WO2018002836A1

WO2018002836A1 - Hiv infections

Info

Publication number: WO2018002836A1
Application number: PCT/IB2017/053861
Authority: WO
Inventors: Masixole Y LUGONGOLO; Sello Lebohang MANOTO; Saturnin OMBINDA-LEMBOUMBA; Patience MTHUNZI-KUFA
Original assignee: Csir
Priority date: 2016-07-01
Filing date: 2017-06-28
Publication date: 2018-01-04
Also published as: ZA201900365B

Abstract

A method of killing HIV-infected cells and/or inhibiting proliferation of HIV-infected cells includes exposing the HIV-infected cells to electromagnetic radiation, or administering electromagnetic radiation to the HIV-infected cells. The electromagnetic radiation has a wavelength of between 500 nm and 1500 nm.

Description

HIV INFECTIONS

THIS INVENTION relates to HIV infections. In particular, the invention relates to a method of killing HIV-infected cells, to use of electromagnetic radiation in killing HIV-infected cells, to an electromagnetic radiation for use in killing HIV-infected cells and/or in inhibiting proliferation of HIV-infected cells, and to a method of treating HIV-infected cells. The HIV/AIDS epidemic is a major challenge to public health, medicine and biological research worldwide. Globally, it is estimated that more than 35.3 million people are living with Human Immunodeficiency Virus (HIV) or AIDS and the number is expected to increase. Two third of people infected with HIV are in sub-Saharan Africa while South and Southeast Asia have the second highest population living with the HIV pandemic. This is followed by the Caribbean, Eastern Europe and Central Asia where 1% of adults were living with HIV in 2011. The use of highly active antiretroviral therapy (HAART) has significantly reduced mortality and morbidity in people infected with HIV-1, resulting in prolonged and improved quality of life, transforming the HIV infection into a chronic manageable disease. Although HAART can reduce plasma HIV-1 RNA levels to less than 50 copies per ml which is below the detection limit of most clinical assays, and although HAART has greatly reduced mortality rates, finding an HIV cure still remains a challenge. An effective vaccine or a treatment that would completely eradicate the virus from the system of infected individuals remains unavailable. Hence new strategies to fight HIV need to be explored. According to one aspect of the invention, there is provided a method of killing HIV- infected cells and/or inhibiting proliferation of HIV-infected cells, which includes exposing the cells to electromagnetic radiation, or administering electromagnetic radiation to the cells, the electromagnetic radiation having a wavelength of between 500 nm and 1500 nm. In one embodiment, the invention thus employs low level laser therapy to kill HIV- infected cells and/or to inhibit proliferation of HIV-infected cells. Low level laser therapy (LLLT) involves the exposure of cells or tissue to low levels of red and near infrared light. LLLT has been widely used in different medical conditions including skin diseases, diabetes and wound healing, but not in HIV infection. The inventors set out to determine the effects of LLLT on HIV infected and uninfected cells. Both HIV infected and uninfected cells were laser irradiated at different fluencies and changes in cellular responses were assessed using cell morphology, viability, proliferation, cytotoxicity and luciferase activity. It was surprisingly demonstrated that laser irradiation in the absence of HIV has no inhibitory effect on cells, while in the presence of HIV infection it induces cell damage in a dose dependent manner. This presents significant possibilities for the treatment of HIV using LLLT.

LLLT is also known as photobiomodulation or biostimulation and makes use of photons to modulate biological activity. LLLT involves the exposure of cells and tissue to red or infrared light (600 nm to 1100 nm) with laser power in the range of 1-1000 mW in order to stimulate cellular functions which lead to beneficial clinical effects. Light absorption induces photochemical effects to induce changes in cells that enhance cell proliferation. The effects of LLLT are not thermal, but rather biochemical, and therefore LLLT cannot cause damage to cells. LLLT has been used in different medical fields to treat skin conditions, wounds, sports injuries, chronic pain and other medical conditions. For LLLT to be effective there are several parameters that need to be considered. These include, but are not limited to fluence, wavelength, and pulse structure. LLLT uses low fluences ranging from 0.04 to 50 J/cm². The choice of parameters to use in LLLT experiments seems to be dependent on the experimenter's choice and experience, not from a consensus statement by an authoritative body. This to some degree makes comparing studies' successes and failures a challenge as different wavelengths and fluences are used. Even if one study could use fluences similar to that of another study, but the wavelength and the target tissue type or cell line is different, the outcomes could be different.

The exact biochemical mechanisms of action of LLLT are not well established, however it is known that photons are absorbed by chromophores, in particular cytochrome c oxidase (unit four in the mitochondria respiratory chain) resulting in an increase in the enzyme activity and electron transport. This then leads to increased production in adenosine triphosphate (ATP), reactive oxygen species (ROS) and nitric oxide (NO). These cytosolic responses can cause changes in cellular redox state and induce transcriptional changes via the activation of transcription factors. Several transcription factors are regulated by changes in cellular redox state such as redox factor-1 (Ref-1), dependent activator protein-1 (AP-1), p53, hypoxia-inducible factor (HIF)-l, nuclear factor kappa B (N FKB), activating transcription factor/cAMP-response element-binding protein (ATF/CREB), and Fos and Jun. These transcription factors cause protein synthesis which triggers an increase in cell proliferation and migration, modulation of growth factors and increased tissue oxygenation. In addition, it has been demonstrated that LLLT results in the elevation of antioxidants in the blood and expression of heat shock proteins. There is strong evidence which suggests that HIV-1 infected patients are under chronic oxidative stress. ROS has been suggested to be responsible for many aspects of HIV-1 pathogenesis such as increased viral replication, reduced immune cell proliferation, loss of immune function, sensitivity to drug toxicity and chronic weight loss. Furthermore, excessive production of ROS can result in oxidation of proteins, peroxidation of lipids and eventually cell death. LLLT can improve the activity of anti-oxidant enzymes through a photochemical process that accelerates the elimination of ROS. This can be achieved at a molecular level by altering the conformation of anti-oxidant enzymes. It has been shown that LLLT (532 nm) can enhance the activity of anti-oxidant enzymes and also induce production of more ROS, with the amount produced dependent on the dose of the laser irradiation.

