Use Of Adenoviruses Research Articles

On the treatment of melanoma: A mathematical model of oncolytic virotherapy

We develop and analyze a mathematical model of oncolytic virotherapy in the treatment of melanoma. We begin with a special, local case of the model, in which we consider the dynamics of the tumour cells in the presence of an oncolytic virus at the primary tumour site. We then consider the more general regional model, in which we incorporate a linear network of lymph nodes through which the tumour cells and the oncolytic virus may spread. The modelling also considers the impact of hypoxia on the disease dynamics. The modelling takes into account both the effects of hypoxia on tumour growth and spreading, as well as the impact of hypoxia on oncolytic virotherapy as a treatment modality. We find that oxygen-rich environments are favourable for the use of adenoviruses as oncolytic agents, potentially suggesting the use of complementary external oxygenation as a key aspect of treatment. Furthermore, the delicate balance between a virus’ infection capabilities and its oncolytic capabilities should be considered when engineering an oncolytic virus. If the virus is too potent at killing tumour cells while not being sufficiently effective at infecting them, the infected tumour cells are destroyed faster than they are able to infect additional tumour cells, leading less favourable clinical results. Numerical simulations are performed in order to support the analytic results and to further investigate the impact of various parameters on the outcomes of treatment. Our modelling provides further evidence indicating the importance of three key factors in treatment outcomes: tumour microenvironment oxygen concentration, viral infection rates, and viral oncolysis rates. The numerical results also provide some estimates on these key model parameters which may be useful in the engineering of oncolytic adenoviruses.

Comparative risk of thrombosis with thrombocytopenia syndrome or thromboembolic events associated with different covid-19 vaccines: international network cohort study from five European countries and the US

ObjectiveTo quantify the comparative risk of thrombosis with thrombocytopenia syndrome or thromboembolic events associated with use of adenovirus based covid-19 vaccines versus mRNA based covid-19 vaccines.DesignInternational network cohort study.SettingRoutinely collected...

A new era of in vivo gene therapy: the applicability of a differentiated HSV-1 based vector platform for redosable medicines

Viral vector-based gene replacement approaches have traditionally focused on the use of adenoviruses, adeno-associated viruses, and lentiviruses for functional gene transmission. Innovation in payload delivery is critical for advancing the boundaries of genetic medicine. While underappreciated, herpes simplex virus type 1 (HSV-1) possesses a number of natural traits that make it an attractive alternative for gene therapy approaches, including episomal delivery, large payload capacity, a broad tissue tropism, and the ability to resist immune clearance via inhibition of innate and adaptive anti-viral immunity. Krystal Biotech has created a proprietary HSV-1-based gene delivery platform leveraging many of the natural properties innate to HSV-1, while engineering it to be replication-incompetent to reduce cytotoxicity. This platform has been validated clinically in dermatology, and its utility is being extended into programs across additional tissue types and organ systems, including initiation of a genetic pulmonary program in cystic fibrosis. This differentiated vector platform provides a broadly applicable, highly versatile gene delivery system for the development of direct and redosable genetically-coded medicines.

Delivery of Anti-IFNAR1 shRNA to Hepatic Cells Decreases IFNAR1 Gene Expression and Improves Adenoviral Transduction and Transgene Expression.

Chronic liver injury leads to advanced fibrosis, cirrhosis, and hepatocellular carcinoma. Genetical cell treatment related to the use of adenovirus (Ads) has proven to be beneficial and efficient in the recovery of hepatic diseases. Nevertheless, they are highly immunogenic and trigger an immune response where interferons type 1 (IFN-I) play a very important role. Three shRNAs against the Interferon-1 receptor (IFNAR1) were designed and cloned in pENTR/U6 plasmid and amplified in DH5α cells. Huh7 cells were transfected with these plasmids in the presence or absence of 1 × 109 viral particles/ml of adenovirus containing the green fluorescent protein gene used as a reporter. Transfection with the shRNA plasmids partially inhibited the IFNAR1 expression. This inhibition substantially decreased antiviral response, demonstrated by the decrease of IFNAR1, IFN-α, and TNF-α gene expression, and the decrease at protein levels of IFNAR1, Protein kinase RNA-activated (PKR), and phosphorylated STAT1, allowing higher adenoviral transduction and transgene expression. Interestingly it was seen shRNA inhibited macrophage activation. These results suggest that the inhibition of the IFN-I pathway could be a strategy to minimize the immune response against Adenoviral vectors allowing higher Adenovirus transduction extending the transgene expression.

