Generation of herpesvirus entry mediator (HVEM)-restricted herpes simplex virus type 1 mutant viruses: resistance of HVEM-expressing cells and identification of mutations that rescue nectin-1 recognition.

Both initial infection and cell-to-cell spread by herpes simplex virus type 1 (HSV-1) require the interaction of the viral glycoprotein D (gD) with an entry receptor on the cell surface. The two major HSV entry receptors, herpesvirus entry mediator (HVEM) and nectin-1, mediate infection independently but are coexpressed on a variety of cells. To determine if both receptors are active in these instances, we have established mutant viruses that are selectively impaired for recognition of one or the other receptor. In plaque assays, these viruses showed approximately 1,000-fold selectivity for the matched receptor over the mismatched receptor. Separate assays showed that each virus is impaired for both infection and spread through the mismatched receptor. We tested several human tumor cell lines for susceptibility to these viruses and observed that HT29 colon carcinoma cells are susceptible to infection by nectin-1-restricted virus but are highly resistant to HVEM-restricted virus infection, despite readily detectable HVEM expression on the cell surface. HVEM cDNA isolated from HT29 cells rendered HSV-resistant cells permissive for infection by the HVEM-restricted virus, suggesting that HT29 cells lack a cofactor for HVEM-mediated infection or express an HVEM-specific inhibitory factor. Passaging of HVEM-restricted virus on nectin-1-expressing cells yielded a set of gD missense mutations that each restored functional recognition of nectin-1. These mutations identify residues that likely play a role in shaping the nectin-1 binding site of gD. Our findings illustrate the utility of these receptor-restricted viruses in studying the early events in HSV infection.

Characterization of soluble glycoprotein D-mediated herpes simplex virus type 1 infection.

Herpes simplex virus type 1 (HSV-1) entry into permissive cells involves attachment to cell-surface glycosaminoglycans (GAGs) and fusion of the virus envelope with the cell membrane triggered by the binding of glycoprotein D (gD) to cognate receptors. In this study, we characterized the observation that soluble forms of the gD ectodomain (sgD) can mediate entry of gD-deficient HSV-1. We examined the efficiency and receptor specificity of this activity and used sequential incubation protocols to determine the order and stability of the initial interactions required for entry. Surprisingly, virus binding to GAGs did not increase the efficiency of sgD-mediated entry and gD-deficient virus was capable of attaching to GAG-deficient cells in the absence of sgD. These observations suggested a novel binding interaction that may play a role in normal HSV infection.

Inactivation of herpes simplex type 1 gene vector on immobilized metal affinity chromatography: oxidative damage by hydroxyl free radicals and its prevention.

Metal catalyzed oxidation (MCO), which typically involves oxygen free radical generation, is an important pathway that leads to the deterioration of many biological molecules in solution. The occurrence of MCO in immobilized metal affinity chromatography (IMAC) systems and its potential for inactivating biological products has not been well recognized. In this study, we report the inactivation of herpes simplex virus type 1 (HSV-1) gene therapy vector on immobilized cobalt affinity chromatography. We observed that purification of KgBHAT, an HSV-1 mutant bearing cobalt affinity tags (HAT) on the surface, on an IDA-Co2+ column using crude supernatant as starting material resulted in signification loss in virus infectivity (<5% recovery). Electron spin resonance (ESR) revealed that the virus inactivation was caused by hydroxyl free radicals generated from the interactions between cellular impurities and the metal ions on the column. Inclusion of 20 mM ascorbate, a free radical scavenger, in the chromatography mobile phase effectively scavenged the hydroxyl radicals and dramatically augmented the infectivity recovery to 70%. This finding is the first demonstration of oxygen free radical-mediated biological inactivation in an actual IMAC purification and the way on how to effectively prevent it.

Delivery of herpes simplex virus-based vectors to stem cells.

