Lab Test Results of HIV
inactivation by electric current from patent US5,139,684
(of Kaali & Schwolsky 8-18-92)
EXPERIMENTAL RESULTS
Overview: A non-flow vessel or cell included a
pair of platinum electrodes 1 mm apart inserted into a well 1.56 mm in
length and 8.32 mm in depth. The non-flow vessel was connected to a
direct current source capable of creating an electric field at a
constant voltage and constant amperage. Into this well was laced a
suspension of the human immunodeficiency virus type 1 (HIV-1) at a
concentration of 1,000,000 infectious particles per ml. An aliquot of
approximately 10 ul of the virus suspension was placed into the well.
Thereafter, the viral suspension was exposed to direct currents ranging
from 0 microamps (uA) for up to 12 minutes, to 100 microamps for up to
6 minutes. Intermediate currents of 25, 50 and 75 microamps were used
to expose similar viral aliquots. After exposure of the viral
suspension to electric currents, the contents of the non-flow vessel
were removed and placed into sterile microtubes. 5 ul of each sample
were removed and diluted with 95 ul tissue culture medium supplemented
with 10% fetal calf serum (FCS. unborn calf blood)
In Experiment 1, the resuspended and treated viral stocks were
incubated with a human T lymphoblastoid cell line named CEM-SS. This
cell line, upon exposure to HIV-1, forms syncytia (giant cells). It is
well documented that the viral titer (amount) used is directly
correlated with the number of syncytia formed. Therefore, evaluation of
infectivity of HIV-1 can be used with this assay. In contrast,
Experiment No. 2 used a different human T lymphoblastoid cell line
named H9. This cell line, in contrast to CEM-SS cells, produces, upon
exposure to HIV-1, many viral particles. The amount of virus produced
is proportional to the amount of virus to which the cells are exposed.
Therefore, quantitation of viral particles, or more commonly associated
viral protein (in this case reverse transcriptase), can be used as an
index of viral infection. In both assays, the CEM syncytia forming
assay and the H9 viral protein assay, similar type results were
obtained. That is, with the CEM cells, although syncytium formation and
quantitation is preferrable, one can quantitate the HIV-1 associated
protein (reverse transcriptase) activity and conversely with the H9
cells, although reverse transcriptase quantitation is preferred, one
can quantitate giant cell (syncytia) formation. Both of these assays
are widely used as reproducible measures of viral infection and can be
used to determine if alterations in viral infectivity as a product of
this electrical treatment can be detected.
Experiment #1
Approximately 100,000 CEM-SS cells per sample were incubated with a
treated or untreated (control) viral aliquot for up to 4 days. The
cells were placed into microtiter plate wells and monitored for
formation of syncytia every 24 hours by microscopic observation. In a
standardized fashion, as it has been reported in the literature and is
currently being conducted in many laboratories, the number of syncytia
at 3 and 4 days was determined. Table 2 summarizes the results from a
representative experiment using this assay. As can be noted, the number
of syncytia formed was inversely proportional to the amount of electric
current. That is, additionally, with increased current (100 vs 50 uA)
there was a reduction in the number of syncytia formed. These results
and those of additional experiments using the CEM-SS cell line indicate
a consistent finding that electrical treatment of the RF strain of
HIV-1 attentuates the virus potential for inducing syncytium formation
in this cell line.
Experiment #2
A separate and independent assay to determine the ability of electric
current to alter HIV-1 infectivity using H9 cells was employed. The
basic strategy was similar to that used for the CEM cells with the
exception that the initial suspension of treated and controlled
(non-treated) viral stock was incubated with 100,000 H9 cells for 2
hours at 37 degrees Celsius. Thereafter, the cell virus suspensions
were further diluted to 5 ml in standard tissue culture medium. The
cell-viral suspensions were then incubated for up to 14 days at 37
degrees Celsius with 5% carbon dioxide. At 3 day intervals (beginning
at day 2), aliquots of cell suspension were removed from each sample.
The aliquots were centrifuged at 1,000 rpm for 5 minutes in order to
pellet the cells. After centrifugation, the supernatant and cell
pellets were separated. The supernatant was cyropreserved for
subsequent reverse transcriptase assay and the cell pellets were
resuspended in fixatives and maintained in a tissue bank for additional
studies employing in situ hybridization and immunocytochemistry to
detect qualitatively and semi-qualitatively viral infection by HIV-1.
At the end of each experiment, the supernatant samples from each of the
tests and time points were examined using standard reverse
transcriptase assay. The results of the representative experiment are
shown in Table 3. The results of this experiment indicate the ability
of HIV-1 to infect H9 cells is attenuated by the magnitude of the
electrical currents to which the virus is exposed. Additionally, at
lower current magnitude, but with prolonged exposure time, attenuation
of viral infectivity is achieved. That is, analogous to the results
observed using syncytium formation and the CEM-SS cell line, either
increased current or increased duration of exposure time was inversely
proportional to the amount of reverse transcriptase produced by the
cell line.
In conclusion, these experiments which have been repeated several
times, and those using the CEM-SS cell line, indicate at a
statistically significant level that direct electrical current at
biocompatible amperages for discrete exposure time intervals can
attenuate the ability of HIV-1 to infect normally healthy cells which
are susceptible to the HIV-1 AIDS virus.
TABLE 2
Syncytium Formation
------------------------------------------
Number of Syncytia
---------------------------------
Time and Electric Current Amount
-----------------------------------
Dilution 6 min 8 min 6 min 4 min 3 min
of virus 0uA 25uA 50uA 75uA 100uA (current applied)
-------- ----- ------ ------ ------ -------
1:160 180 44 9 9 2
1:320 115 28 4 6 0
1:640 70 10 0 2 0
----------------------------------------------
Percent reduction in infectivity
Dilution ---------------------------------
of virus 25uA 50uA 75uA 100uA (current applied)
-------- ------ ------ ------ -------
1:160 75.5% 95.0% 95.0% 98.9%
1:320 75.0% 96.5% 94.8% 100.%
--------------------------------------------
(derived from Table 2 above)
TABLE 3
Reverse Transcriptase Activity
(count per million x .001)
-----------------------------------------
after 4 days
microAmps/Time incubation % reduction (assuming an avg 12.8 untreated)
---------------- ----------- -----------
0uA/6min 13.8
0uA/12min 11.7
50uA/3min 9.1 29%
50uA/6min 9.1 29%
50uA/12min 4.8 62%
100uA/3min 5.7 55%
100uA/6min 3.6 72%
------------------------------------
("0uA" is just their way of saying a sample not treated with electricity,
to be used as a point of comparison.)
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