Patent Grant
10336817
The Applicant hereby incorporates by reference the sequence listing contained in the ASCII text file titled 32490.44_Sequence_Listing_ST25, created on Jul. 3, 2017, and having 64 KB of data.
This application claims the benefit of South African Patent Application No. 2016/07084, filed on Oct. 14, 2016.
A therapeutic composition of camel milk relates to a composition useful for medical treatment, and particularly, to a composition useful for the treatment of HIV/AIDS.
Infection with Human Immunodeficiency Virus (HIV), a pathogenic retrovirus, can cause Acquired Immunodeficiency Syndrome (AIDS). AIDS is a major global health emergency, and is the sixth leading cause of early death in the world and the third in low-income countries. AIDS is considered an important cause of morbidity and mortality among young people. Moreover, it has reduced the life expectancy at birth in the African continent to 49 years from 53 years.
AIDS was first recognized in the United States in 1981 and has become one of the biggest problems facing the world today. A total of 36.9 million people were infected by HIV by the end of 2014. It has also been observed that the annual rate of new infection with HIV in the entire human population is not declining as 2 million people became newly infected with HIV in 2014 globally.
There are more than 21 million people who died from AIDS in the last decade. Although the rate of death due to AIDS has begun to drop in some nations, by an annualized rate of 2.8% between 2000 and 2013 as in the United States, primarily through the recent use of combination drug therapies against HIV infection and/or through the strict religious and moral codes. However, it is estimated that 16,000 people worldwide are being infected daily with HIV. Treatment success also has been limited by poor tolerance of the treatments by patients and the emergence of resistant strains of HIV. Thus, a need exists for an effective HIV treatment that is well tolerated, relatively inexpensive, and easily accessible.
Although great efforts have been dedicated to effective remedial and preventive methods for many years, there is no working vaccine or cure for HIV/AIDS yet. An ideal vaccine should be innocuous and capable of inducing neutralizing antibodies as well as persistent immune responses in the mucous membrane and blood. Many HIV vaccines currently developed in the world are still in the stages of animal trials. Although vaccines against HIV membrane proteins gp160 and gp120 have already moved into first, second, or third stages of clinical trials, the results of the trials are disappointing. Moreover, many vaccines that are effective to prevent HIV infection in small laboratory animals are not necessarily effective in humans. Although the subsequent or the second generation DNA vaccines seem to influence towards both humoral and cellular immune responses regardless of animal models used, researchers suggested that new modified DNA vaccines can be more efficient by broadly activate CD8+ cytotoxic T lymphocytes (CTLs) in larger animal models, compared with previous approved DNA methods. The fact that scientists are making little progress in HIV vaccine research could be attributed to the complexity and variability of HIV genetic materials. Although the use of combination drug therapies against HIV has proven to be effective in many patients, the current drug regimens are far from ideal due to several drawbacks and side effects documented in the literature.
Currently novel therapeutic approaches are being investigated at an encouraging rate and have the potential to improve the odds against the virus. Among them, immunotherapeutic approaches are one of the exciting areas and can be considered as adjunct to combined antiretroviral therapy for improving immune competence and can provide a sustained control of HIV replication.
Understanding the immune responses against HIV infection is essential for immunotherapy and the treatment of HIV/AIDS. Immunotherapy is meant to help the natural immune system in accomplishing control over viral infection. Different immunotherapy configurations have been assessed in either therapy-naive or therapy-experienced HIV-tainted patients throughout the last 20 years. These arrangements or configurations included non-antigen specific approaches, for example, cytokines that invigorate immunity or stifle the viral replication, and additionally antibodies that piece negative regulatory pathways. Various HIV-specific therapeutic vaccinations have additionally been proposed, utilizing inactivated virus in vivo, plasmid DNA encoding HIV antigens, or recombinant viral vectors containing HIV genes. These findings may give an objective and rational for immunotherapy in persons with progressive infection and have inspired an expanding effort to develop new strategies to enhance immunity.
Thus, a therapeutic composition solving the aforementioned problems is desired.
A therapeutic composition of camel milk can include an herbal composition having solid material or liquid extracts from the solid material of at least one of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne. The solid material may include one or more of parts or the whole of the stem, the bark, the flowers and the roots of one or more, but preferably all of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne. The therapeutic composition can include camel milk. The camel milk can include camel's milk having anti-HIV antibodies therein (HIV immunized camel milk). The therapeutic composition can include the herbal composition mixed with the camel milk to provide a treated camel milk. The HIV immunized camel milk can be obtained from a camel immunized against HIV. The camel can be immunized against HIV by administering to the camel the modified DNA plasmids of SEQ. ID No.: 3 and SEQ ID No.: 4.
