Click Here for PDF File
http://bloodjournal.hematologylibrary.org/content/78/7/1882.full.pdf+html
Video - International Ozone Research, Information & Interviews - Canadian Government Scientists, Canadian Department of Nation Defence, U.K. Doctors & German Ozone Doctors
"This research was originally published in Blood. bloodjournal.hematologylibrary.org 1991 78: 1882-1890
© the American Society of Hematology."
KH Wells, J Latino, J Gavalchin and BJ Poies
________________________________________ |
Inactivation of (HIV) Human Immunodeficiency Virus Type 1 by O3 Ozone in vitro
Abstract:
A device was designed to deliver a constant source of given concentrations of ozone to fluids containing human immuno- deficiency virus type 1 (HIV-1).
Ozone was found to inactivate HIV-1 virions in a dose-dependent manner. Greater than 11 log inactivation was achieved within 2 hours at a concentration of 1,200 ppm ozone.
Similar concentrations of ozone had minimal effect on factor Vlll activity in both plasma and immunoaffinity-purified preparations of factor Vlll treated for the same time period.
The data indicate that the antiviral effects of ozone include viral particle disruption, reverse transcriptase inactivation, and/or a perturbation of the ability of the virus to bind to its receptor on target cells.
Ozone treatment offers promise as a means to inactivate human retroviruses in human body fluids and blood product preparations.
© 1991 by The American Society of Hemetology.
________________________________________ |
Introduction:
UMAN immunodeficiency virus type 1 (HIV-1), the H etiologic agent of acquired immunodeficiency syndrome (AIDS), is a lentivirus that completes its replicative cycle by budding through the host cell membrane, acquiring host-derived and virus-encoded components in the process.
Subsequent rounds of replication require an intact lipid envelope containing the virally encoded envelope proteins necessary for receptor binding.’ It has been suggested that perturbation of the HIV-1 envelope may be a suitable approach to inactivating HIV-l.* Compounds that fluidize
membranes by removing cholesterol (AL721)3 or by complexing membrane cholesterol (amphotericin B methylester) 4 can inhibit HIV-1 replication in vitro.
Although their precise mode of action has yet to be defined, these compounds may reduce HIV-1 infectivity by perturbing the envelope of HIV-1. We therefore investigated the activity of another membrane active agent, ozone, on HIV-1 infectivity in vitro.
Ozone, the triatomic allotrope of oxygen, is an extremely potent oxidant that has been shown to possess broad spectrum antimicrobial a~tivity.’.~ It has been widely used in sewage treatment, in water purification, and in medicine.’.’”
In particular, ozone has been shown to be effective against a number of enveloped and nonenveloped viral species, with enveloped viruses being more susceptible to ozone inactiva-
tion than those lacking lipid envelopes?”’”*
We report on the in vitro inactivation of HIV-1 in cell culture media and deliberately infected factor VI11 preparations at concentrations of ozone that are not toxic to target cells, while
maintaining the biologic activity of factor VIII. We also investigated the mechanism by which ozone mediates its antiviral effect.
________________________________________ |
DISCUSSION
Reducing the infectious risk of human blood products is an area of intense research. Inactivation of infectious agents in blood products under conditions that retain their intrinsic protein biologic activities has undergone extensive study. Thermal inactivation of viruses in blood derivatives
has been marginally successful, due in part to the thermolability of these components."- 25
Methods ranging from gamma-irradiation:6 porous membrane filtration? and solvent fdetergent mixtures zE3' have been investigated. To date, tri (n-buty1) phosphate (TNBP) detergent mixtures have demonstrated a minimum 4 to 5 log 10 reduction in a variety of lipid-enveloped viruses, including HIV-1, vesicular stomatitis virus, and Sindbis virus, with a concomitant high recovery rate of protein biologic activity.
Analogous work in chimpanzees with TNBP-treated, exogenously viral-spiked blood samples with hepatitis B and non-A, non-B (Hutchinson strain) viruses has shown similar log reductions. However, methods eclipsing a 6 log viral reduction with a similar protein recovery are still being explored. In addition, methods that obviate the necessity for removal of the inactivating agent (ie, Sephadex G-25) and/or that are compatible with cellular systems would ultimately be preferable.