A New Look at Vaccine Strategies Against PPRV Focused on Adenoviral Candidates.

Peste des petits ruminants virus (PPRV) is a virus that mainly infects goats and sheep causing significant economic loss in Africa and Asia, but also posing a serious threat to Europe, as recent outbreaks in Georgia (2016) and Bulgaria (2018) have been reported. In order to carry out the eradication of PPRV, an objective set for 2030 by the Office International des Epizooties (OIE) and the Food and Agriculture Organization of the United Nations (FAO), close collaboration between governments, pharmaceutical companies, farmers and researchers, among others, is needed. Today, more than ever, as seen in the response to the SARS-CoV2 pandemic that we are currently experiencing, these goals are feasible. We summarize in this review the current vaccination approaches against PPRV in the field, discussing their advantages and shortfalls, as well as the development and generation of new vaccination strategies, focusing on the potential use of adenovirus as vaccine platform against PPRV and more broadly against other ruminant pathogens.

Genetic updating and marks of cellular lines

Abstract. Despite great advances in the biology of stem cells, there are still many dark spots. Genetic modification techniques, which can be used to track the lines of different cells, primarily stem cells, help to solve this problem. Various methods of biotechnology research are considered, allowing to evaluate the options of introducing new genes into cells and even whole organisms, as well as methods of controlling their expression in time and space, their activation, differentiation and decrease in functional activity, expression of several target genes. Options with multi-cystron vectors encoding several proteins are described. Options for introducing genes using plasmids, electroportation of their disadvantages and advantages are characterized. The most promising and the safest is a retroviral vector using lentivirus vectors capable of generating additional copies of itself, which is very important in the field of biotechnology security. A line of packing cells, usually 293T cells, is used to produce a viral vector. Prospects for the use of adenovirus and adenoassociated vectors are characterized. The achievement of modern biotechnology methods is the system of short palindrome repetitions located in groups, which is a unique tool for genome editing. At the heart of this system is the process of cutting out sequences of deoxyribonucleic acid, which are permanent and which are supported by cells regardless of subsequent divisions or changes in condition. The system allows geneticists and medical researchers to edit parts of the genome by removing, adding or modifying successive sites of deoxyribonucleic acid. An important problem with biotechnology methods is how to control the expression of transgenes. Today, it is quite effective to control expression with a factor present in the gene delivery vector itself and which is only active in a certain type of cell. Endonuclease bacteriophage P1 is used to regulate transgene expression, which cuts deoxyribonucleic acid only at specific sites. This system is introduced in both eukaryotic and prokaryotic systems.

MECHANISMS AND CLINICAL EFFECTS OF DRUGS BASED ON ONCOLYTIC ADENOVIRUSES, HERPES VIRUSES, REOVIRUSES AND MEASLES VIRUS

Despite all the advances in pharmacology, many types of oncological diseases remain incurable. To that end, the demand for the development of new effective methods with high safety still exists. Based on mechanisms that differ from the action of current anticancer drugs, oncolytic viruses expand the possibilities of treating tumors of various localizations. In this review, recent scientific data on the general characteristics of oncolytic viruses are systematized. The factors providing the selectivity and the reasons limiting the efficacy of virotherapy are discussed. The influence of oncolytic viruses on antitumor immunity is considered. The mechanisms of action and the most prospective directions for the use of adenoviruses, herpesviruses, reoviruses and measles virus are analyzed.