These and other features of the present invention will become readily apparent upon further review of the following specification and drawings.
Similar reference characters denote corresponding features consistently throughout the attached drawings.
A therapeutic composition of camel milk can include an herbal composition having solid material or liquid extracts from the solid material of at least one of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne. The solid material may include one or more of parts or the whole of the stem, the bark, the flowers and the roots of one or more, but preferably all of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne. For example, the solid material may include parts or the whole of the stem and the bark of Saussurea acrophila Diels and Saussurea ceratocarpa, and parts or the whole of the roots of Aucklandia lappa Decne. The solid material may be in finely divided particulate form and may be dry or wet, e.g. in suspension. The composition may include a mixture of solid material of two or more of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne.
The therapeutic composition can include camel milk. The camel milk can include camel's milk having anti-HIV antibodies therein (HIV-immunized camel milk). The therapeutic composition can include treated camel's milk. The treated camel's milk can include a mixture of camel milk and the herbal composition. The treated camel's milk can include a concentration of about 800 g of the herbal composition per 5-6 liters of the camel milk.
The liquid extract may include liquid solvent extracts from the solid material of one or more, but preferably all of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne. The extracts may be obtained by treatment of solid material of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne with a solvent. The solvent may be ethanol. The ethanol may be about 96% ethanol, more preferably 99.9% ethanol. In the case of the composition comprising liquid extracts, the composition may comprise a mixture of liquid solvent extracts from solid material of two or more of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne.
The solid material may comprise one or more of parts or the whole of the stem, the bark, the flowers and the roots of one or more but preferably all of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne. More preferably, the solid material may comprise parts or the whole of the stem and the bark of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne, and parts or the whole of the roots of Aucklandia lappa Decne. The solid material can be previously harvested. The harvested material can be formed into finely divided particulate, e.g., powdered, form, e.g., by grinding.
On a dry basis, the herbal composition may comprise from about 10-20% by weight, preferably 14% by weight, of the solid material of Saussurea acrophila Diels or liquid extract thereof, from about 10-20% by weight, preferably 14% by weight, of the solid material of Saussurea ceratocarpa or liquid extract thereof and about 45-75% by weight, preferably 72% by weight, of the solid material of Aucklandia lappa Decne or liquid extract thereof.
The herbal composition may be provided in an aqueous form for use, particularly for use as a medicament. The aqueous form may, for example, comprise from about 0.5 g to about 1 g, preferably about 0.8 g of extract per ml water, i.e. preferably 800 g of extract per liter of water. Of the 800 g of extract, about 112 g is extract of Saussurea acrophila Diels, about 112 g is extract of Saussurea ceratocarpa, and about 576 g is extract of Aucklandia lappa Decne. The water is preferably distilled water. The quantity can be scaled-up according to the requirements of a subject to which it is to be administered, or to the need of the manufacturer.
An effective amount of the therapeutic composition can be administered to a patient in need thereof for medical treatment. The medical treatment can include treatment of human immunodeficiency virus (HIV), HIV/AIDS or prevention of HIV infection. The medical treatment can include treatment for or prevention of any one or more of viral infections, including, but not limited to Epstein Barr virus, Herpes simplex, Herpes zoster, influenza, and cancer. The medical treatment can include administering to a patient in need thereof an effective amount of treated camel milk. The treated camel milk can include about 6.8 g of extract of Saussurea acrophila Diels, about 6.8 g of extract of Saussurea ceratocarpa, about 34.6 g of extract of Aucklandia lappa Decne, and about 300 ml of camel milk.
The composition need not strictly comprise the liquid extracts. It may, in one embodiment, comprise an emulsion or a mixture of solid material of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne.
For commercial and industrialized purposes the composition can be further processed by spray-drying, or other similar drying techniques, and subsequent granulation thereof into particles which can be further encapsulated, tableted or filled into sachets or blister packets as required.
The method of preparing the composition may include combining the solid material of at least one but preferably all of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne. The method may include forming a mixture of solid material of at least one but preferably all of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne. The method may include a prior step of harvesting the solid material. The method may include another prior step of forming the harvested solid material into finely divided particulate, powdered, form, e.g. by grinding. Thus, the composition of solid material may be one or a mixture of the harvested solid material in finely divided particulate form.