Preferably, the electromagnetic radiation has a wavelength of between about 500 nm and about 1500 nm, and more preferably between about 600 nm and about 1100 nm.

The electromagnetic radiation may be produced mechanically and/or electrically and/or electronically. The electromagnetic radiation may be provided by a light beam, a light ray, light emissions, light radiation, or the like. More preferably, the electromagnetic radiation is provided by a laser beam. The laser beam will thus have a wavelength of 500 nm - 1500 nm and will thus lie in the near infrared or visible range of the light spectrum. Still more particularly, the laser beam may be a low level laser beam having a wavelength as hereinbefore described, and in particular in the range of 600 nm to 1100 nm. The cells may be treated with or exposed to an antiretroviral drug prior to exposure to or administering of the electromagnetic radiation. The antiretroviral drug may be selected from the group consisting of Efavirenz, Abacavir, Atazanavir, Atripla, Darunavir, Descovy, Dolutegravir, Elvitegravir, Emtricitabine, Etravirine, Eviplera, Evotaz, Fosamprenavir, Genvoya, Kivexa, Lamivudine, Lopinavir, Maraviroc, Nevarapine, Odefsey, Raltegravir, Rezolsta, Rilpivirine, Ritonavir, Stribild, Tenofovir, Triumeq, Truvada and Zidovudine.

The invention thus also proposes the combination of LLLT and HAART to aid in eliminating HIV in infected cells that have been treated with an antiretroviral drug. The non- nucleoside reverse transcriptase inhibitor drug, efavirenz, is preferred. This antiretroviral drug interferes with HIV replication by interfering with the functioning of the viral reverse transcriptase enzyme, which is responsible for transcribing viral RNA to DNA. The increase in CD4 positive cells boosts the immune system which in turn would vigorously eliminate the virus. The inventors have surprisingly found that the LLLT enhances the antiretroviral activity of the drug by inducing cellular damage in the HIV-infected cells. This presents a further attractive possibility to use LLLT in combination with antiretroviral drug therapy to kill HIV-infected cells or decrease the proliferation of HIV-infected cells.

In this specification, the term "killing HIV-infected cells" may include increasing the rate at which the HIV-infected cells are killed off.

In this specification, the term "inhibiting proliferation of HIV-infected cells" may include decreasing said proliferation. In this specification, "a low level laser beam" is a laser beam with laser power in the range of between about 1 mW and about 1000 mW. Preferably, the laser power is in the range of between about 1 mW and about 200 mW, more preferably between about 1 mW and about 100 mW, e.g. about 67 mW. The electromagnetic radiation can thus, in certain aspects or embodiments of the invention, constitute a light therapy or light regime to which the cells are exposed or which is administered to the cells. The electromagnetic radiation will thus serve to kill HIV-infected cells or inhibit proliferation of HIV-infected cells.

Killing of the cells or inhibiting cell proliferation may correlate with a varying dosage of the electromagnetic radiation.

The cell deaths or inhibited cell proliferation of HIV-infected cells may occur at an optimal dosage of the electromagnetic radiation, below or above which HIV-infected cell death may decrease and cell proliferation of HIV-infected cells may increase.

According to another aspect of the invention, there is provided the use of electromagnetic radiation in killing HIV-infected cells and/or in inhibiting proliferation of HIV- infected cells, the electromagnetic radiation having a wavelength of between 500 nm and 1500 nm.

The electromagnetic radiation is typically administered to the cells, or the cells are typically exposed to the electromagnetic radiation.

The electromagnetic radiation may be as hereinbefore described, and/or may be provided by a low level laser beam having a wavelength in the range of 600 nm to 1100 nm.

The electromagnetic radiation or low level laser beam may be provided by a laser generator or laser generating means, and the invention thus extends to use of a laser generator or laser generating means configured to generate electromagnetic radiation having a wavelength of between 500 nm and 1500 nm, in killing HIV-infected cells and/or inhibiting proliferation of HIV-infected cells.

The HIV-infected cells may be treated with or exposed to an antiretroviral drug prior to use of the electromagnetic radiation.

The antiretroviral drug may be as hereinbefore described. According to further aspect of the invention, there is provided electromagnetic radiation, having a wavelength of between 500 nm and 1500 nm, for use in killing HIV-infected cells and/or in inhibiting proliferation of HIV-infected cells.

The electromagnetic radiation or low level laser beam may be provided by a laser generator or laser generating means, and the invention thus extends to a laser generator or laser generating means configured to generate electromagnetic radiation having a wavelength of between 500 nm and 1500 nm, for use in killing HIV-infected cells and/or inhibiting proliferation of HIV-infected cells. The HIV-infected cells may be treated with or exposed to an antiretroviral drug prior to use of the electromagnetic radiation.

The antiretroviral drug may be as hereinbefore described. According to yet another aspect of the invention, there is provided a method of treating HIV-infected cells which includes exposing the cells to electromagnetic radiation or administering electromagnetic radiation to the cells, thereby killing HIV-infected cells or inhibiting proliferation of HIV-infected cells, the electromagnetic radiation having a wavelength of between 500 nm and 1500 nm.

The antiretroviral drug may be as hereinbefore described. Cell death or inhibiting cell proliferation of the HIV-infected cells may correlate to a varying dosage of the electromagnetic radiation. According to yet a further aspect of the invention, there is provided a use of electromagnetic radiation to kill HIV-infected cells or inhibit proliferation of HIV-infected cells, by administering the electromagnetic radiation to the cells, or exposing the cells to the electromagnetic radiation, the electromagnetic radiation having a wavelength of between 500 nm and 1500 nm.

The antiretroviral drug may be as hereinbefore described.

