WO1991003264A1

WO1991003264A1 - Process for rendering inactive infectious and parasitic agents in biological media using ultrasound and its applications

Info

Publication number: WO1991003264A1
Application number: PCT/FR1990/000644
Authority: WO
Inventors: Léandre Pourcelot; Frédéric PATAT; Laurence Boucher
Original assignee: Fondation Nationale De Transfusion Sanguine
Priority date: 1989-09-07
Filing date: 1990-09-05
Publication date: 1991-03-21
Also published as: FR2651438A1

Abstract

The invention relates to a process for the treatment of a liquid biological medium which can be contaminated by at least one infectious agent of a viral, parasitic or bacterial nature, which is characterised by the fact that a coherent ultrasound beam is applied to said medium at a frequency between about 0.1 and 100 MHz, such that the infectious agent or agents are rendered inactive. Application: treatment of blood products and their derivates, sperm, and various biological and food products.

Description

METHOD FOR ULTRASONIC INACTIVATION OF INFECTIOUS AGENTS OR

PARASITARIES IN BIOLOGICAL MEDIA AND APPLICATIONS OF THE PROCESS

The present invention relates to the field of in vitro inactivation of infectious rents of viral, bacteriological or even parasitic nature capable of contaminating biological liquid media, and relates more particularly to a new method of treatment of such a biological medium for less partially inactivate the infectious agents that it may contain.

The seriousness of the contamination problems of recipients of blood transfusions or blood derivatives is currently being brought to light in connection with the epidemic development of AIDS. However, there are many viruses which can be transmitted from one patient to another by blood transfusion, and in particular the HIV, HTLV1 and CMV viruses, hepatitis viruses, etc.

Likewise, the safety of transfusion products with regard to diseases such as malaria or

Chagas is a major concern of transfusion centers in endemic areas.

Thus, in addition to the usual screening precautions, which are not capable of completely solving these problems, it is necessary to use techniques intended to further reduce the risk of contamination by transfusion. However, at present there is no known effective means for inactivating the infectious agents potentially present in erythrocyte concentrates while retaining the quality of these concentrates.

In addition, there is currently an increasing demand addressed to the centers of sperm conservation and to the centers of in vitro fertilization by men seropositive vis-a-vis the virus of the AIDS virus HIV, men who would like to have children but not can do so without risk of infecting their partner and their child. And these two types of organizations want legitimately better guard against the risks of contamination linked to the use of donor sperm.

On a more general level, there is a strong demand to inactivate in vitro any infectious agents present in a biological fluid intended for in vivo use (by transfusion, insemination, ingestion ...) without degrading the useful properties of such a liquid, and in particular the capacity for keeping red cells for the blood, fertilizing power for sperm, lifespan, etc.

And currently there is no technique for processing such products which is capable of effectively meeting this demand.

Furthermore, it is already known in the prior art to use ultrasound on an experimental basis for:

- release a virus (such as the flu virus) from host cells;

_ homogenize a viral suspension;

- dissociate Ag-Ac complexes;

- disaggregate viruses to separate the various constituents; or

- cause alterations of various cells, and in particular the phenomenon of hemolysis.

Furthermore, with regard to the field of viral inactivation, ultrasound has already been used occasionally and anecdotally not for the inactivation of viruses, but for the purely experimental study of viral properties or even for the implementation at the point of virological techniques (in particular extraction of virions from their host cells).

In addition, all this research on this subject made use of low frequency ultrasonic fields (from 10 to 50 kHz) generated by cleaning or cell grinding tanks. The most representative document of the prior art is "Changes in light scattering, absorption, fluorescence and biological characteristics of influenza virus A (H1N1) exposed to sonification", CN ZAHARDA and AL PETRFSCU, Rev. Roum. Med. - Virol., 35. 2, 105-107, 1984. This document teaches the application of an ultrasonic field with a frequency of 20 kHz in a 600 W tank, by simply describing the morphological anomalies observed under the electron microscope , which appeared during the exhibition.