A composition comprising liquid extracts from solid material of at least one of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne can be prepared by forming a mixture of liquid extracts from solid material of at least one of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne. This method may include preparing the liquid extracts by treating solid material of at least one of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne with a solvent. The solvent may be ethanol, typically about 96% ethanol, or about 99.9% ethanol.
The solid material used to produce the liquid extracts may comprise one or more of parts or the whole of the stem, the bark, the flowers and the roots of one or more but preferably all of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne. The solid material may comprise parts or the whole of the stem and the bark of Saussurea acrophila Diels and Saussurea ceratocarpa, and parts or the whole of the roots of Aucklandia lappa Decne.
Treatment of the solid material with the solvent may be in a mass ratio of 2:1 solvent:material, particularly when the solvent is ethanol.
The liquid extracts may be individually prepared by treating the solid material of one or more of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne separately. The composition may then comprise one of the extracts, or may be prepared by mixing two or more of the extracts.
The liquid extracts may be prepared as a combination of extracts, by preparing a mixture of solid material of two or more of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne and treating the mixture of solid material with the solvent. On a dry basis, the mixture may comprise from about 10-20% by weight, or about 14% by weight, of the solid material of Saussurea acrophila Diels, from about 10-20% by weight, or about 14% by weight, of the solid material of Saussurea ceratocarpa and about 45-75% by weight, or about 72% by weight, of the solid material of Aucklandia lappa Decne.
The therapeutic composition can include a medicament or a precursor to a medicament. The medicament may be treated camel's milk or HIV-immunized camel's milk, i.e., camel's milk obtained from a camel that was immunized using modified DNA inserts of HIV pWT/BaL (SEQ ID NO.: 3) and HIV pNL4-3 (SEQ ID NO.: 4). The HIV-immunized may include milk obtained from a camel that is caused to digest the herbal composition which comprises solid material or liquid extracts of solid material from one or more of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne, and immunized using modified DNA inserts of HIV pWT/BaL (SEQ ID NO.: 3) and HIV pNL4-3 (SEQ ID NO.: 4). This may be accomplished by supplementing the camel's feed, or by intubating the camel with 500-1000 g, preferably 800 g, of the solid material or liquid extracts of solid material from one or more of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne. Preferably, immunization occurs prior to causing the camel to digest the herbal composition. About 300 ml of the HIV-immunized camel's milk can be administered to a patient in need thereof three times a day.
The immunized camel's milk may include anti-HIV heavy chain IgG antibodies (hcIgG). hcIgG antibodies are rare, small antibodies, known to be produced by camels. These antibodies have unique features due to their smaller size, the absence of heavy chain constant regions, and the complete absence of the light chain. Due to their smaller size and unique features, hcIgG antibodies may possess improved bioactivity when compared with normal IgG antibodies.
The therapeutic composition may be used in medical treatment, by administering it to a person in need thereof. The therapeutic composition can be processed and formulated in a form suitable for oral, parenteral, or intravenous administration to a patient.
The medical treatment may be medical treatment for treatment of HIV/AIDS or prevention of human immunodeficiency virus (HIV) infection.
The medical treatment may also be medical treatment for treatment or prevention of any one or more of viral infections including but not limited to Epstein Barr virus, Herpes simplex, Herpes zoster, influenza, and cancer.
The treated camel's milk can be formed by mixing the solid material or liquid extracts of solid material from one or more of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne. The treated camel's milk may comprise a concentration of about 800 g of the solid material or liquid extracts of solid material from one or more of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne, per about 5-6 liters of the camel's milk. Other concentrations may also find application.
The medical treatment may involve administering the treated camel's milk to a subject in need thereof. The treated camel's milk can be prepared by mixing about 6.8 g of extract of Saussurea acrophila Diels, about 6.8 g of extract of Saussurea ceratocarpa, and about 34.6 g of extract of Aucklandia lappa Decne with about 300 ml camel's milk. The medical treatment may include administering 300 ml of the treated camel's milk to a subject three times a day.