The invention will now be described in more detail, with reference to the following non-limiting example or study and the accompanying drawings.

In the drawings

FIGURE 1 shows, for the Example, an experimental setup for low level laser therapy; FIGURE 2A shows, for the Example, morphology of HIV uninfected (top panel) and HIV infected (bottom panel) TZM-bl cells irradiated with fluencies of 0, 2, 4, 6, 8 and 10 J/cm²;

FIGURE 2B shows for the Example, cell morphology of irradiated and non-irradiated uninfected, HIV infected and HIV infected with the drug TZM-bl cells;

FIGURE 3 shows for the Example, trypan blue assay to evaluate the percentage viability of HIV uninfected TZM-bl cells irradiated with fluencies of 0, 2, 4, 6, 8 and 10 J/cm² with significant differences between the controls and their respective experimental groups represented in the graph as (*) P<0.05, (**) P<0.01 and (***) P<0.001; FIGURE 4 shows for the Example, ATP luminescence assay to measure cellular viability of HIV infected and uninfected TZM-bl cells irradiated with fluencies of 0, 2, 4, 6, 8 and 10 J/cm2 with significant differences between controls and their respective experimental groups represented in the graph as (*) P<0.05, (**) P<0.01 and (***) P<0.001;

FIGURE 5 shows for the Example, MTT assay to evaluate cellular proliferation of (A) HIV infected and uninfected TZM-bl cells irradiated with fluencies of 0, 2, 4, 6, 8 and 10 J/cm² ; (B) uninfected and HIV infected TZM-bl cells irradiated at 2 to 10 J/cm² and the controls (NC, PC and DC), with significant differences between the controls and their respective experimental groups represented in the graph as (*) P<0.05 and (**) P<0.01, error bars represent the standard error of the mean where n = 3;

FIGURE 6 shows for the Example, flow cytometric analysis of uninfected unirradiated TZM-bl (A) and cells irradiated at different fluences of 2 (B), 4 (C), 6 (D), 8 (E) and 10 (F) J/cm² showing the distribution of different cell populations (live - bottom left quadrant), necrotic - top left quadrant, apoptotic - bottom right quadrant and dead - top right quadrant) detected by annexin V-FITC and PI staining;

FIGURE 7 shows for the Example, flow cytometric analysis of infected unirradiated TZM- bl cells (A) and irradiated at different fluences of 2 (B), 4 (C), 6 (D), 8 (E) and 10 J/cm² (F) showing the distribution of different cell populations (live - bottom left quadrant, necrotic - top left quadrant, apoptotic - bottom right quadrant and dead - top right quadrant) detected by annexin V-FITC and PI staining;

FIGURE 8 shows for the Example, LDH assay of (A) HIV infected and uninfected TZM-bl cells irradiated with fluencies of 0, 2, 4, 6, 8 and 10 J/cm² , (B) uninfected, infected and infected with drug TZM-bl cells irradiated at 2 - 10 J/cm² and the controls (NC, PC and DC), where significant differences between the controls and their respective experimental groups are represented in the graphs as (*) P<0.05, (**) P<0.01) and (***) P<0.001, and error bars represent the standard error of the mean where n = 3; and

FIGURE 9 shows for the Example, luciferase activity assay to monitor infection of (A) HIV infected and uninfected TZM-bl cells irradiated with fluencies of 0, 2, 4, 6, 8 and 10 J/cm2, (B) uninfected, HIV infected and infected with drug TZM-bl cells irradiated at 2 - 10 J/cm², where significant differences between controls (NC, PC and DC) and their respective experimental groups are represented on the column bars as (**) = P < 0.01 and (***) = p < 0.001. EXAMPLE

Materials and methods Cell lines

The TZM-bl cell line (ATCC, PTA-5659) and 293T/17 cells (ATCC, CRL, 11268) were used. Both cell lines are adherent and they were maintained in Dulbeco's minimal essential medium (DMEM) growth medium (Sigma-Aldrich, D5796) containing 10% fetal bovine serum (FBS, FBS Superior, S 0615), 0.5% L-Glutamine-Penicillin-Streptomycin (Sigma-Aldrich, G6784). Since both cell lines were adherent, trypsin-EDTA solution (Sigma-Aldrich, T4049) was used for cell harvesting experiments as it causes the cells to detach from the flask surface and causes the cells to be in suspension so that they could be counted and transferred from one flask to another. This process was mainly used for cell maintenance and also when cells were to be used in experiments.

Cells were harvested using trypsin/EDTA solution (Sigma-Aldrich, T4049). The cell lines were maintained at 37°C in 5% C0₂ and 85% humidity. The 293T/17 cell line was used only for the production of the pseudovirus which was used to infect the TZM-bl cell line used in all the experiments.

HIV-1 ZM53 Env pseudovirus production and titration

Stocks of HIV-1 Env pseudovirus were produced by co-transfecting 293T/17 cells (3 x 10⁶ cells per 75cm² flask) with 4 μg of an HIV-1 ZM53 env expression plasmid and 8 μg of an env deficient HIV-1 backbone plasmid (pSG3 Env). The Superfect transfection reagent (Qiagen, 310305) was added in the mixture of the two plasmids in order to facilitate the entry of DNA into cells with ease due to the interaction between the cell surface charges and charges on the Superfect reagent. Psuedovirus-containing supernatant was harvested 48 hours following transfection and clarified by 0.45μιη filtration. The 50% tissue culture infectious dose (TCID50) for the pseudovirus was determined by infection of TZM-bl cells to determine the infectious titre as previously described (Li M, Gao F, Mascola JR, Stamatatos L, Polonis VR, Koutsoukos M, Voss G, Goepfert P, Gilbert P, Greene KM, Bilska M, Kothe DL, Salazar-Gonzalez JF, Wei X, Decker JM, Hahn BH and Montefiori DC (2005) Human immunodeficiency virus type 1 env clones from acute and early subtype B infections for standardized assessments of vaccine-elicited neutralizing antibodies. J. Virol. 79:10108-10125.). The plasmids were a donation from Prof Maria Papathanasopoulos, Director of the HIV Pathogenesis Research Unit at the University of the Witwatersrand, Johannesburg, South Africa.