The Association and Significance of p53 in Gynecologic Cancers: The Potential of Targeted Therapy.

Dysfunction of p53 is observed in the many malignant tumors. In cervical cancer, p53 is inactivated by degradation through the complex with human papilloma virus (HPV) oncoprotein E6 and E6-associated protein (E6AP), an E3 ubiquitin protein ligase. In endometrial cancer, overexpression of p53 in immunohistochemistry is a significant prognostic factor. A discrepancy between p53 overexpression and TP53 mutations is observed in endometrioid endometrial cancer, indicating that the accumulation of p53 protein can be explained by not only gene mutations but also dysregulation of the factors such as ERβ and MDM2. Furthermore, the double-positive expression of immunoreactive estrogen receptor (ER) β and p53 proteins is closely associated with the incidence of metastasis and/or recurrence. High-grade serous ovarian carcinoma (HGSC) arises from secretary cells in the fallopian tube. The secretary cell outgrowth (SCOUT) with TP53 mutations progresses to HGSC via the p53 signature, serous intraepithelial lesion (STIL), and serous intraepithelial carcinoma (STIC), indicating that TP53 mutation is associated with carcinogenesis of HGSC. Clinical application targeting p53 has been approved for some malignant tumors. Gene therapy by the adenovirus-mediated p53 gene transfer system is performed for head and neck cancer. A clinical phase III trial using MDM2/X inhibitors, idasanutlin (RG7388) combined with cytarabine, is being performed involving relapse/refractory acute myeloid leukemia patients. The use of adenoviruses as live vectors which encode wild-type p53 has given promising results in cervical cancer patients.

The application of naked DNA plasmid (DrZP3) and recombinant adenovirus (Ad-rZP3) in rat animal model to determine comparative efficacy of ZP3-Immunocontraceptive vaccines

The common physical and chemical methods for controlling rat pest are less than satisfactory and inhumane. Immunocontraception approach has been considered more humane and it can be accomplished by inducing the relevant host immune response that block further development of reproductive gametes. ZP3 proteins are known to play very important role during sperm-ovum fertilization. It is a self-antigen and only localized in female ovaries. Therefore, an immunization with ZP3 protein elsewhere will induce a generalize host immune response against ZP3 protein. This study employed rat ZP3 (rZP3) gene prepared from its cDNA of Rattus rattus diardii. It was delivered and expressed in vivo by naked plamid DNA (DrZP3) or recombinant ZP3-Adenovirus (Ad-rZP3). Expression studies in vitro with DrZP3 or Ad-ZP3 showed rZP3 proteins were successfully expressed in Vero cells. Hyperimmune serum against rZP3 that were prepared by immunizing several rats with purified rZP3-pichia yeast fusion protein showed it blocked sperms from binding DrZP3-transfected Vero cells. Female Sprague Dawley rats immunized with DrZP3 demonstrated a long-term effect for significant reduction of fertility up to 92.6%. Ovaries from rats immunized with DrZP3 were severely atrophied with disappearance of primordial follicles from ovarian cortex with an increased in the amount of oocyte-free cell clusters. Female rats immunized with Ad-rZP3 demonstrated 27% reduction of fertility. The infertility induced by Ad-rZP3 is comparatively low and ineffective. This could be due to a strong host immune response that suppresses the recombinant virus itself resulted in minimum rZP3 protein presentation to the host immune system. As a result, low antibody titers produced against rZP3 is insufficient to block oocytes from maturity and fertilization. Therefore, immunization with DrZP3 for immunocontraception is more effective than Ad-rZP3 recombinant adenovirus. It is proposed to explore further on the use of adenovirus or other alternative viruses to deliver ZP3 protein and for the development of enhanced expression of rZP3 in target host.

Assessment of diversity of adenovirus DNA polymerase gene in recreational waters facilitated by ultracentrifugal concentration.