The dosage of the compositions of the invention to be ingested will vary, depending on factors such as severity of the conditions, age, physical condition and body weight of the patient, diet, etc. Subjects may vary considerably depending on the factors indicated above. However, one benefit of the composition of the present invention is that the dosage is not “critical” since the edible compositions of the present invention are holistic in nature and represent dietary supplements in their own right, “over dosing” is not a problem. The individual patient with a particular body weight and life style may readily determine the proper dosage by starting out the general dosage.
As a general guide, it is expected that a camel with a body weight in the range of about 600 to about 1000 Kg would ingest about 500-1000 g/day of the solid material or liquid extracts from the solid material of at least one of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne. Typically, a camel would ingest about 0.18 g of the solid material or liquid extracts from the solid material of at least one of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne per Kg of body weight. It is to be understood that these consumption amounts are only general guides and the proper amounts for individual camels may vary. The amount ingested by a camel has been found to be most effective when administered at a dosage of about 750 g per day, with an effective range of about 500-800 g per day. This is based on a pilot study where a subject is administered about 300 ml of the medicament or HIV-immunized camel's milk three times a day, in which case the camel was intubated with about 800 g of the solid material or liquid extracts from the solid material of at least one of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne per day. However, it will be appreciated that subjects may be given a higher dosage according to their status and life style with no expected harm or side effects that may result.
The intubated solution administered to the camel may comprise an ethanolic extract of the stem/bark of Saussurea acrophila Diels and Saussurea ceratocarpa in the range of about 10-20% respectively, and preferably about 14% respectively, in water. The intubated solution administered to the camel may further comprise an aqueous extract of the root of Aucklandia lappa Decne in the range of about 45%-75%, and preferably about 72% in water.
The stem and bark of Saussurea acrophila Diels and Saussurea ceratocarpa, and the root of Aucklandia lappa Decne were harvested, weighed and thoroughly washed in cold water to remove any dirt, soil and other undesirable contaminants.
The washed, harvested material was pre-treated with distilled or de-ionized water in an about 16:1 dry weight ratio of water:harvested material and subsequently dried in an oven. The dried material was ground into a finely divided particulate, powdered form, to provide a powder mixture comprising about 28% (280 g) of Saussurea acrophila Diels and Saussurea ceratocarpa, present in equal amounts (about 14% each), and about 72% (720 g) of Aucklandia lappa Decne. The powder mixture was soaked in about 99.9% ethanol, as extraction solvent, and homogenized for about 10 minutes to provide a solvent/powder mixture.
The solvent/powder mixture was incubated at room temperature for 4 days. The solvent/powder mixture, after incubation, was filtered twice using vacuum to provide a first filtrate and a first retentate. Ethanol was allowed to evaporate from the first filtrate, or extract solution, at room temperature.
The first filtrate was subjected to re-extraction with a re-extraction solvent of about 99.9% ethanol using a shaking water-bath at about 75° C. for about 6 hours, thereby obtaining a re-extraction mixture of the first filtrate and the re-extraction solvent. The re-extraction mixture was filtered, thereby obtaining a second filtrate, or extract solution, and a second retentate. Ethanol was allowed to evaporate from the second filtrate at room temperature.
The yield of this two-step extraction method provided a filtered herbal extract. The filtered herbal extract was yellowish brown.
The filtered herbal extract was kept at about 4° C. The concentration of this filtered herbal extract was adjusted accordingly, e.g. about 800 g thereof was added to one liter of distilled water.
Two different species of animals were the subjects of the experiment: rabbits (group-A) and WKY rats (group-B). The animals were obtained from Small Animal House, SQU, Oman. Group-A Rabbits, included ten (10) male rabbits and five (5) female rabbits, each weighing between 800 g to 1300 g. Group-B WKY rats included nine (9) males and six (6) females, each weighing between 300 g to 600 g. Both rabbits and rats were randomly divided to investigate the lethal dosage.
The filtered herbal extract, prepared as described in Example 1, was administered orally to the animals using antistatic at variable dosages to reach a maximum of about 16 g/Kg for each animal group. The concentration of each dose of fluid containing the filtered herbal extract was about 1 g/ml. Accordingly, the higher dosage group at 15 g/Kg had a concentration of about 5 ml of 15 g/Kg, which could be calibrated to suit bigger animals, including humans.
The animals were observed for behavior continuously for a period of two weeks after administration. Observation was conducted hourly at day 1; and during the following days, observation was conducted four to six (4-6) times per day. At the end of the observation period, animals were sacrificed and dissected to examine the eyes, liver, lung, and spleen for any adverse effects.