The present invention is based on the experimental discovery that, under certain conditions, ultrasound irradiation is capable of effectively inactivating various infectious agents, that is to say of definitively preventing them from replicating without degrading organic products thus treated.

Thus, the present invention relates to a method for treating a liquid biological medium capable of being contaminated with at least one infectious agent of viral, parasitic or bacteriological nature, characterized in that a coherent beam of ultrasound is applied to the medium at a frequency between about 0.1 and 100 MHz, so as to inactivate the infectious agent or agents.

Preferably, the ultrasound beam is applied for a period of between 1 and 10,000 seconds. In addition, an ultrasonic beam with a surface power of between 0.5 and 50 W / cm ² is particularly advantageous.

The exact mechanism of viral inactivation observed by implementing the process of the present invention has not yet been identified with certainty. We can nevertheless mention the following hypotheses:

- the ultrasonic field creates at these frequencies an intense phenomenon of transient cavitation thanks to which, at the time of the collapse of the bubbles, it is generated high local pressures and temperatures as well as shock waves. These phenomena create free radicals in large numbers, whose chemical activity can be a key to the phenomenon of inactivation observed;

- ultrasound is at the origin of molecular relaxations such that certain proteins or the nucleic acid of the infectious agent come to be modified; or:

- the ultrasound causes the medium to vibrate at a high frequency to such a degree that the mechanical structure of the viral capsid breaks.

It may be noted here that the analysis of the prior art concerning the behavior of cells, and in particular red blood cells, under the effect of ultrasound irradiation, although all the studies described therein involve low frequency ultrasound, of the order of 10 to 80 kHz, and despite disparate conditions of implementation, leads to favor the hypothesis according to which the cavitation phenomenon, either directly by mechanical effect in the vicinity of the collapse, or indirectly by the production of radicals free or shock waves, is responsible for the observed inactivation effect.

Other aspects, aims and advantages of the present invention will appear better on reading the following detailed description of preferred embodiments of apparatus for implementing the method, given by way of nonlimiting example and given with reference to the accompanying drawings, in which:

FIG. 1 is a schematic elevational view of an experimental device for implementing the method of the invention,

FIG. 2 is a view in axial vertical section of an ultrasonic irradiation tank according to the invention, and - Figure 3 is a diagram illustrating the results obtained in a particular case with the method of the invention.

It will first be indicated that, from one figure to another, identical or similar elements or parts are designated by the same reference numbers.

Referring firstly to FIG. 1, an irradiation apparatus for implementing the method according to the invention comprises a tank 100, preferably made of aluminum, of parallelepiped shape. In a side wall 101 of the tank 100 is formed an opening 102 to the right of which is rigidly fixed, for example by bonding, a ceramic 110, protected by a waterproof housing 120. For example, the tank 100 may have a volume of 80 cm ³ and ceramic 110 an area of 9 cm ² . On the wall 102 of the tank which is opposite to the wall 101 is fixed, on the inside, a reflector 130 of ultrasonic waves, so as to create in the tank a stationary field of ultrasonic waves.

It should be noted that, in an alternative embodiment, the reflector 130 can be replaced by a material absorbing the ultrasound at the frequencies considered, so as to create in this case a progressive field of ultrasound.

Means are provided for ensuring in the tank

100 a circulation of chilled water. These means comprise a tank 140 das which are arranged with water and ice cubes. A tube 141 connects the tank 140 to the inlet of a water pump 142, the outlet of which is connected by a second tube 143 to an inlet fitting 103 of the tank 100. An outlet fitting 104 of the tank is connected by a third tube 144 to tank 140.

The supply part of the ceramic consists of a high frequency generator 150 whose output is connected on the one hand to a high frequency amplifier 160 and on the other hand to an oscilloscope 170. The frequency and amplitude of the output signal from generator 150 can be adjusted.

The output of the amplifier 160 is connected, by an appropriate coaxial cable 161, to the ceramic 110.

It is thus possible to create in the tank 100, by means of the ceramic 110, an ultrasonic field of determined frequency and intensity, these conditions being displayed on the oscilloscope.