Adenoviruses (AdV) are related to respiratory and gastrointestinal diseases in animals and human beings. Their wide genetic diversity in water bodies and their resistance to environmental conditions allow the use of AdV as a reliable marker for detection of fecal contamination. In this work, the diversity of AdV along Belo Stream - in the city of Caxias do Sul, Rio Grande do Sul, Brazil - was evaluated. Samples were compared in both concentrated and unconcentrated forms. The identification of different AdV species was performed by amplifying a partial sequence of the DNA polymerase gene. AdV was detected in 24 out of 55 concentrated samples (43.6%) and the following species were identified: human adenovirus (HAdV) species C (4/55; 7.2%), D (6/55; 10.9%), E (2/55; 3.6%), and F (9/55; 16.3%). AdV related to other mammalian hosts, such as bovine adenovirus (1/55, 1.8%) and murine adenovirus (2/55, 3.6%), have also been identified; 23.6% (13/55) of the unconcentrated samples were positive, and identified as HAdV species C (6/55, 10.9%), D (1/55, 1.8%), and F (6/55, 10.9%). Results obtained evidenced the presence and the great diversity of AdV, mainly of human origin, circulating in Belo Stream. As expected, the concentration step performed helped to detect AdV in more samples.

Recombinant Adenoviruses Displaying Matrix 2 Ectodomain Epitopes on Their Fiber Proteins as Universal Influenza Vaccines.

Influenza is a zoonotic disease that poses severe threats to public health and the global economy. Reemerging influenza pandemics highlight the demand for universal influenza vaccines. We developed a novel virus platform using extracellular domain IV of the matrix 2 protein (M2e), AdC68-F3M2e, by introducing three conserved M2e epitopes into the HI loop of the chimpanzee adenovirus (AdV) fiber protein. The M2e epitopes were expressed sufficiently on the AdV virion surface without affecting fiber trimerization. Additionally, one recombinant adenovirus, AdC68-F3M2e(H1-H5-H7), induced robust M2e-specific antibody responses in BALB/c mice after two sequential vaccinations and conferred efficient protection against homologous and heterologous influenza virus (IV) challenges. We found that the use of AdV with tandem M2e epitopes in fiber is a potential strategy for influenza prevention.IMPORTANCE Influenza epidemics and pandemics severely threaten public health. Universal influenza vaccines have increasingly attracted interest in recent years. Here, we describe a new strategy that incorporates triple M2e epitopes into the fiber protein of chimpanzee adenovirus 68. We optimized the process of inserting foreign genes into the AdC68 structural protein by one-step isothermal assembly and demonstrated that this 225-bp HI loop insertion could be well tolerated. Furthermore, two doses of adjuvant-free fiber-modified AdC68 could confer sufficient protection against homologous and heterologous influenza virus infections in mice. Our results show that AdC68-F3M2e could be pursued as a novel universal influenza vaccine.

A nonviral vector with transfection activity comparable with adenoviral transduction.

Viral vectors are used commonly in gene therapy trials, but their potential toxic effects are a serious concern. Identification of highly efficient nonviral vectors may alleviate these effects. Results & methodology: We compared the abilities of TransfeX, TransIT-LT1 and adenovirus to deliver the firefly luciferase and green fluorescent protein genes into HeLa cervical carcinoma, and HSC-3 and H357 oral squamous cell carcinoma cells. TransfeX mediated fourfold higher gene expression in HeLa cells than adenovirus, even at the highest multiplicity of infection. Flow cytometry indicated that a population of transfected cells expresses higher levels of green fluorescent protein than transduced cells. TransfeX may be useful for gene therapy applications, particularly where the use of adenovirus is contraindicated.