In contrast to traditional drugs that generally act by altering existing gene product function, gene therapy aims to target the root cause of the disease by altering the genetic makeup of the cell to treat the disease. Researchers have adapted several classes of viruses as gene-transfer vectors, taking advantage of natural viral mechanisms designed to efficiently and effectively deliver DNA to the host-cell nucleus. Among these, the human herpesviruses are excellent candidate vectors for a variety of applications. Herpes simplex virus type 1 (HSV-1) is a particularly attractive gene-transfer vehicle because natural infection in humans includes a latent state in which the viral genome persists in a nonintegrated form without causing disease in an immune-competent host. HSV-1 is a large DNA virus with a broad host range that can be engineered to accommodate multiple or large therapeutic transgenes (4). HSV vectors may be generally useful for gene transfer to a variety of tissues in which short-term or extended transgene expression of therapeutic transgenes achieve a therapeutic effect. We have used therapeutic vectors to successfully treat human disease models in animals, including cancer, Parkinson's disease, and nerve damage (5-10).

Evaluation of infection parameters in the production of replication-defective HSV-1 viral vectors.

Herpes simplex virus type-1 (HSV-1) is a neurotrophic human pathogen that establishes life-long latency in the nervous system. Our laboratory has extensively engineered this virus to retain the ability to persist in neurons without expression of lytic genes or disease phenotype. Highly defective, replication-incompetent HSV mutants are thus potentially ideal for transfer of therapeutic transgenes to human nerves where long-term therapy of nervous system disease may be provided. A prerequisite for using recombinant HSV vectors for therapeutic gene delivery to humans is the development of methods for large-scale manufacture of HSV vectors. Here we report studies to identify infection parameters that result in high-yield production of immediate early gene deletion mutant HSV vectors in complementing cells that supply the deleted essential viral functions in trans. Virus yield was correlated with various culture media conditions that included pH, glucose metabolism, and serum levels. The results demonstrated that systematic media exchange to remove lactate derived from high-level glucose consumption, maintenance of tissue culture pH at 6.8, and the use of 5% fetal bovine serum gave the highest yield of infectious virus. The data indicate that these are important parameters to consider for high-yield, large-scale virus production.

Effect of genetic background and culture conditions on the production of herpesvirus-based gene therapy vectors.

Herpes simplex virus type-1 (HSV-1) represents a unique vehicle for the introduction of foreign DNA to cells of a variety of tissues. The nature of the vector, the cell line used for propagation of the vector, and the culture conditions will significantly impact vector yield. An ideal vector should be deficient in genes essential for replication as well as those that contribute to its cytotoxicity. Advances in the engineering of replication-defective HSV-1 vectors to reduce vector-associated cytotoxicity and attain sustained expression of target genes make HSV-1 an attractive gene-delivery vehicle. However, the yield of the less-cytotoxic vectors produced in standard tissue-culture systems is at least three order of magnitudes lower than that achieved with wild-type virus. The low overall yield and the complexity involved in the preparation of HSV vectors at high concentrations represent major obstacles in use of replication-defective HSV-derived vectors in gene therapy applications. In this work, the dependence of the vector yield on the genetic background of the virus is examined. In addition, we investigated the production of the least toxic, lowest-yield vector in a CellCube bioreactor system. After initial optimization of the operational parameters of the cellcube system, we were able to attain virus yields similar to or better than those values attained using the tissue culture flask system for vector production with significant savings with respect to time, labor, and cost.

Effect of temperature, medium composition, and cell passage on production of herpes-based viral vectors.

Our work uses replication-defective genomic herpes simplex virus type-1 (HSV-1)-based vectors to transfer therapeutic genes into cells of the central nervous system and other tissues. Obtaining highly purified high-titer vector stocks is one of the major obstacles remaining in the use of these vectors in gene therapy applications. We have examined the effects of temperature and media conditions on the half-life of HSV-1 vectors. The results reveal that HSV stability is 2.5-fold greater at 33 degrees C than at 37 degrees C and is further stabilized at 4 degrees C. Additionally, a significantly higher half-life was measured for the vector in infection culture conditioned serum medium compared to fresh medium with or without serum. Synchronous infections incubated at 33 degrees C produced 2-fold higher amounts of vector than infected cells incubated at 37 degrees C, but with a lag of 16-24 h. Vector production yielded 3-fold higher titers and remained stable at peak levels for a longer period of time in cultures incubated at 33 degrees C than 37 degrees C. A pronounced negative effect of increased cell passage number on vector yield was observed. Vector production at 33 degrees C yielded similar levels regardless of passage number but was reduced at 37 degrees C as passage number increased. Together, these results contribute to improved methods for high-titer HSV vector production.