Erratum in print version of "Toward a More Accurate Quantitation of the Activity of Recombinant Retroviruses: Alternatives to Titer and Multiplicity of Infection"

Abstract

In this paper, we present a mathematical model with experimental support of how several key parameters govern the adsorption of active retrovirus particles onto the surface of adherent cells. These parameters, including time of adsorption, volume of virus, and the number, size, and type of target cells, as well as the intrinsic properties of the virus, diffusion coefficient, and half-life (t 1/2 ), have been incorporated into a mathematical expression that describes the rate at which active virus particles adsorb to the cell surface. From this expression, we have obtained estimates of C vo , the starting concentration of active retrovirus particles. In contrast to titer, C vo is independent of the specific conditions of the assay. The relatively slow diffusion (D ‫؍‬ 2 ؋ 10 ؊8 cm 2 /s) and rapid decay (t 1/2 ‫؍‬ 6 to 7 h) of retrovirus particles explain why C vo values are significantly higher than titer values. Values of C vo also indicate that the number of defective particles in a retrovirus stock is much lower than previously thought, which has implications especially for the use of retroviruses for in vivo gene therapy. With this expression, we have also computed AVC (active viruses/cell), the number of active retrovirus particles that would adsorb per cell during a given adsorption time. In contrast to multiplicity of infection, which is based on titer and is subject to the same inaccuracies, AVC is based on the physicochemical parameters of the transduction assay and so is a more reliable alternative.Recombinant retroviruses are promising vehicles for the transfer of genes into mammalian cells for the purpose of gene therapy and have been tested clinically for the treatment of a variety of diseases, including cancer and AIDS (8). As the number of clinical studies increases, the quantitation of stocks of retrovirus and comparisons between different laboratories and clinical trials will become increasingly important. This task can be facilitated by a quantitative understanding of the steps of retrovirus-mediated gene transfer. These analyses can provide insight into the effects that key physicochemical factors have on transduction and suggest new strategies to improve the efficiency of retrovirus-mediated gene transfer.Stocks of recombinant retroviruses are typically quantitated by measuring titer, the number of gene transfer events per unit volume of retrovirus solution. To determine titer, the virus stock is first diluted a few 1,000-fold and then used to transduce target cells. The titer, expressed as the number of CFU/ milliliter, is the number of colonies of transduced cells multiplied by the dilution factor and divided by the volume of retrovirus applied to the target cells. The visualization and quantitation of transduced cells are achieved by the use of retrovirus vectors that carry reporter genes, such as the lacZ gene, or antibiotic resistance genes, such as the neo gene.Although titer is a conventional measure of retrovirus bioactivity, it suffers from certain problems and inconsistencies.

Molecular Determinants of the Ratio of Inert to Infectious Virus Particles

“…Thus, increasing the volume and keeping the concentration constant adds only marginally to the infection, whereas keeping the amount of virus constant and increasing the volume reduces infection proportionately, as illustrated both for retrovirus and adenovirus. 15,17,39,40 The slight deviations from these sketched ideal relationships, i.e. strict concentration dependence and volume independence might be explained by a weak tendency for virions to sediment, i.e., the same tendency that is vastly augmented by spinoculation; as mentioned, the density of retrovirus particles is only marginally above that of the medium, 15 but denser viruses might show somewhat greater influence of gravitation on the degree of infection.…”

“…By only measuring the infectivity in the first culture, we would underestimate the total infectivity 20- to 100-fold and hence overestimate the corresponding P/IU ratios to the same extent. 36,40 …”

“…17,20,40 But even improved estimates of the total infectious content in a suspension does not predict how many cells will be infected by how many virions. That relationship requires a more complex analysis, which also has a long history.…”

“…17,40,45; cf., “L’État, c’est MOI!,” attributed to Louis XIV). But I suggest that there are two distinct, indeed incompatible, prevalent uses of MOI; and that is how the concept acquired its ill repute.…”

“…But practical use of viruses for gene transduction will benefit from other measurements, such as the number of active virions that will adsorb per cell and the extrapolated total infectious content. 17,20,40 New sophisticated techniques on the horizon may change our outlook further.…”