Albumin-binding adenoviruses circumvent pre-existing neutralizing antibodies upon systemic delivery

Recombinant adenoviruses are used as vaccines, gene therapy vectors, and oncolytic viruses. However, the efficacy of such therapies is limited by pre-existing neutralizing antibodies (NAbs), especially when the virus is administered systemically for a wider biodistribution or to reach multiple metastases. To protect adenovirus against NAbs we inserted an albumin-binding domain (ABD) in the main adenovirus capsid protein, the hexon. This domain binds serum albumin to shield the virus upon systemic administration. The ABD-modified adenoviruses bind human and mouse albumin and maintain the infectivity and replication capacity in presence of NAbs. In pre-immunized mice non-modified viruses are completely neutralized, whereas ABD-modified viruses preserve the ability to transduce target organs, induce oncolysis, or generate immune responses to expressed proteins. Our results indicate that albumin coating of the virus capsid represents an effective approach to evade pre-existing NAbs. This strategy has translational relevance in the use of adenovirus for gene therapy, cancer virotherapy, and vaccination.

467. Assay Conditions Significantly Alter AAV Neutralizing Antibody Determination

A major limitation of vectors based on current AAVs is the wide prevalence of memory B and T cells reactive to the AAV capsid in human populations arisen following natural AAV infection. The Neutralizing Antibody (NAB) assay guides experimentation and determines inclusion or exclusion of subjects in clinical studies in order to predict in vivo neutralization. Furthermore, evidence of a memory B-cell response to AAV may correlate with anti-capsid memory T-cell populations implicated in the safety and efficacy outcome of clinical AAV liver studies. Based on previously reported findings, NAB assay protocols in current use are unable to robustly predict in vivo neutralization in monkeys (Zinn et al. Cell Reports, 2015 & Wang et al. Mol Ther, 2010) leading to false negatives. In a clinical setting, the enrollment of a seropositive individual exposes the subject to undue risk, and generates outcome variability of the often small clinical studies. Here, we sought to permutate several of the NAB assay conditions in order to evaluate their impact on the NAB titer readout. Specifically, we varied the following variables: cell line, transgene detection, use of adenovirus, and AAV amount. Prior to evaluating these assay permutation, we sought to increase the sensitivity of the readout for transgene expression in the assay in order to evaluate at all conditions both high and low transducing AAV serotypes. This optimization increased our sensitivity by ~200-fold. With this increased sensitivity of readout, we first sought to evaluate the compliance of standard AAVNAB assays with the percentage law, formulated by Andrewes and Elford in 1933, a standard for virological NAB assays, which seeks stoichiometric conditions of NAB and viral load that enables measurement of NAB titers independent of viral input in the assay. Our data shows that at the conditions in common use in the field the percentage law is not upheld, and NAB titers are highly dependent on input viral load of the assay, directly impacting sensitivity and specificity, and leading to the occurrence of false negatives. Indeed, quantitative assessment of seroprevalence in primate populations with assays that reduce the amount of AAV to within percentage law range affects assay outcome qualitatively and quantitatively. We also independently assessed other assay parameters to improve sensitivity, robustness and reproducibility, and to study whether they may also have qualitative effects on titer outcomes. Our studies highlight the importance of pre-existing immunity in AAV gene therapy and limitations of the current methodologies to measure it in a robust and predictive manner. Data indicates the potential for false positive and negative readout for commonly used assay protocols. An optimized protocol was developed for further validation and evaluation. These studies may impact translational and clinical AAV gene therapy studies.

Further Developments In the Electrophysiology of Induced Stem-Cell Derived Human Cardiomyocytes

Production of human cardiac cells by genetic reprogramming offers exciting possibilities for tissue repair, artificial organs and the testing of new drugs. The electrophysiological behavior of the artificial cells is important and must be understood for all of these applications. This review summarizes the findings of a large number of experimental papers that have appeared in the last ten years. Recurring themes in this review are the use of tissue culture factors to program the specialized differentiation of cardiac cells, the use of adenoviruses to add specific genes to the cardiomyocyte transcriptome, and the ability of transplanted cells to cause favorable and lasting paracrine even when the transplanted cells to do not permanently survive within the host tissue. Electrophysiological effects can include up-regulation of ion channels that hyperpolarize the membrane, which reduces beat frequency, or up-regulation of depolarizing ion channels, which increases beat frequency. Evidence that transplanted myocytes can sometimes align and couple with the host myocytes, and thereby augment contractile force is discussed. Most of the studies reviewed here involve animal research, although promising clinical studies have already been reported.