In vitro infection

There were two experimental groups. In group 1, cells were infected with the pseudovirus. In group 2, cells were infected with the pseudovirus and, after 48 hours incubation, the drug efavirenz was added. A pseudovirus with a volume corresponding preferably from about 5000 RLU (relative luminescence units) to about 50 000 RLU, more preferably from about 10 000 RLU to about 30 000 RLU, and most preferably from about 15 000 RLU to about 25 000 RLU) was used. For example, a pseudovirus with a volume of 250 μΙ was placed in a 2.3 cm diameter tissue culture dish. Subsequently cell suspension of TZM-bl cells (2 x 10⁵) containing 25 μg/ml of DEAE dextran (Sigma-Aldrich, D9885) was added to the tissue culture dish. The culture dish was then incubated at 37°C in 5% C0₂ and 85% humidity for 48 hours. After 48 hours incubation, efavirenz to a final concentration of 20 ug/ml was added and incubated for 30 minutes at 37°C in 5% C0₂ and 85% humidity.

Optical setup and laser irradiation

Figure 1 depicts the low laser level therapy setup used. Briefly, a continuous wave (CW) diode laser (Cube Coherent L00610901) operating at, for example, 660 nm, with a maximum output power 100 mW and beam size of about 1.2 mm was used for cells irradiation. Light from the diode laser is magnified about 20x using a telescope system comprising of two optical lenses (LI and L2) in order to overfill the entire sample area of 4.2 cm² in a petri dish with a diameter of 23.5 mm. The magnified laser beam is then reflected by a totally reflective silver mirror (Ml) to a petri dish where the sample is placed. The laser light power at the sample point was, in this instance, 67 mW. Certain irradiation parameters of the laser were considered. Table 1 shows the summary of the irradiation parameters. Prior to laser irradiation, the growth medium from the previous 48 hr incubation was removed and cells were washed twice with Hank's Balance Salt Solution (HBSS; Gibco, Life Technologies, 14170-088) and 1 ml of fresh growth medium was added. Cells were then irradiated with the diode laser and all laser irradiations were conducted in the dark. Post irradiation culture dishes were incubated for 24 hours at 37°C in 5% C0₂ and 85%, before biological assays were conducted.

Table 1: Laser irradiation parameters

Variable Parameters

Wavelength 660 nm

Power at the sample 67 mW

Pulsed or continuous wave Continuous wave

Illuminated area 4.2 cm²

Irradiation time 125 s, 250 s, 376 s, 501s and 626s

Energy density 0, 2, 4, 6, 8 and 10 J/cm²

Beam profile Gaussian

Irradiation times were determined using this formula:

t = 0xA/P (1)

where t is the irradiation time, A is the irradiated area, P is the power of the beam on the sample, 0 is fluence. A higher power of the beam on the sample requires less irradiation time to obtain a predetermined fluence as per a predetermined area, and vice versa.

Fluence was determined using the following formula:

Fluence = J/cm² (2) where J is laser pulse energy in joules and cm² is the effective focal spot area.

Prior to laser irradiation, cells were washed twice with HBSS and 1 ml of fresh growth medium was added. Cells were then irradiated with the diode laser and all laser irradiations were conducted in the dark to prevent contamination from stray light. Post laser irradiation culture dishes were incubated for 24 hours at 37°C in 5% CO² and 85% humidity, before biological assays were conducted.

Cell morphology

Changes in cell morphology were assessed using an inverted light microscope (CKX41, Olympus) attached to a digital camera. Once digital pictures were taken, cells were trypsinized and resuspended to perform further assays.

Trypan blue ossoy

The Trypan blue assay (Sigma-Aldrich, T8154) was used to determine the percentage viability of cells. In this assay, viable cells with an intact cellular membrane do not take up the dye and maintain a clear appearance under the microscope while damaged nonviable cells are stained blue as they take up dye. A 1:2 dilution was done by carefully mixing an equal volume of 0.4% Trypan blue reagent and cell suspension. This was then transferred onto a Neubauer hemacytometer counting chamber and cells in the 1 mm² central square and 4 outer squares were counted. Percentage viability was determined by multiplying the viable cell number by 100 then dividing the obtained number by the total cell number (blue and clear).

Adenosine triphosphate assay

The CellTiter-Glo^® luminescent cell viability assay (Promega, Anatech, G7573) is a homogenous method for determining the number of viable cells based on the quantification of the ATP present in metabolically active cells. Equal volumes of cell suspension and reconstituted reagent were added together and mixed to induce cell lysis. The mixture was allowed to stabilize at room temperature for 10 minutes in the dark. The luminescence was recorded using the GloMax^® Discover System (Promega, Anatech) in relative light units (RLUs). MTT assay

The CellTiter 96^® non-radioactive cell proliferation assay (Anatech, Promega) is a rapid and convenient method of determining viable cell number in proliferation. This assay is based on the cellular conversion of tetrazolium salt into a formazan product. Fifteen microlitres of the Dye solution was added to 100 μΙ of the cell suspension and incubated at 37°C for 4 hours for the conversion to take place. After incubation, 100 μΙ of the Solubilization Solution/Stop Mix was added and the plate was left at room temperature for 1 hour. To get a uniformly coloured solution, the contents were mixed using a multichannel pipette, avoiding bubble formation. The absorbance was read at 560 nm using the GloMax^® Discover System.

Lactate dehydrogenase assay

The Lactate Dehydrogenase (LDH) assay (Sigma-Aldrich, TOX7) is a means of measuring membrane integrity as a function of the amount of cytoplasmic LDH released into the medium. It is used to evaluate the presence of cell membrane damage and cytoxicity. The supernatant (culture media) was removed before the cells were trypsinized and spun down at 2200 rpm. Hundred microlitres of the supernatant was added to a 96 well plate and 200 μΙ of the LDH assay mixture was then added. The 96 well plate was incubated in the dark for 30 minutes at room temperature and the reaction was terminated by the addition of IN Hydrochloric acid. Absorbance was measured at 490nm using GloMax^® Discover System.