Ozone has previously been shown to possess potent antiviral activity, especially when used against lipid- enveloped viruses. For this reason we investigated the use of ozone as a potential anti-HIV-1 agent. We used a hollow fiber delivery system that maximizes the surface area available for ozone to interact with the fluid material of interest. The system also allows for the precise regulation of
concentrations of ozone to be delivered into the hollow fiber cartridge.
Afferent as well as efferent concentrations of ozone can also be monitored to determine if saturating levels of ozone are achieved in the treated material. With regard to laboratory safety concerns, this closed system has proven to be safe and leak-proof, thereby reducing possible
exposure of laboratory personnel to ozone and human retroviruses to an absolute minimum.
We first examined the ability of ozone to inactivate cell-free HIV-1 in cell-free CM. These results indicate that ozone has potent anti-HIV-1 activity. Preliminary experiments using an escalating dose regimen indicated that a 1,200 ppm dose of ozone achieved a 2 2 log inactivation of
virus.
The data show that neither incubation of the virus preparation at room temperature and at atmospheric conditions for the duration of the experiment nor mechanical shear created by pumping the virus through the system inactivate the virus to any great extent.
However, there was a significant inactivation of the virus due to exposure of virus-containing CM to the stream of carrier nitrogen. Exposure of the virus to pure nitrogen for 6 hours results in
an almost 85% inactivation of virus.
During exposure to nitrogen for 6 hours, conditions are completely anoxic and a maximal pH of 8.1 is achieved, whereas with exposure to room air the oxygen concentration is 21% and the maximal p H is 7.58. It is possible that anoxic conditions or p H changes seen with 6 hours of nitrogen exposure contribute to HIV-1 inactivation.
Because some HIV-1 inactivation was observed with N, alone, it was necessary to determine the proportion of virus inactivation attributable to ozone over the course of a 6-hour treatment. Comparison of the time-dependent inactivation of virus seen with 1,200 ppm ozone with that seen with pure nitrogen clearly indicates that ozone plays the predominant role in achieving virus inactivation for periods up to 6 hours.
Ozone inactivation of HIV-1 occurs relatively quickly compared with any inactivation observed with N, alone. Inactivation of HIV-1 at 45 minutes is 2 2 log when 1,200 ppm ozone is used and only 25% when nitrogen alone is administered.
Investigation of the degree of inactivation of HIV-1 in CM or human plasma by 1,200 ppm ozone for 2 hours indicates a minimum 11 log reduction in viral infectivity, as determined by a 28-day culture assay. Therefore, the effects of temperature, N,, and mechanical shear, which yield an
approximate 0.22 log reduction in HIV-1 infectivity, are certainly eclipsed by the 11 log reduction in infectivity achieved by ozone.
We examined the ability of ozone to render blood products, such as factor VIII plasma preparations, free of HIV-1 without seriously damaging their biologic activity. The escalating dose study indicated that, at 1,200 ppm, ozone can achieve a 90% inactivation of HIV-1 in human
plasma with only a corresponding 25% decrease in factor VIII biologic activity.
Measurement of exhaust ozone levels indicated that saturating levels of ozone in the factor VIIIpreparations were not achieved in this experiment (data not shown) and, thus, longer exposures of HIV-l-containing factor VIII preparations at the higher ozone concentrations were used to eliminate the virus entirely. Use of 1,200 ppm ozone for a period of 2 hours achieved saturation and resulted in an 11 log inactivation of HIV-1 in the human plasma preparation.
Under the same conditions, factor VIII biologic activity was diminished only 10%. Similarly, 2 2 log HIV-1 inactivation in immunoaffinity-purified factor VI11 preparations occurred within 1 hour of exposure to 1,200 ppm of ozone with an approximately 10% loss of factor VIII.
Comparison of the results of inactivation of HIV-1 by high-dose ozone (1,200 ppm) in cell culture media, monoclate, and factor VI11 plasma preparations indicated that a longer exposure time was necessary to achieve total virus inactivation in the plasma factor VI11 preparation.
The reason for the apparent protection of HIV-1 from the effects of ozone in this experiment is most likely the increased protein levels in the factor VI11 preparation. Such increased protein levels and other plasma components may act to protect the virus from the effects of ozone.32
________________________________________ |
OZONE INACTIVATION OF HIV-I 1889
The exact mechanism by which ozone mediates the inactivation of HIV-1 remains to be elucidated.