Applicability of integrated cell culture quantitative PCR (ICC-qPCR) for the detection of infectious adenovirus type 2 in UV disinfection studies

Practical difficulties of the traditional adenovirus infectivity assay such as intensive labor requirements and longer turnaround period limit the direct use of adenovirus as a testing microorganism for systematic, comprehensive disinfection studies. In this study, we attempted to validate the applicability of integrated cell culture quantitative PCR (ICC-qPCR) as an alternative to the traditional cell culture method with human adenovirus type 2 (HAdV2) in a low-pressure UV disinfection study and to further optimize the procedures of ICC-qPCR for 24-well plate format. The relatively high stability of the hexon gene of HAdV2 was observed after exposure to UV radiation, resulting in a maximum gene copy reduction of 0.5 log10 at 280 mJ cm−2. Two-day post-inoculation incubation period and a maximum spiking level of 105 MPN mL−1 were selected as optimum conditions of ICC-qPCR with the tested HAdV2. An approximate 1:1 correlation of virus quantities by the traditional and ICC-qPCR cell culture based methods suggested that ICC-qPCR is a satisfactory alternative for practical application in HAdV2 disinfection studies. ICC-qPCR results, coupled with a first-order kinetic model (i.e., the inactivation rate constant of 0.0232 cm2 mJ−1), showed that an UV dose of 172 mJ cm−2 achieved a 4-log inactivation credit for HAdV2. This estimate is comparable to other studies with HAdV2 and other adenovirus respiratory types. The newly optimized ICC-qPCR shows much promise for further study on its applicability of other slow replicating viruses in disinfection studies.

A combinatory use of adenoviruses expressing melanoma differentiation-associated gene-7 and replication-competent adenoviruses produces synergistic effects on pancreatic carcinoma cells.

Type 5 adenoviruses expressing mda-7 gene (Ad-mda-7) induced cell death in various kinds of human tumors, but pancreatic carcinoma cells were relatively resistant to Ad-mda-7-mediated cytotoxicity. We then examined whether infection of Ad-mda-7 together with replication-competent Ad produced combinatory cytotoxic effects. We prepared replication-competent Ad, defective of the E1B55kDa gene or activated by a transcriptional regulatory region of the midkine or the survivin gene of which the expression was up-regulated in human tumors. Type 5 Ad bearing the exogenous regulatory region were further modified by replacing the fiber-knob region with that of type 35 Ad. Pancreatic carcinoma cells were infected with replication-incompetent Ad-mda-7 and the replication-competent Ad. Combinatory effects were examined with the CalcuSyn software and cell cycle analyses. Ad-mda-7 and the replication-competent Ad achieved cytotoxicity to pancreatic carcinoma. A combinatory use of Ad-mda-7 and either Ad defective of the E1B55kDa gene or Ad activated by the regulatory region produced synergistic cytotoxic effects. Cell cycle analyses demonstrated that the combination increased sub-G1 populations. These data collectively suggest that expression of MDA-7 augments cytotoxicity of replication-competent Ad and achieves adjuvant effects on Ad-mediated cell death.