A test tube 200 containing the sample of biological liquid to be irradiated is immersed in the water contained in the tank 100, through a suitable opening (not shown) formed in the upper wall 105 of the tank.

There is shown in Figure 2 an alternative embodiment of the assembly of the tank 100. The tank here comprises a cylindrical side wall 106, a bottom 107 also forming a support plate and a cover 108, assembled together by suitable mechanical means , with suitable seals. The tank can advantageously be made of duralumin.

The ultrasound here is generated by a ceramic 110 of circular shape arranged horizontally and produced for example from a ferroelectric ceramic. The ceramic 110 is fixed in an opening 107a made in the bottom wall 107. The core 161a of the power cable 161 is welded to the underside of the ceramic, while its shield is welded to the bottom 107. Furthermore, a peripheral weld bead 107b between the upper face of the ceramic and the wall 107 constitutes the other supply connection for the ceramic.

The sample of biological fluid to be treated is contained in a vertical sample tube 200, also made of duralumin, passing through an opening provided in the cover 108. The tube 200 is closed at its upper end by a plug 201 and at its lower end by a membrane 202 in "Kapton" (registered trademark), permeable to ultrasonic irradiation.

As in the case of FIG. 1, refrigerated water is made to circulate in the tank 100, using the fittings 103 and 104.

By having in the upper region of the tank 100 an appropriate ultrasonic reflector (not shown), this establishes when the ceramic 110 is supplied a stationary field of ultrasound in the tank 100, to which field the biological liquid contained in the tube 200.

According to an essential aspect of the invention, the ultrasonic field has a frequency between 0.1 and 100 MHz, preferably between 1 and 20 MHz. The surface intensity of the ultrasonic field is preferably between 0.5 and 50 W / cm ₂ , and the optimal irradiation time, which varies according to the frequency and intensity of the field and the type of infectious agent. to deactivate, can be between 1 and 10,000 seconds.

Example 1

We will describe below an experiment of viral inactivation of a type II herpesvirus strain in suspension in a biological conservation medium.

Samples were subjected to an ultrasonic field in the device shown in Figure 2.

The infectious power of the virus was assessed by cell culture for four days, on an MRC5 cell. The titration was carried out by calculating the infecting dose 50 (ID 50%), by the method of Reed and Muench; it may be recalled here that, according to this procedure, the 10 ⁿ dilution which would have infected 50% of the cell culture is calculated. A 4 log decrease in the titer of viruses exposed to ultrasound compared to that witness virus was retained here as a criterion for inactivation of this viral strain.

This experiment has been summarized in Table I below, which indicates the results obtained with various ultrasonic energies I and various ultrasonic frequencies F.

These results are symbolized as follows:

+ indicates complete inactivation of the virus;

(+) indicates partial inactivation of the virus (decrease in viral titer, but less than 4 log); and indicates an absence of modification of the viral titer, that is to say no inactivation.

In addition, the minimum duration of irradiation necessary to achieve the indicated result has been indicated in each case (in seconds).

It can be observed in Table I that a partial or total inactivation is obtained for each of the ultrasound frequencies used. We also note that at the lowest frequencies (around 1 MHz), inactivation is carried out with low ultrasonic intensities. At 3 and 5 MHz, a higher intensity, of the order of 20 W / cm ² , is required. Finally at the highest frequencies (7 and 11 MHz) and with high intensity, inactivation can be carried out with the shortest durations.

Example 2

The second experiment consisted in studying the inactivation of an echovirus strain, still in suspension in a biological conservation medium, under the same conditions and with the same methods as for example 1.

The results are presented in Table II below.

It can be seen from this table that the conditions of exposure to ultrasound allowing inactivation are close to those observed for herpesvirus type II, but the treatment durations are higher.

We also note that the highest frequencies (7 and 11 MHz) seem to be the most favorable.

