Controlled inactivation of recombinant viruses with vitamin B2.

Inactivated viruses are important tools for vaccine development and gene transfer. 8-Methoxypsoralen (8-MOP) and long-wavelength ultraviolet irradiation (LWUVI) inactivates many viruses. Toxicity limits its use in animals and humans. Toxicological and photosensitizing properties of riboflavin make it suitable for virus inactivation in preparations for biological use. Viruses expressing beta-galactosidase were mixed with either 8-MOP (1.5mM) or riboflavin (50 microM) and exposed to LWUVI (365 nm) for 2 h. Virus activity was determined by limiting dilution. The half-life of the adenovirus preparation treated with 8-MOP was 8.28 ns(-1) and 36.5 ns(-1) after treatment with riboflavin. Despite the difference in half-life, both preparations were completely inactivated within 45 min. In contrast, the half-lives for adeno-associated virus (AAV) preparations were similar (63 ns(-1) 8-MOP vs. 67 ns(-1) riboflavin). Each AAV preparation was fully inactivated within 90 min. The half-life of lentivirus was 193.4 ns(-1) after treatment with 8-MOP and 208 ns(-1) after exposure to riboflavin. Virus treated with riboflavin was inactivated within 20 min. Virus exposed to 8-MOP was inactivated in 90 min. DNA and RNA viruses can be inactivated by riboflavin and LWUVI and used in physiological systems sensitive to other photochemicals.

Comparative thermal stabilities of recombinant adenoviruses and hexon protein.

Differential scanning calorimetry was used to identify the thermal stability profile of the replication deficient and protein IX deleted recombinant adenovirus type 5 that contains the p53 transgene (rAd/p53) in phosphate buffered saline (vPBS) or 10% glycerol (TRIS/phosphate buffer). The wildtype adenovirus (Ad/WT) and purified hexon protein (major capsid protein) were also evaluated in 10% glycerol (TRIS/phosphate buffer) as controls. The thermal profile of rAd/p53 revealed three endothermic transitions (T1, T2 and T3) occurring between 25 degrees C and 90 degrees C. T1, which occurred at 46.7 degrees C in vPBS and 49.4 degrees C in TRIS/PO4 10% glycerol buffer, was irreversible following repeated scanning and attributed to the degradation of the intact vector. The latter two endothermic transitions, T2 and T3, occurring at 69 degrees C and 78 degrees C, respectively, corresponded with the two transitions of purified hexon in temperature and amount of heat absorbed. The thermal profile of Ad/WT revealed four endothermic transitions at 51.5 degrees C (T1), 70.5 degrees C (T2A), 73.6 degrees C (T2B), and 77.4 degrees C (T3). The higher temperature of degradation as well as additional transition was attributed to the presence of protein IX associated with the hexon. The positions and excess molar heat capacities of the intact rAds were found to be affected by pH, glycerol, vector concentration and the presence or absence of protein IX in the capsid. Irreversibility of T1 implied that the degradation of the intact virus may follow first-order kinetics. The thermal scan rate dependence of T1 further confirmed that degradation of the intact virus may be first-order. The apparent activation energies for the degradation of the intact vectors were determined from the scan rate dependence of T1 and shown to be affected by protein IX in the capsid and solution conditions. Analysis of rAd samples incubated at 45 degrees C by Field Emission Electron Microscopy (FESEM) confirmed that loss of single particles was first-order. Although aggregates were observed in the samples, degradation appeared to be the dominant reaction leading to disappearance of single virions from the aqueous matrix. Based on thermal and FESEM analysis, an empirical model was proposed that accounted for the disappearance of single rAd particles. At or near T1, degradation of rAd particles followed a unidirectional, pseudo-first order reaction. However, at lower temperatures, disappearance of single virions resulted from competing irreversible degradation and aggregation reactions.

The use of field emission scanning electron microscopy to assess recombinant adenovirus stability.

A field emission scanning electron microscopy (FESEM) method was developed to assess the stability of a recombinant adenovirus (rAd). This method was designed to simultaneously sort, count, and size the total number of rAd viral species observed within an image field. To test the method, a preparation of p53 transgene-expressing recombinant adenovirus (rAd/p53) was incubated at 37 degrees C and the viral particles were evaluated by number, structure, and degree of aggregation as a function of time. Transmission electron microscopy (TEM) was also used to obtain ultrastructural detail. In addition, the infectious activity of the incubated rAd/p53 samples was determined using flow cytometry. FESEM image-analysis revealed that incubation at 37 degrees C resulted in a time-dependent decrease in the total number of detectable single rAd/p53 virus particles and an increase in apparent aggregates composed of more than three adenovirus particles. There was also an observed decrease in both the diameter and perimeter of the single rAd/p53 viral particles. TEM further revealed the accumulation of damaged single particles with time at 37 degrees C. The results of this study demonstrate that FESEM, coupled with sophisticated image analysis, may be an important tool in quantifying the distribution of aggregated species and assessing the overall stability of rAd samples.

Use of an automated image processing program to quantify recombinant adenovirus particles.

Electron microscopy has a pivotal role as an analytical tool in pharmaceutical research. However, digital image data have proven to be too large for efficient quantitative analysis. We describe here the development and application of an automated image processing (AIP) program that rapidly quantifies shape measurements of recombinant adenovirus (rAd) obtained from digitized field emission scanning electron microscope (FESEM) images. The program was written using the macro-recording features within Image-Pro Plus software. The macro program, which is linked to a Microsoft Excel spreadsheet, consists of a series of subroutines designed to automatically measure rAd vector objects from the FESEM images. The application and utility of this macro program has enabled us to rapidly and efficiently analyze very large data sets of rAd samples while minimizing operator time.