Luciferase assay Luciferase activity was assessed using Bright-Glo™ luciferase assay system (E2610,

Promega, Anatech) to evaluate infection efficiency in TZM-bl cells. TZM-bl cells have the firefly luciferase reporter gene under the control of HIV-1 LTR, which is expressed in the presence of HIV infection. In the absence of HIV infection, the luciferase gene will not be expressed. Hundred microlitres of the luciferase reagent was added to an equal volume of the cell suspension and incubated in the dark for 2 minutes. The luciferase activity was quantified using the GloMax^® Discover System. The RLUs are directly proportional to the number of infectious virus particles present in the initial inoculum. Flow cytometry

The flow cytometry experiments using Fluorescein isothiocyanate (FITC) Annexin V Apoptosis Detection Kit I (BD Biosciences, Johannesburg, South Africa) were performed to determine the type of programmed cell death experienced by cells following HIV infection. These experiments were conducted on cells not treated with HIV drugs but exposed to LLLT. Following irradiation at different fluences, the cells were incubated for further 24 hours, thereafter flow cytometry experiments were completed. During flow cytometry experiments, the cell culture medium was removed and placed in a 15 ml centrifuge tube. Following this, cells in the culture dish were rinsed with HBSS, and 500 μΙ of trypsin was added in each culture dish and placed in the incubator for 5 minutes to allow cells to detach from the culture dish, leaving them in suspension. Once successfully in solution, 1 ml of growth medium was added to the cells and this mixture transferred to a 15 ml tube containing the culture medium and centrifuged for 10 minutes at 2200 rpm.

The cell pellet was then rinsed twice with cold PBS and re-suspended by adding 500 μΙ of IX annexin V binding buffer. From the 500 μΙ cell suspension, 100 μΙ was stained in 5 ml tubes with 5 μΙ of annexin-V and 5 μΙ of propidium iodide (PI) and incubated on ice for 30 minutes. The stained cells were suspended in 400 μΙ of annexin V binding buffer before flow cytometric analysis. For positive controls, apoptosis and necrosis were induced using Dimethyl sulfoxide (DMSO) and ice cold methanol, respectively. The samples were sorted using the BD Accuri C6 cytometer. Statistics

The TZM-bl cells between passages 8 and 17 were used. Each experiment was repeated four times (n=4). Biochemical assays were done in triplicates, and the average of the results was used. Untreated cells were included in the study and comparisons were made between the treated and the untreated to determine the statistical difference. Statistical analysis was performed using Sigma Plot version 11.0 and the mean, standard deviation and standard error were obtained. Statistical significances between the controls and their respective experimental groups are shown in the graphs as P<0.05 (*), P<0.01 (**) and P<0.001 (***). The non-irradiated cells, uninfected, infected and infected with drug were used as controls in the study, with uninfected cells being the negative control (NC), infected cells representing the positive control (PC) and the cells infected with drug considered the drug control (DC).

Results

Cell Morphology Cell morphology changes of TZM-bl cells that were uninfected, infected, irradiated and those that were exposed to efavirenz were assessed using light microscopy (Figure 2A, 2B). The top row has controls (NC = negative control, which is uninfected and non-irradiated cells, PC = positive control, which is non-irradiated infected cells and the DC = drug control, which is non- irradiated infected cells in the presence of efavirenz), while the bottom row has cells that were laser irradiated. The negative control (NC - cells with no infection and no drug) and all the uninfected cells irradiated at different fluences showed no changes in cellular morphology and appeared similar to untreated control cells, with healthy cells growing as a monolayer sheet of cells (Figure 2A). This correlates with what was observed in keratinocytes in another study that monitored the bio-stimulatory effects of laser irradiation on cells in vitro. The infected cells and those with drug looked stressed as there was an increase in the number of round cells (Figure 2B). Rounding of cells indicate that the infecting virus has cytopathic effects on the virus permissive cells. Non-irradiated infected cells became round indicating cell stress. These changes correlate with those of cells undergoing cell death after HIV infection. Infected cells irradiated with 2, 4, 6, 8 and 10 J/cm² showed a dose dependent increase in cell stress as indicated by an increase in the number of round cells and the presence of floating cells. This is due to the cytopathic effects of HIV infection on cells which causes the rounding of infected cells, fusion with adjacent cells to form syncytia and the appearance of nuclear or cytoplasmic inclusion bodies. Trypan blue ossoy

The trypan blue assay was done to evaluate the percentage viability of cells (Figure 3). Uninfected cells irradiated with 2, 4, 6, 8 and 10 J/cm² showed no significant differences in cellular viability when compared to non-irradiated uninfected cells. Infected cells (untreated with the drug) irradiated with 2, 4, 6, and 8 J/cm² also showed no changes in cellular viability in comparison to non-irradiated infected cells. This is not in agreement with the cell morphology results where cell stress was seen with an increase in fluence. This could be attributed to the principle of trypan blue assay which considers cells that are no longer active as viable cells because the cell membrane would still be intact thereby increasing the percentage of viable cells. Infected cells irradiated with 10 J/cm² showed a significant decrease in viability as compared to non-irradiated infected cells. There were no statistical significant differences noted between uninfected and infected cells except for infected cells irradiated with 10 J/cm². This correlates with cell morphology of infected cells irradiated with 10 J/cm².