A separate experiment was performed in parallel to the above animal groups where, a medicament of camel's milk as described below, was also given to each group of animals to evaluate the toxicity status thereof. The volume of the medicament of camel's milk was given according to the animal's capacity on a daily basis for a period of two weeks.
No abnormal behavior was observed in either groups of animals during the observation period. All animals were alive after the two weeks of having been given 16 g/Kg or the medicament of camel's milk. All animals showed normal body weight increase during the two weeks period. Biochemical analysis showed normal range of ALT, AST, CBC, and GGT. Inspection of the eyes, liver, lung, and spleen (after scarification and dissection) showed no extraordinary signs. The results when compared to a general acute toxicity index were normal and no acute toxicity was observed. Based on the results of the toxicity experiment, the weight range of the filtered herbal extract or diffused composition (i.e. a mixture, or solution, of the composition of the extract in water) is preferred to be 10-20 g/Kg of animal weight for each extract.
In the tables below, “composition” means the filtered herbal extract described in Example 1, on a dry mass basis dissolved in distilled water at about 973.5 g/5000 ml.
For generation of anti-mouse HIV antibody, conventional immunization was performed in parallel with DNA immunization using fifty (50) Balb-C mice. Mice were boosted 3 times at 2 weeks interval. Blood was collected before and after boosting for seropositivity analysis using ELISA kit.
For generation of anti-camel HIV antibody, conventional immunization was performed in parallel with DNA immunization using female camels. Each camel was boosted 6 times at two week intervals, using different delivery routes. Blood was collected before and after boosting for seropositivity analysis using ELISA kit.
For analysis of the generated anti-HIV camel antibodies, anti-HIV camel antibodies secreted in the camel milk and orally taken by HIV/AIDS patients was analyzed. Assessment of the impact of the treatment modality, virus load, and CD4+T cell count of HIV/AIDS patients was made for three months.
Both HIV/pwT/Bal vector (SEQ ID NO.:2) and HIV/pNL/4-3 vector (SEQ ID NO.:1), were donated from the NIH-AIDS Research and Reference Reagent Program, USA. Prior to the amplification step, both vectors' DNA insets were modified using techniques generally known in the art. All experiments were performed using the modified vectors “HIV/pwT/Bal/Mod” and “HIV/pNL/4-3/Mod” (corresponding to SEQ ID NO.:3 and SEQ ID NO.:4, respectively). Transformed “one shot chemically competent” E. coli colonies were amplified in about 500 ml LB cultures and the plasmid DNA constructs were purified chromatographically using Qiagen Megaprep kits, according to manufacturer's instructions (MegaPrep; Qiagen, Hilden, Germany). The purified DNA was stored at −20° C. until ready for use.
Before proceeding to DNA immunization, the ability of the HIV/pwT/Bal/Mod vector (SEQ ID NO.:3) and HIV/pNL/4-3/Mod vector (SEQ ID NO.:4) to express recombinant HIV Provirus was confirmed in vitro by transfection of mammalian COS-7 and 293 T cells (ATCCC, Germany) with DNA or vector vaccines alone (Control plasmids lacked the HIV inserts). The main reagents used in this transfection/expression procedure were FUGENE 6 (Roche) and/or Lipofectamine 2000 (Invitrogen, CA). The transfection procedure was performed according to manufacturer instructions.
Monitoring of virus production was performed every 2 days by measuring p24 antigen concentration in the culture fluid starting on day 5 using the p24 antigen ELISA kit. Culture medium was changed fully and regularly every two days after day 5 when completing the p24 antigen measurement. Three or more harvests were performed when the p24 concentration reached greater than about 10 ng p24 per milliliter. Each harvested culture fluid sample was saved, filtered through a 0.45-μm pore filter, and treated with about 50 units of Benzonase per ml of filtered HIV viral stock for about 25 minutes at about 37° C. to remove contaminating plasmid DNA. Previous studies proposed that the use of Benzonase has no impact on the viral ability to infect or to bind to its specific targets, and therefore any effect of Benzonase on infectivity would be only slight. Virus stocks were frozen at −80° C. to halt Benzonase activity and/or until use.
Viral titers were calculated using the TCID50 assay, according to the method of Reed and Muench (1938). Samples with less than about 0.2 ng p24/ml were considered negative for the calculation.
