In vitro efficacy of 0.2% and 0.4% sodium oxychlorosene against meticillin-resistant Staphylococcus pseudintermedius.

BACKGROUND: There is a need for alternative topical therapies as a consequence of the increased prevalence of meticillin-resistant Staphylococcus pseudintermedius (MRSP) skin infections in dogs. Sodium oxychlorosene has been used as a topical antibacterial agent in human medicine since 1955. OBJECTIVES: To determine whether 0.2% and 0.4% sodium oxychlorosene solutions have a bactericidal effect (>3-log reduction) on MRSP strains isolated from canine skin infections. METHODS AND MATERIALS: A genetically heterogeneous collection of MRSP isolates from dogs was assembled from laboratories across the United States. Time-kill assays were performed with 0.2% and 0.4% sodium oxychlorosene on a 0.5 McFarland standard [approximately 108 colony-forming units (cfu/ml)] suspension of each strain. The average bacterial counts (cfu/ml) of each MRSP strain then were determined at 5, 10, 20 and 60 s after exposure to sodium oxychlorosene; cfu/ml data were converted to log10 scale to calculate microbial reduction. RESULTS: The average bacterial counts following exposure to the 0.2% solution at 5, 10, 20 and 60 s were 6.94 × 104 , 5.63 × 103 , 2.96 × 102 and 1.48 × 102  cfu/ml, respectively. For the 0.4% solution, the average bacterial count at 5 s was 2.12 × 103  cfu/ml. No bacterial growth was observed for any MRSP strain by 10 s. The greatest reduction in cfu/ml occurred within 5 s following exposure to each solution 3.4-log and 4.9-log reduction for 0.2% and 0.4%, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: 0.2% and 0.4% sodium oxychlorosene solutions have a bactericidal effect (>99.9% reduction) against MRSP in vitro. Further in vivo studies are necessary to determine whether it is an appropriate alternative therapy for canine pyoderma.

Detection of replication competent retrovirus and lentivirus.

Retroviral vectors based on murine leukemia viruses (MuLV) have been used in clinical investigations for over a decade. Alternative retroviruses, most notably vectors based on HIV-1 and other lentiviruses, are now entering into clinical trials. Although vectors are designed to be replication defective, recombination events during vector production could lead to the generation of replication competent retroviruses (RCR) or replication competent lentiviruses (RCL). Careful screening of vector prior to human use must insure that patients are not inadvertently exposed to RCR or RCL. We describe methods capable of detecting low levels of virus contamination and discuss the current regulatory guidelines for screening gene therapy products intended for human use.

Product-enhanced reverse transcriptase assay for replication-competent retrovirus and lentivirus detection.

The product-enhanced reverse transcriptase (PERT) assay has been used to detect reverse transcriptase (RT) activity associated with retroviruses. Although the PERT assay has been proposed as a method for detection of replication-competent retrovirus (RCR) and lentivirus (RCL), it has not been rigorously compared with existing methods for RCR and RCL detection. We have assessed the PERT assay for detection of RCL and RCR that may contaminate lentiviral and retroviral vectors and compared it with published methods for RCL (p24gag ELISA/gag PCR) and RCR (S+/L-) detection. Our results suggest that the PERT assay is as sensitive as p24gag ELISA and gag PCR for detection of replication-competent HIV-1 in an RCL detection assay. Comparison of detection of replication-competent retroviruses, GALV and RD114, by extended S+/L- and PERT assays indicates that both assays can detect 1 IU of each virus. Our findings suggest that the PERT assay can be used for RCL and RCR testing of a variety of retroviral vectors regardless of the structure, sequence, and envelope of the vectors.

Evaluation of plasmid DNA removal from lentiviral vectors by benzonase treatment.

To improve the purity of lentiviral vector supernatants for clinical studies we have evaluated plasmid DNA removal from lentiviral vectors and also the extent of plasmid DNA associated with transduced CD34 cells in an ex vivo transduction protocol. Optimal conditions of plasmid DNA removal by benzonase treatment were established by varying the temperature, time, and benzonase concentrations in the reaction mix and were determined to be 50 units of benzonase per milliliter of vector supernatant at 37 degrees C, for 15 min. No plasmid DNA was detected, suggesting efficient plasmid degradation was achieved under these experimental conditions. The infectious titer of benzonase-treated lentiviral vector (RRL-CMV-GFP) was nearly identical to the titer of untreated vector (2.3 +/- 0.3 x 10(6) transduction units per milliliter (TU/ml) and 2.7 +/- 0.3 x 10(6) TU/ml, respectively). Analysis of plasmid DNA in concentrated lentiviral vectors shows that concentration substantially decreases the amount of DNA per TU. Analysis of the extent of plasmid DNA associated with transduced CD34 cells in an ex vivo transduction protocol suggests that a minimal amount of plasmid is transferred to transduced cells if the vector supernatant was not previously treated with benzonase. In conclusion, benzonase treatment is effective in eliminating plasmid DNA from vector supernatants and treatment does not affect infectious titers. However, because there is minimal transfer of plasmid DNA to transduced cells under ex vivo transduction conditions, DNA removal from lentiviral vectors may not be essential for all ex vivo clinical applications.

G2 cell cycle arrest and cyclophilin A in lentiviral gene transfer.

Lentiviral vectors derived from the human immunodeficiency virus-1 (HIV-1) have a higher propensity to transduce nondividing cells compared to vectors based on oncoretroviruses. We report here that genistein, a previously known protein tyrosine kinase (PTK) inhibitor and G2 cell cycle arrest inducer, significantly enhanced lentiviral transduction in a dose-dependent manner. Increased transduction, as measured by vector expression, was seen in a variety of human cell lines, murine primary lymphocytes, and primary human CD34(+) peripheral blood progenitor cells as well. Increased vector expression was also associated with an increase in vector DNA copy number, as assessed by quantitative PCR. Genistein-mediated G2 cell cycle arrest, rather than PTK inhibition, appears to be the major factor responsible for increased gene transfer. Genistein also increases cyclophilin A (CypA) protein, a cellular protein important for efficient HIV-1 infection. While we show that CypA(-/-) Jurkat cells transduce poorly with lentiviral vectors, genistein does increase gene transfer in CypA-deficient cells. CypA and G2 cell cycle arrest appear to be two independent factors important for efficient lentiviral gene transfer. The role of genistein and other G2-arresting agents may be useful for improving the efficiency of lentiviral gene therapy.

Certification assays for HIV-1-based vectors: frequent passage of gag sequences without evidence of replication-competent viruses.

A principal concern regarding the safety of HIV-1-based vectors is replication-competent lentivirus (RCL). We have developed two PCR assays for detecting RCL; the first detects recombination between gag regions in the transfer vector and the packaging construct (sensitivity of detection approximately 10-100 copies of target sequence). The second assay uses real-time PCR to detect vesicular stomatitis virus glycoprotein (VSVG) envelope DNA (sensitivity approximately 5-50 VSVG sequences). In an attempt to amplify any RCL, test vectors were used to transduce C8166 and 293 cells, which were then screened weekly for 3 weeks. Psi-gag recombinants were routinely detected (20 of 21 analyses) in four transductions using the RRL-CMV-GFP vector. In contrast, VSVG sequences were detected only once in 21 analyses. Interestingly, p24 levels (as measured by ELISA) were occasionally detectable after 3 weeks of culture. To determine if a true RCL was present, 21-day cell-free medium was used to transduce naïve cells. No evidence of psi-gag or VSVG transfer was detected, indicating that the recombination events were insufficient to reconstitute a true RCL. These findings have important implications for the design and safety of HIV-1-based vectors intended for clinical applications.