Adenosine triphosphate assay

The ATP assay was used to assess viability of cells (Figure 4). The cells here were not treated with the antiretroviral drug. Uninfected cells irradiated with 2, 6, 8 and 10 J/cm² showed no significant differences in cellular viability when compared to non-irradiated uninfected cells. However, uninfected cells irradiated with 4 J/cm² showed a significant increase in cell viability as compared to non-irradiated uninfected cells (P<0.001). This result suggests that 4 J/cm² stimulates ATP production in HIV uninfected TZM-bl cells. Infected cells irradiated with 2, 4, and 6 J/cm² showed a significant decrease in cell viability when compared to non-irradiated infected cells (P<0.001; P<0.01 and P<0.01 respectively) while infected cells irradiated with 8 and 10 J/cm² showed no significant differences.

Non-irradiated infected cells showed a significant increase in cell viability as compared to non-irradiated uninfected cells (P<0.001). The significantly high ATP levels seen in non-irradiated infected cells could be attributed to the function of the HIV p2 peptide of the Gag protein, which has been previously shown to increase ATP content in the early stage of HIV infection so that efficient reverse transcription occurs, thereby producing mature and infectious virions. Infected cells irradiated with 2, 4 and 6 J/cm² showed a decrease in cellular viability as compared to their respective uninfected cells (P<0.05; P<0.001 and P=0.293). On the contrary, infected cells irradiated with 8 and 10 J/cm2 showed an increase in cellular viability (P=0.193 and P<0.001 respectively). The difference in the cellular viability at different fluences could be attributed to the biphasic dose response.

MTT assay

The MTT assay was used to evaluate cell proliferation (Figure 5A, 5B). Uninfected cells (NC) irradiated with 2, 4, 6, 8 and 10 J/cm² showed no significant changes in cell proliferation as compared to non-irradiated uninfected cells. Uninfected cells (NC) showed a significantly higher proliferation compared to the infected (PC) and the infected cells with the drug (DC) controls (Figure 5B). Even after cells have been irradiated, uninfected cells proliferated better than the infected cells and cells with the drug. Infected cells with drug proliferated poorly compared to the uninfected and infected cells only, except at fluences 2 and 10 J/cm² where they proliferated similar to the infected ones. Similarly to that of uninfected cells, no significant differences were noted between infected cells irradiated with 2, 4, 6, 8, and 10 J/cm² and non- irradiated infected cells. Infected cells (PC) irradiated with 0, 2, 4, 6, 8 and 10 J/cm² showed a decrease in cell proliferation as compared to their respective uninfected cells. This is due to the cytopathic effects of HIV infection and this correlates with what was observed in cell morphology. HIV can cause destruction of cells by direct cytotoxicity of the infected cells. The only statistically significant decrease in cell proliferation between infected and uninfected cells was noted in cells irradiated with 4, 6 and 8 J/cm² (P<0.05; P<0.01 and P<0.05). Flow cytometry

During flow cytometry, the cells were sorted to acquire unmixed populations of cells that were either viable, necrotic, apoptotic or dead. Using flow cytometry, it was observed that uninfected cells were viable and remained unstained, early apoptotic cells were positively stained with annexin V, necrotic cells were positively stained with propidium iodide and already dead cells were stained positively with both annexin-V and propidium iodide. The apoptosis positive control was prepared by adding DMSO to uninfected TZM-bl cells that had been incubated for 48 hours and were 80-90% confluent at the time of adding DMSO. After incubating cells with DMSO overnight, the cells were then treated the same way as the experimental cells in preparation for the flow cytometry experiments. Contrarily the necrosis positive control was also prepared in a similar fashion, whilst replacing DMSO with ice cold methanol employed to induce both mechanical and chemical stress.

The uninfected cells (Figure 6) had a high population of live cells as shown by different percentages 88.2 % (A), 96.9 % (B), 94 % (C), 88.4% (D), 92.3% (E) and 88.9% (F) for the unirradiated and those irradiated at fluences 2 to 10 J/cm², respectively. The live cells are those cells that did not take up any of the dyes (PI negative and annexin V negative) and they are in the bottom left quadrant. The uninfected cells irradiated at 4 (C) and at 10 J/cm² (F) had 0 % dead cells, while the unirradiated cells (A) and those irradiated at 2 (B), 6 (D) and 8 (E) J/cm² had 4 %, 0.1 %, 5.3 %, 3.4 % of dead cells, respectively. The dead cells are those cells which took up both dyes (PI positive and annexin V positive) and they are in the top right quadrant. For all the samples, there were necrotic cells present 4.9 % (A), 2 % (B), 4.2 % (C), 3.5 % (D), 2.9 % (E) and 11.1 % (F), respectively. The necrotic cells are those cells that took up only the PI stain (PI positive and annexin V negative) and they are in the top left quadrant. The uninfected cells irradiated at 10 J/cm² (F) did not have apoptotic cells, while the unirradiated cells (A) and those irradiated at 2 to 8 J/cm² had 2.8 % (A), 1 % (B), 1.8 % (C), 2.8 % (D) and 1.4 % (E) population of apoptotic cells. Apoptotic cells are those cells that took up only the annexin V-FITC (annexin V positive and PI negative), they are in the bottom right quadrant.

The infected cells (Figure 7) had a small population of live cells as shown by different percentages 5.4 % (A), 1.5 % (B), 0 % (C), 0 % (D), 0 % ( E) and 1.7 % (F) for the unirradiated and those irradiated at fluences 2 to 10 J/cm², respectively. The live cells did not take up any of the dyes (PI negative and annexin V negative) and they are in the bottom left quadrant. The infected cells unirradiated and those irradiated at 2 to 10 J/cm² had 36.6 % (A) , 20.4 % (B), 42.1 % (C), 90.5 % (D), 93.5 % (E) and 62.1 % (F) of dead cells, respectively. The dead cells are those cells that took up both dyes (PI positive and annexin V positive) and they are in the top right quadrant. For all samples, there were necrotic cells present at 58.8 % (A), 77.6 % (B), 57.9 % (C), 9.5 % (D), 6.5 % (D), and 24.1 % (F), respectively. The necrotic cells are those cells which took up only PI stain (PI positive and annexin V negative) and they are in the top left quadrant. The infected cells irradiated at 4 to 8 J/cm² did not have apoptotic cells, while the unirradiated and those irradiated at fluences of 2 and 10 J/cm² had 0.1 % (A), 0.5 % (B) and 12.1 % (F) apoptotic population, respectively. Lactate dehydrogenase assay