Female BALB/c Mice [a total of 10 mice] were immunized by intramuscular injection with about 1.0×108 infection units (equivalent to 1×106 TCID50 of concentrated vector stocks) of HIV recombinant viruses over a time course of 4 weeks post-immunization and boosted 1 month later with the same dose of recombinant viruses. The control group (a total of 10 BALB/c mice), were immunized with the same dose but with vectors alone. For seroconversion analysis, blood samples were collected via the periorbital route 4 weeks after the first inoculation, then at 4 weeks after boosting.
The HIV/pwT/Bal/Mod (SEQ ID NO.:3) and HIV/pNL/4-3/Mod (SEQ ID NO.:4) vectors and the control pwT/Bal and pNL/4-3 plasmids were precipitated onto 1.6 μm gold beads and loaded into half-inch lengths of plastic tubing according to the manufacturer's instructions (BioRad, Hercules, Calif.). The amount of gold powder and DNA were acclimated to give pieces of tubing (‘shots’) containing 1 mg DNA/0.5 mg gold. The abdomens of thirty anaesthetized, 8±10-week-old female BALB/c were shaved and subjected to three ‘shots’ expelled under a burst of helium gas at about 350 psi into the epidermal layer using the Helios GeneGun (BioRad). Mice were divided into three groups of 10 mice in each group, immunized with about 3 mg of the HIV/pwT/Bal/Mod (SEQ ID NO.:3) and HIV/pNL/4-3/Mod (SEQ ID NO.:4) DNA constructs or the vectors alone, on three occasions, two weeks apart and their sera were examined four weeks later.
For each of the HIV/pwT/Bal/Mod (SEQ ID NO.:3) and HIV/pNL/4-3/Mod (SEQ ID NO.:4) DNA constructs and the empty plasmids vectors, about 0.5 mg of each in saline was adjusted to about 100 μg with distilled water and about 25 μl was injected into each rectus femoris muscle of 30 8±10-week-old female BALB/c mice with a 25 gauge needle on three occasions, two weeks apart. Clinical parameters for each animal were inspected, including weight of animals, temperature and swelling of lymph nodes. Blood samples were drawn prior to each vaccination, then at week six and week eight. The immunization sites were examined for any adverse reactions.
Evaluation of antibody seroconversion was conducted using ELISA. Ninety-six-well plates (ICN, Costa Mesa, Calif.) coated with recombinant gp140 (0.5-1 μg/ml in PBS) or gp120 (1 μg/ml) in 0.05 M carbonate were buffered overnight at 4° C. The plates were washed 3 times with TST (Tris (0′01 m, pH 8.5), saline (NaCl, 0.15 m) and Tween 20 (0.1%)) and blocked for 1 hour with 5% fat-free dried milk (Carnation, Wirral, UK) in TST at 37° C. Individual sera from all inoculated BALB/c mice, including the control groups, were diluted 1:500 with 5% milk and applied, in duplicate, to the plates overnight at 4° C. The plates were washed 3 times with TST and horseradish peroxidase-(HRP)-conjugated anti-mouse immunoglobulin reagents (Nordic, Tilburg, The Netherlands), diluted to 1:1000 with TST, and then added for 2 hours at 37° C. The plates were washed 3 times and the assay developed with a 0.02% solution of the chromogenic substrate 2,2′-azino-bis (2-ethylbenzthiazoline-6-sulphonic acid; Sigma, Poole, UK) in phosphate-citrate buffer (pH 4.0) containing 0.015% hydrogen peroxide and the optical density (OD) were read at 405 nm.
Immunoblotting results were analyzed using one and two-dimensional SDS-PAGE immunoblotting assay. Western blot or protein immunoblot from the above gels were used to transfer HIV fractionated recombinant proteins to nitrocellulose and molecular weight markers visualized by reversible staining with Ponceau S.
The filters were blocked with 5% non-fat milk for 1 hour at room temperature, washed 3 times with TST and diluted (5% milk) sera are added overnight at 4° C. The filters were washed 3 times with TST and incubated for 2 hours at room temperature with HRP- or alkaline phosphatase-conjugated goat anti-mouse IgG, or anti-rabbit IgG (1:1000; Sigma Aldrich). Unbound secondary antibody was washed off and the specific antigen-bound antibody was visualized with the appropriate substrate solution.
Seroconversion of the anti-HIV antibodies was confirmed. DNA immunization with the HIV Provirus DNA constructs was found to generate higher titer seroconversion than immunization with the recombinant HIV Provirus of Example 4.
