis would be very low. This reassuring point of view was challenged last year when the firstever case of iatrogenic malignant transformation was reported in a gene therapy patient treated for Xlinked severe combined immune deficiency (SCID). The consternation brought upon by this serious adverse event (SAE), which shook the medical community and the concerned regulatory agencies, was compounded, but also mitigated, when a second case was reported 6 months later – in the same trial. These two SAEs imposed a reevaluation of the safety concerns associated with gene transfer and of the risk benefit ratio of gene therapy in the context of SCID as well as other diseases. Following a temporary hold on gene therapy trials using retroviral vectors to transduce hematopoietic stem cells, the US Food and Drug Administration and the NIH Recombinant DNA Advisory Committee judiciously concluded that this pair of SAEs did not constitute ground for the cessation of gene therapy investigation. The interrupted trials were therefore allowed to reopen, pending the case-by-case re-evaluation of the risk/benefit ratio for every study and the appropriate revision of the monitoring plans and of the information provided to prospective patients before obtaining informed consent. The prompt resumption of these trials is good news for the field, but the need to reassess the safety of the many gene transfer strategies under development remains. Our understanding of the biological mechanisms that determine, in qualitative and quantitative terms, the risk of clonal expansion and transformation, is still fragmentary. However, two recent reports provide valuable insight into the transforming mechanisms that may have operated in the two SCID patients who developed clonal T-cell expansions. Based on these observations, we can begin to identify possible factors that increase the risk that insertional mutagenesis will turn into oncogenesis. While prospective studies are direly needed to analyze and quantify the role of these risk factors, the reports by HaceinBey-Abina et al and Dave et al provide important clues for taking steps to investigate and reduce the risk of oncogene trans-activation by integrating gene transfer vectors. Gene therapy of X-linked SCID: landmark success and two SAEs

950. Production of Replication-Defective HSV-1 Viral Vectors and Development of Precise Assay Systems To Analyze the Quality of Clinical-Grade HSV Vectors

950. Production of Replication-Defective HSV-1 Viral Vectors and Development of Precise Assay Systems To Analyze the Quality of Clinical-Grade HSV Vectors