LDH assay was used to determine cell membrane damage (Figure 8A, 8B). LDH is a cytosolic enzyme which is released by cells with damaged cell membranes, making it a suitable marker for necrosis. Uninfected cells irradiated with 2, 4, 6, 8 and 10 J/cm² showed no significant differences in cell membrane damage when compared to non-irradiated uninfected cells. Infected cells irradiated with 2, 6, 8 and 10 J/cm² showed a dose dependent increase in cell membrane damage as compared to non-irradiated infected cells (P<0.01; P<0.01, P<0.001 and P<0.001) (Figure 8A). This is in agreement with morphological changes observed in infected cells after laser irradiation with fluencies of 2, 4, 6, 8 and 10 J/cm². There was no statistical significance in cell membrane damage between non-irradiated infected cells and those irradiated at 4 J/cm². Infected cells irradiated with 0, 2, 4, 6, 8 and 10 J/cm² showed an increase in cell membrane damage as compared to their respective uninfected cells. However, the only statistically significant increase in cell membrane damage was observed in infected cells irradiated with 2, 6, 8 and 10 J/cm² (P<0.01; P<0.01; P<0.001 and P<0.001).

There were notable differences in the controls in the drug treated cells. PC and DC showed an increase in LDH levels as compared to NC (Figure 8B). The same pattern was maintained in irradiated cells. No significant changes were observed when NC was compared to uninfected irradiated cells. Infected cells irradiated with 6 and 8 J/cm2 showed a significant increase in LDH levels as compared to PC (P < 0.05 and P < 0.01 respectively). Infected cells with the drug irradiated with 2, 4, 6, 8, and 10 J/cm2 showed a significant increase in LDH levels compared to DC (P < 0.01, P < 0.01, P < 0.05, P < 0.001, P < 0.001). According to these observations, irradiation had no detrimental effect on the cell membrane of the uninfected cells. LDH levels were significantly elevated at all fluencies in the presence of a drug. Luciferase ossoy

The luciferase assay was used to monitor HIV infection (Figure 9A, 9B). Uninfected cells irradiated with 2, 4, 6, 8 and 10 J/cm² showed significant decrease (P<0.001, P<0.05, P<0.01, P<0.001 and P<0.01 respectively) in luciferase luminescence when compared to non-irradiated uninfected cells (Figure 9A). Essentially, the uninfected cells are not expected to demonstrate luciferase activity, as the luciferase gene in TZM-bl cells is only expressed when HIV infection has taken place. However, the luciferase activity seen in uninfected cells could be as a result of endogenous bioluminescence. Non-irradiated infected TZM-bl cells in the absence of efavirenz showed a significant increase in luciferase luminescence as compared to non-irradiated uninfected cells (P<0.001). Infected cells irradiated with 2, 4, 6, 8, and 10 J/cm² also showed a statistically significant increase in HIV infection when compared with uninfected cells (P<0.001). This result indicates that TZM-bl cells are susceptible to HIV infection and the pseudovirus used in this study successfully infected the cells. TZM-bl cells express CD4, CCR5 and CXCR4 which are cell surface receptors that HIV uses to gain entry into target cells. These cells contain the firefly luciferase gene under the control of HIV-1 LTR which is expressed when HIV infection takes place. Due to the nature of these cells, the significantly high luciferase luminescence in HIV infected cells was expected. There was a significant decrease in luciferase luminescence in infected cells irradiated with 2, 4, 6, 8 and 10 J/cm² as compared to non-irradiated HIV infected cells (P<0.01, P<0.05, P<0.05, P<0.01 and P<0.05, respectively). The significant reduction in luciferase luminescence in irradiated cells is a clear indication that laser irradiation interfered with HIV infection. The mechanisms in which laser irradiation interfered with HIV infection is not clear.

In drug-treated cells, PC and DC showed an increase in luciferase activity when compared to NC (Figure 9B). The same trend was seen in all the groups except for infected cells with the drug irradiated at fluencies of 6 and 10 J/cm². No significant changes were noted when NC was compared to uninfected irradiated cells. Infected irradiated cells showed a significant decrease in luciferase activity when compared to PC (P < 0.001). Irradiated infected cells treated with the drug showed a significant reduction in luciferase activity when compared to DC (P < 0.01 and P< 0.001). The results show that HIV infection and efavirenz have undesirable effects on the cell viability, which compromises cell integrity as shown by cell shape change via cell rounding in Figure 2, poor cell proliferation in Figure 5 and increased LDH levels in Figure 8. It is known that HIV can cause direct cytotoxicity on the infected cells. On the other hand, efavirenz has been shown to induce oxidative stress in many cell types which results in the induction of heat shock proteins, endoplasmic reticulum (ER) stress, autophagy and apoptosis.

Furthermore, the reduction in cell proliferation and an increase in LDH levels in both the infected cells and the infected cells with efavirenz at some fluences, demonstrates that irradiation in the presence of HIV infection and efavirenz in TZM-bl cells kills cells. Cell death was generally observed as the fluences increased. The different cell responses to laser irradiation at different fluences are not unusual. It is known that there is a biphasic dose response associated with LLLT. At lower doses laser irradiation has beneficial effects, while it has damaging effects at higher doses. Various substances or molecules (e.g. DNA, proteins) in the cells and tissues being irradiated and wavelengths used can influence the response of the cells to laser irradiation. There is also evidence which suggests that in the presence of HIV infection there is oxidative stress, which hinders the ability of cells to repair and detoxify when there is damage. Both HIV infection and efavirenz induce oxidative stress in cells, hence poor cell proliferation and high LDH levels.