Our preliminary data indicate that, at the concentrations tested, ozone results in the solubilization of virions. However, the reduction in infectious virus (about 10 to the power of 11) was far greater than the observed reduction in detectable virions (about 85%). This finding suggested that, in addition to the physical destruction of HIV-1 virions, there was a far greater functional impairment of these virions in the infectivity assay.
Our data do not suggest that there was any obvious effect of ozone-treated CM on the viability and CD4a levels of the HUT 78 target cells used in the assay. Nor do our results indicate any preferential quantitative effect on HIV-1 envelope proteins and reverse transcriptase levels in the
virions. However, there was some suggestion of a deleterious effect on virus binding to cell surface receptors.
Ozone may react with the unsaturated fatty acids in the lipid envelope of the virus, thereby increasing membrane fluidity and resulting in viral inactivation. Alternatively, ozone may react with other components in the virus preparation (serum proteins, fatty acids, etc) to produce
secondary reaction products, such as hydroxyperoxides, that in turn mediate the actual viral inactivation.
Far more extensive analyses of all components of the HIV-1 life cycle will be required to fully elucidate ozone's antiretroviral activity. Ozone has potent anti-HIV-1 activity in cell culture
media and factor VI11 preparations. Although the exact mechanism by which ozone mediates its effect remains unclear at this time, it is readily apparent that ozone may be of use in rendering factor VI11 and possibly other blood products, both proteinaceous and cellular, free of HIV-1
and other infectious agents.
________________________________________ |
REFERENCES
1. Poiesz BJ, Ehrlich GD, Papsidero L, Sninsky JJ: Detection of human retroviruses, in DeVita V, Hellman S, Rosenberg S (eds): AIDS: Etiology, Diagnosis, Treatment and Prevention. Philadelphia, PA, Lippincott, 1988, p 137
2. Reimund E Envelope perturbation and AIDS. Lancet 2:1159, 1986
3. Sarin PS, Gallo RC, Schur DI, Cuws F, Lippa AS: Effects of a novel compound (AL 721) on HTLV-I11 infectivity in vitro. N Engl J Med 313:1289,1986
4. Schafher CP, Plescia OJ, Pontani D, Sun D, Thornton A, Pandey RC, Sarin PS: Antiviral activity of amphotericin B methyl ester: Inhibition of HTLV-111 replication in cell culture. Biochem
Pharmacol35:4110,1986
5 . Akey DH, Walton TE: Liquid phase study of ozone inactivation of Venezuelan equine encephalomyelitis virus. Appl Environ Microbiol50:882,1985
6. Burleson GR, Murray TM, Pollard M: Inactivation of viruses and bacteria by ozone with and without sonication. Appl Microbiol 29:340,1975
7. Katzenelson E, Klether B, Sherval HI: Inactivation kinetics of viruses and bacteria in water by ozone. J Am Water Works Assoc 66:725,1974
8. Domique EL, Tyndall RL, Mayberry WR, Pancorbo OC: Effects of three oxidizing biocides on Legionella pneumophilla serogroup I. Appl Environ Microbiol54:741,1988
9. Clarke NA, Breman D: Disinfection of drinking water, swimming-pool water and treated sewage emuents, in Block SS (ed): Disinfection, Sterilization and Preservation. Philadelphia, PA, Lea and Febiger, 1983, p 524
10. Rilling S, Viebahn R: The Use of Ozone in Medicine (2nd rev ed). Heidelberg, Germany, Haug, 1987
11. Bolton DC, Zee YC, Osebold JW: The biological effects of ozone on representative members of five groups of animal viruses. Environ Res 27:476,1982
12. Roy D, Wong PKY, Engelbrecht RS, Chian ESK Mechanism of enteroviral inactivation by ozone. Appl Environ Microbiol 41:718,1981
13. Gazdar AF, Carney DN, Bunn PA, Russell EK, Jaffe ES, Schecter GP, Guccion JG: Mitogen requirements for the in vitro propagation of cutaneous T-cell lymphomas. Blood 55:409,1980
14. Kwok S, Mack DH, Mullis KB, Poiesz B, Ehrlich G, Blair D, Friedman-Kien A, Sninsky J: Identification of HIV viral sequences using in vitro enzymatic amplification and oligomer cleavage