The methods for DNA immunization of Example 5 were followed to immunize female camels with slight modifications based on the vector/recombinant HIV Provirus and the period of immunization. Seven female well-milking camels (healthy and infection free, as checked by veterinary doctor) were used. The first 5 camels were immunized with about 10 mg each of the HIV/pwT/Bal/Mod (SEQ ID NO. 3) and HIV/pNL/4-3/Mod DNA (SEQ ID NO.: 4) constructs, using the GeneGun protocol, on six occasions, two weeks apart. Their sera and milk were then examined 12 weeks later. The same group was further injected “i.m.” with 1 ml of both the HIV/pwT/Bal/Mod (SEQ ID NO. 3) and HIV/pNL/4-3/Mod DNA (SEQ ID NO. 4) constructs adjusted to about 20 mg in distilled water, into each rectus femoris muscle of the same camel with an 18 gauge needle. The same camels were used for the two routes to emphasize the delivery intake of the vector/DNA constructs and to initiate different orientation of the Th1 and Th2 immune responses. The second group comprising the last two female camels was non-immunized and used as a control group.
Confirmation of seroconversion of the anti-HIV camelid antibodies was analyzed in the same manner as in Example 5. HIV neutralization assays were also performed as described below (prior to performing the clinical trial) as an efficacy procedure of the generated anti-HIV camelid antibodies. Both the camel serum and camel milk were used for these analyses. Furthermore, techniques generally known in the art were used to generate a secondary antibody against the generated anti-HIV camel IgG antibodies (for use in the ELISA and immunoblotting assays, conjugated with a proper substrate such as HRP). The secondary antibody was generated by using serum from an unimmunized camel to immunize a goat, and isolating the goat anti-camel antibodies.
In vitro HIV neutralization assessment was performed using the indicator cell line HEK293T. Briefly, HEK293T cells were seeded into 24-well plates (20,000 cells per well) and incubated for 24 hours at 37° C. in DMEM-10% FCS supplemented with hygromycin plus G418 to select and maintain the eukaryotic cells. Culture medium was changed with 30 μl of DMEM-10% FCS-DEAE Dextran (10 μg/ml) and cells were then incubated further for 30 minutes at 37° C. HIV Provirus (about 0.5 ng per well) was then incubated with 1:30 diluted camel milk and/or sera in 30 μl of FCS-free culture medium at 37° C. for 45 minutes and the mixtures virus-sera/milk were added to the cells in duplicate. Two hours later 100 μl of DMEM-10% FCS was added to each well. Subsequently wells were incubated for 48 hours and then β-galactosidase activity was measured using a chemiluminescent reporter gene assay (Roche). The mean neutralization for milk and sera tested were compared and reported to the value recorded in wells of the control containing serum of non-immunized camel at the same dilution that served as reference for 100% infection.
These tests confirmed that (i) DNA immunization is the most efficient DNA-delivery route in contrast with the recombinant HIV immunization and (ii) also showed that satisfactory anti-HIV titers are only achieved after the final immunization. The generated anti-HIV antibodies were found to be significant and showed 100% efficacy to neutralize the HIV Provirus.
Seventeen (17) patients of Arab, Indian and African origin [excluding +Ve and −Ve controls] were studied and followed up for six months [two months of which are under the invented therapy]: Patients were excluded if they had administered any investigational drug less than 6 weeks prior to the first dose of the medicament of the invention. The selected patients had never been prescribed any HIV/AIDS drugs. Patients' immune function was abnormal (i.e., the CD4+ cells to CD8+ cells ratio is under 1, the number of CD4+ T cells is under 200/mm3). The average age of each patient is above 16 years. No pregnant women were chosen. The patients agreed voluntarily to use the medicament of the invention.
Patients were required to take an ELISA test to indicate the presence of the HIV virus. Patients underwent testing with monoclonal antibody reagent and flow cytometry (FAC-SCAN) before and subsequently every two, four weeks and then after two and six months and after treatment ceased to calculate the rate of CD4+ to CD8+ cells, and the quantity of CD4+ cells (in cm3) as index to measure the immune function of patients under the treatment. Complete diagnostic records were recorded, including physical sign and pulse condition.