Formulation and in vitro and in vivo evaluation of a cationic emulsion as a vehicle for improving adenoviral gene transfer

Advancements in the use of adenoviral vectors in gene therapy have been limited by the need for specific receptors on targeted cell types, immunogenicity and hepatotoxicity following systemic administration. In an effort to overcome the current limitations of adenovirus-mediated gene transfer, cationic emulsions were explored as a vehicle to improve adenoviral vector-mediated gene transfer. Complexation of adenovirus with emulsions containing the cationic lipid 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) enhanced the potency of adenoviral gene transfer as compared to DOTAP liposomes. Among the various emulsion formulations examined, those containing the iodized oil, Lipiodol, as an inner core and stabilized by DOTAP/cholesterol/1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(poly-ethylene glycol)-5000 most efficiently enhanced adenovirus-mediated gene transfer. Optimized Lipiodol-containing emulsions appear to be more strongly associated with adenoviral particles, exhibiting higher complex stability compared to other formulations. They provide the adenovirus with an additional cellular entry mechanism through caveolae-dependent endocytosis, thereby increasing adenovirus entry into cells. Furthermore, adenovirus–emulsion complexation significantly reduced transgene expression in the liver following systemic administration. These findings indicate that emulsion complexation may be a promising strategy for overcoming many of the challenges associated with the use of adenoviruses in gene therapy. Additionally, the observation of increased transgene expression in lung together with reduced expression in liver demonstrates that the adenovirus–emulsion complex may act as a lung-targeting adenoviral gene delivery system.

Use of adenovirus as a model system to illustrate a simple method using standard equipment and inexpensive excipients to remove live virus dependence on the cold-chain

Thermolability of complex biological molecules is a major consideration for the long-term maintenance of titer during periods of storage.The development of a simple, cost effective method for long term storage of virus samples, which maintains viral titer would prove useful for a wide variety of applications including the preservation of viral vaccines, and is paramount for alleviating the reliance upon the cold chain.We have investigated the potential use of a method adapted for this purpose originating from a natural mechanism used by plants which helps to maintain the integrity of seeds, enabling them to overcome extensive periods of temperature elevation and desiccation.As maturation of a seed progresses, many complex biological macromolecules are laid down which maintain the germination potential. Sucrose and raffinose (in addition to other oligosaccharides) are commonly found to accumulate. In addition highly charged protein molecules accumulate, Late Embryogenesis Abundant (LEA) proteins, reaching their maximal level when the seed is most desiccation and thermally tolerant, and indeed are among the first molecules to be lost when germination is initiated.We have examined the potential use of sucrose and raffinose in concert with chemical replacements for the LEA, which when dried with the active product forms an amorphous solid able to maintain the titer of infectious Adenovirus at elevated temperatures for extended periods, in the case of lyophilized presentations several months at 37°C, or as liquid, stability for several weeks at 37°C was achieved.We demonstrate that after embedding the active product in the matrix, the viral titer is preserved even at temperatures for relatively extended periods at temperatures significantly greater than ambient.In addition we believe that these results could open the way for a new type of vaccine which we refer to as a hybrid stability vaccine, whereby for the first time the same excipient components are used to confer stability in both liquid and solid forms (albeit at different concentrations) which may ultimately result in a stable vaccine which has a very high stability index whilst dry, whereas upon reconstitution using nothing more than WFI at proscribed volumes, the vaccine would benefit from having much improved stability during the administration procedures.This paper describes the use of Adenovirus (itself fast becoming a vector of choice for a new generation of vaccines) as a model system, and identifies non-toxic, inexpensive excipients which are compatible with current manufacturing processes which could be instrumental in removing the dependence upon the cold chain.

Oncolytic Adenoviruses: Design, Generation, and Experimental Procedures

Oncolytic adenoviruses are designed to take advantage of the virus' native ability to replicate in cancer cells to induce oncolysis. Subsequently, the released viral progeny spread and kill the neighboring cancer cells. These characteristics, together with the ability of adenovirus to infect a broad spectrum of cells, its well understood replication machinery, and relative ease of manufacture have led to the intensive use of adenovirus as an anticancer agent. This unit describes cloning strategies, procedures to turn the intended design into virus, and quality analyses of resultant adenoviral vectors. Most of these procedures were optimized especially for oncolytic adenoviral vectors.