detection. J Virol61:1690,1987
15. Hardisty RM, MacPherson JC: A one-stage factor VIII (anti-hemophilic globulin) assay and its use on venous and capillary plasma. Thromb Diath Hemorrhag 7:215,1962
16. Zee YC, Bolton DC: Ozone decontamination of blood and blood products: US patent No. 4,632,980. Official Gazette of US Patent and Trademarks Office. Dec 30,1986
17. Amin RM, Dean MT, Zaumetzer LE, Poiesz BJ: Virucidal efficacy of various lens cleaning and disinfecting solutions on HIV-1 contaminated contact lenses. AIDS Res Hum Retroviruses
7403,1991
18. Poiesz BJ, Ruscetti FW, Gazdar AF, Bunn PA, Minna JD, Gallo R C Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous
T-cell lymphoma. Proc Natl Acad Sci USA 777415,1980
19. Kirchbaum K, Poiesz BJ, Keller P, Ehrlich G, Gavalchin J, Davis BH, Moore J L Specific absorption of HTLV-I to various target human and animal cells. Blood 701301,1987
20. Gomperts ED: Inactivation of viruses in clotting factor concentrates, in Roberts HH (ed): Proceedings of the Symposium on Biotechnology and the Promise of Pure FVIII. Brussels,
Belgium, Baxter Healthcare Publications, 1989, p 41
21. Meyer D: Obtainment of new factor VI11 concentrates by monoclonal antibody affinity chromatography, in Roberts HH (ed): Proceedings of the Symposium on Biotechnology and the Promise of Pure FVIII. Brussels, Belgium, Baxter Healthcare Publications, 1989, p 61
22. Limentani SA, Furie BC, Poiesz BJ, Montagna R, Wells K, Furie B: Separation of human plasma factor IX from HTLV-I or HIV by immunoaffinity chromatography using conformation specific antibodies. Blood 701312,1987 From bloodjournal.hematologylibrary.org by guest on April 23, 2012. For personal use only.1890 WELLS ET AL
23. Colombo M, Carnelli V, Gazengel C, Mannucci PM, Savidge GF, Schimpf K Transmission of non-A, non-B hepatitis by heat-treated factor VIII concentrate. Lancet 2:1,1985
24. Preston FE, Hay CRM, Dewar MS, Greaves M, Triger D R Non-A, non-B hepatitis and heat-treated factor VI11 concentrates. Lancet 2213,1985
25. Rouzioux C, Chamaret S, Montagnier L, Camelli V, Rolland G, Mannucci PM: Absence of antibodies to AIDS virus in haemophiliacs treated with heat-treated factor VIII concentrate.
Lancet 1:271,1985
26. Kitchen AD, Mann GF, Harrison JF, Zuckerman AJ: Effect of gamma irradiation on the human immunodeficiency virus and human coagulation proteins. Vox Sang 56:223,1989
27. Hamamoto Y, Harada S, Kobayashi S, Yamaguchi K, Iijima H, Manabe S, Tsurumi T, Aizawa H, Yamamoto N: A novel method for the removal of human immunodeficiency virus: Filtration with porous polymeric membranes. Vox Sang 56:230,1989
28. Prince AM, Horowitz B, Brotman B, Huima T, Richardson L, van den Ende M C Inactivation of hepatitis B and Hutchinson strain non-A, non-B hepatitis viruses by exposure to tween 80 and
ether. Vox Sang 46:36,1984
29. Horowitz B, Wiebe ME, Lippin A, Stryker M H Inactivation of viruses in labile blood derivatives: Disruption of lipid-enveloped viruses by tri(n-buty1)phosphate detergent combinations. Transfu-
sion 25:516,1985
30. Prince AM, Horowitz B, Brotman B: Sterilization of hepatitis and HTLV-111 viruses by exposure to tri(n-butyl) phosphate and sodium cholate. Lancet 1:706,1986
31. Edwards CA, Piet MPJ, Chin S, Horowitz B: Tri(n-butyl) phosphate/detergent treatment of licensed therapeutic and experimental blood derivatives. Vox Sang 52:53,1987
32. Mudd JB, Levitt R, Ongun A, McManus ?T: Reaction of ozone with amino acids and proteins. Atmos Environ 3:669,1969
________________________________________ |
"This research was originally published in Blood. bloodjournal.hematologylibrary.org 1991 78: 1882-1890
© the American Society of Hematology."
http://bloodjournal.hematologylibrary.org/content/78/7/1882.full.pdf+html
Click Here for PDF File
Video - International Ozone Research, Information & Interviews - Canadian Government Scientists, Canadian Department of Nation Defence, U.K. Doctors & German Ozone Doctors
|