Example 3 The inactivating effect of ultrasound on the HIV1 virus (strain HTLVIII-RF) was studied. This virus was suspended in a culture supernatant consisting of RPMI 1640 medium supplemented with 20% of decomplemented fetal calf serum, 1% of glutamine and 1% of antibiotic and antifungal mixture. This supernatant was distributed in five tubes, the first of which was kept as a control. The other four tubes were subjected to an ultrasonic treatment at a frequency of 4.7 MHz with a power density of 30 W / cm ² for 2000 s for two of them and 5000 s for the other two. A viral titration was then carried out by observation of the cytopathogenic effect after 5 days on Sup T1 lymphocyte cells.

The results are presented in FIG. 3 of the drawings, with the abscissa the duration of exposure to ultrasound and the ordinate the title (in logarithm to base 10) of the infectious dose 50%.

A decrease in viral activity of more than 3 log is observed for the media treated for 2000 s and by more than 5 log for the media treated for 5000 s.

We conclude that, in a low protein medium like the one studied, containing 20% fetal calf serum, an inactivation of the order of 4 log can be obtained by treating the medium for a period of between about half an hour and one o'clock. Of course, the present invention is in no way limited to the embodiment described above and shown in the drawings, but a person skilled in the art will know how to make any variant or modification in accordance with his spirit.

In particular, although experimental apparatus has been described above for irradiating samples of biological liquids, those skilled in the art will know how to design apparatus capable of treating biological liquids in large quantities, by multiplying the capacity of the tank and the size and power of the device generating the ultrasound field or otherwise. In this regard, it is possible to envisage an overall treatment of a liquid contained in a container such as a tube, a pocket or a tray, and this immediately after the sampling or deferred. It is also possible to envisage applying the ultrasonic field to a part of a blood collection tube in the case of blood donations.

Furthermore, the ultrasonic field can be, as indicated above, stationary or progressive, and the ultrasonic waves can be emitted continuously or pulsed, the most suitable modes of implementation being determined by experience in the case. not case.

In addition, it is clear that the ultrasound treatment described above can be combined with any other treatment capable of amplifying the effect of ultrasound. For example, detergents of the deoxycholate or sodium dodecylsulfate type can be used, or alternatively products which bind to nucleic acids, such as acridine and its derivatives, porphyrins, chlorines, cyclic compounds, etc. These substances are preferably incorporated into the biological medium to be treated before exposure to ultrasound. Finally, genetic engineering and cell engineering processes now make it possible to manufacture injectable therapeutic products (in particular insulin, specific antibodies, vaccines, etc.) requiring procedures, or derived from transformed cells, likely to contain viruses. human or animal origin. The invention is of course also applicable to the treatment of these products in order to ensure viral safety.

Claims

1. Method for treating a liquid biological medium such as blood, sperm, bone marrow samples, or blood derivatives such as blood cells, plasma or protein fractions, which may be contaminated with at least one infectious agent of viral, parasitic or bacterial nature, characterized in that a coherent beam of ultrasound is applied to the medium at a frequency between about 0.1 and 100 MHz, so as to inactivate the infectious agent (s).

2. Method according to claim 1, characterized in that the ultrasound beam is applied for a period of between 1 and 10,000 seconds.

3. Method according to one of claims 1 and 2, characterized in that an ultrasonic beam with a surface power of between 0.5 and 50 W / cm ² is applied.

4. Method according to one of the preceding claims, characterized in that before the application of the ultrasound beam, at least one amplifying substance chosen from detergents and products which bind to nucleic acids is added to the biological medium.

5. Method according to one of the preceding claims, characterized in that the ultrasound beam has a frequency between 1 and 20 MHz.

6. Application of the method according to one of claims 1 to 5 to the viral inactivation of blood products and their derivatives.

7. Application of the method according to one of claims 1 to 5 to viral inactivation of sperm.

8. Application of the method according to one of claims 1 to 5 to the viral inactivation of bone marrow samples.

9. Application of the method according to one of claims 1 to 5 to the viral inactivation of therapeutic products obtained by the techniques of genetic engineering and cell engineering.