Uninfected irradiated and non-irradiated cells showed no significant differences in luciferase activity (Figure 9), which is an indication that irradiation did not have any influence on the expression of the luciferase gene in the absence of an infection. Luciferase activity in infected cells confirmed that TZM-bl cells are permissive to HIV infection; the presence of CD4 and CCR5 on the surface of TZM-bl cells enables HIV entry into these cells. These cells also contain a firefly luciferase gene under the control of HIV LTR, which is expressed when HIV infection takes place. The significant reduction in luciferase activity in irradiated infected cells is an indication that irradiation interferes with HIV infection, however the mechanism by which irradiation reduces infection in TZM-bl is still unknown. Further reduction in luciferase activity in infected cells treated with the drug confirms that efavirenz inhibits HIV replication . Efavirenz is an antiretroviral drug used for the treatment of HIV. It is generally used in combination with other antiretrovirals in the form of HAART. It is a non-nucleoside reverse transcriptase inhibitor, which blocks the functioning of the reverse transcriptase. Reverse transcriptase is an essential viral enzyme that transcribes viral RNA into DNA. Efavirenz blocks the functioning on the viral reverse transcriptase enzyme by binding to a distinct site away from the enzyme's active site, thereby altering the active site. The further reduction of HIV infection in irradiated cells in the presence of efavirenz is an indication that the combination of the two therapies has a great potential against HIV infection.

Although HAART have dramaticaly improved the outcomes of HIV treatment by shutting down virus production, HIV infection remains incurable. HIV continues to spread worldwide and currently the number of infections is reported to be approximately 2.6 million per year. New therapeutic approaches that can fight HIV/AIDS and overcome the need of lifelong adherence to antiretroviral drugs needs to be explored. In the study, as described herein, the reduction of HIV infection in laser irradiated in cells was demonstrated. Further reduction in HIV infection was demonstrated when laser irradiation was combined with an antiretroviral drug. In addition, it was shown that uninfected TZM-bl cells were stimulated by laser irradiation, while the effects of both HIV infection and irradiation had detrimental effects on HIV infected cells, both treated and untreated with antiretrovirals, at specific doses of irradiation.

More particularly, the study has shown that laser irradiation in the absence of HIV infection does not have any inhibitory effects in TZM-bl cells. However, it shows stimulatory effects by increased ATP production when using 4 J/cm² and significant cell proliferation. On the contrary, irradiating cells in the presence of HIV infection, both in cells that were treated and untreated with antiretrovirals, surprisingly induce stress as exhibited by cell rounding and high levels of LDH released. Laser irradiation therefore reduces HIV infection in TZM-bl cells by inhibiting proliferation and increasing cell death. It appears from the results that the combination of both HAART and LLLT against HIV infection could aid in the elimination of the infection.

Claims

CLAIMS:

1. A method of killing HIV-infected cells and/or inhibiting proliferation of HIV- infected cells, the method including exposing the HIV-infected cells to electromagnetic radiation, or administering electromagnetic radiation to the HIV-infected cells, wherein the electromagnetic radiation has a wavelength of between 500 nm and 1500 nm.

2. The method of claim 1, wherein the electromagnetic radiation is provided by a low level laser beam having a wavelength in the range of 600 nm to 1100 nm.

3. The method of claim 1, wherein the HIV-infected cells have been treated with or exposed to an antiretroviral drug prior to exposure to or administering of the electromagnetic radiation.

4. The method of claim 3, wherein the antiretroviral drug is selected from the group consisting of Efavirenz, Abacavir, Atazanavir, Atripla, Darunavir, Descovy, Dolutegravir, Elvitegravir, Emtricitabine, Etravirine, Eviplera, Evotaz, Fosamprenavir, Genvoya, Kivexa, Lamivudine, Lopinavir, Maraviroc, Nevarapine, Odefsey, Raltegravir, Rezolsta, Rilpivirine, Ritonavir, Stribild, Tenofovir, Triumeq, Truvada and Zidovudine.

5. Use of electromagnetic radiation in killing HIV-infected cells and/or in inhibiting proliferation of HIV-infected cells, wherein the electromagnetic radiation has a wavelength of between 500 nm and 1500 nm.

6. The use of claim 5, wherein the electromagnetic radiation is provided by a low level laser beam having a wavelength in the range of 600 nm to 1100 nm.

7. The use of claim 5, wherein the HIV-infected cells have been treated with or exposed to an antiretroviral drug prior to use of the electromagnetic radiation.

8. The use of claim 7, wherein the antiretroviral drug is selected from the group consisting of Efavirenz, Abacavir, Atazanavir, Atripla, Darunavir, Descovy, Dolutegravir, Elvitegravir, Emtricitabine, Etravirine, Eviplera, Evotaz, Fosamprenavir, Genvoya, Kivexa, Lamivudine, Lopinavir, Maraviroc, Nevarapine, Odefsey, Raltegravir, Rezolsta, Rilpivirine, Ritonavir, Stribild, Tenofovir, Triumeq, Truvada and Zidovudine.

9. An electromagnetic radiation for use in killing HIV-infected cells and/or in inhibiting proliferation of HIV-infected cells, wherein the electromagnetic radiation has a wavelength of between 500 nm and 1500 nm.

10. The electromagnetic radiation for use of claim 9, wherein the electromagnetic radiation is provided by a low level laser beam having a wavelength in the range of 600 nm to 1100 nm.

11. A method of treating HIV-infected cells, the method including exposing the HIV- infected cells to electromagnetic radiation, or administering electromagnetic radiation to the HIV-infected cells, thereby killing the HIV-infected cells or inhibiting proliferation of the HIV- infected cells, wherein the electromagnetic radiation has a wavelength of between 500 nm and 1500 nm.

12. The method of claim 11, wherein the electromagnetic radiation is provided by a low level laser beam having a wavelength in the range of 600 nm to 1100 nm.

13. The method of claim 11, wherein the HIV-infected cells have been treated with or exposed to an antiretroviral drug prior to exposure to or administering of electromagnetic radiation.