The criteria for measuring curative effect were: if testing PCR and/or RT-PCR is negative, the immune function increased and/or recovers to normal (i.e., ratio of CD4+ to CD8+>1 and/or the percentage of the CD4+ T cells are increased in correlation to the total WBC, and if there are no symptoms, no physical signs and no opportunistic infections. The criteria for measuring evident effect were: if HIV appears negative or undetectable, the immune function improved dramatically (i.e., ratio of CD4+ and CD8+>0.2 but the number of CD4+ T cells is ≥200/mm3), if opportunistic infections were basically removed (disappeared) and the symptoms and physical signs recovered fundamentally back to normal. The criteria for measuring some effect were: if testing PCR for HIV appeared positive, the immune function improved (ratio CD4+ to CD8+>0.2, the quantity of CD4+ T cells is beyond 40/mm3), opportunistic infections improved and the symptoms and physical signs were relieved. The criteria for measuring no effect were: if there were no dramatic changes to the index of immune function, or if there was a decrease of immune function in the treatment.
A comprehensive analysis of the curative effect according to the clinical symptoms and immune state before and after treatment are studied and recoded. The total number of patients was 17 (4 females and 13 males). Their infection was confirmed by both ELISA and Quantitative PCR. The average age was about 33 years, with the oldest being 55 years old, and the youngest being 24 years old.
Pretreatment status was determined by physical examination. All patients showed common symptoms of depression, weakness, stegnosis and weight loss associated with loss of appetite, and some patients occasionally developed fever.
In accordance with the present techniques, camel's milk was used from a camel which had been DNA immunized against HIV and which had ingested, via intubation, the filtered herbal extract of Example 1. The immunized camel's milk from these camels was the only source of treatment administered to the patients. Each patient consumed about 300 ml of the immunized milk three times a day on a daily basis. Immunized milk was administered until HIV viral load was undetected for two-tested occasions or when a −Ve viral load was shown. Patients were then asked to stop taking the medicament of the invention for 16 weeks and were tested for HIV from the start of the 16 week period. Two patients “as control group 1” were administered the same dose of milk but from an unimmunized camel with no intake of the composition of the invention.
Detailed hematological and biochemical tests were conducted on each patient. Table 2 shows the most significant clinical data obtained from the pilot studies on the effectiveness of the herbal composition and camel milk in protecting HIV/AIDS patients from the debilitating effects of the disease.
Ten (10) ml blood samples were taken from the patients each time before, during, and after the treatment and further tested using ELISA, Viral Load (VL), CD4+ cells, CD8+ cells, White Blood cells (WBC) and other screening profile such as full blood count (FBC), liver function, renal function, fasting lipid profile, blood glucose. Moreover, physical examination including recording weight, was performed each time before, during, and after the treatment.
Of the 17 patients, the mean CD4+/CD8+ ratio (ratio of helper T cells to cytotoxic T cells) at the commencement of treatment was 0.42±0.05 (the ratio in a healthy population is 1.0-3.5). After a treatment period ranging from 2-8 weeks the CD4+/CD8+ cells ratio had significantly increased by over two-fold to 0.90±0.02 (p<0.001). Concurrently with this change, an improvement in the response to skin hypersensitivity tests and a general increase in well-being were observed. This latter effect is demonstrated by the overall restoration of body weight, an increasing average from 67.9 to 88.1 Kg, a weight gain of 10 Kg (p<0.003).
Table 3 shows the percentage elevation in the CD4+ T cell populations of peripheral blood cells from HIV infected patients upon receiving the immunized camel's milk within the period of 2 months. It also shows the CD4+ T cells after 6 months from which patients stopped the treatment. The CD4+ percentages reported in Table 3 represent the percentage of white blood cells that are CD4+ cells. For HIV negative adults, the average CD4+ percentage is about 45% (This can range from 24% to 64%). In adults, a CD4+ percentage of about 12%-15% is considered similar to a CD4+ count of 200 cells/mm3. A percentage of about 15%-20% is considered similar to an absolute CD4+ cell count of 300 cells/mm3. Missing data points were due to patients lacking the financial means to travel to the checkup location (Sana'a City in the Republic of Yemen).
Table 4 shows the elevation in the Total White Blood Cells population of peripheral blood from HIV infected patients upon receiving the immunized camel's milk within the period of 2 months. The data reported in Table 4 represents the total white blood cells per cubic millimeter (mm3). Normal white blood cell counts of healthy adults are within the range of about 5,000-10,000 mm3. Missing data points were again due to patients lacking the financial means to travel to the checkup location (Sana'a City in the Republic of Yemen).
Table 5 shows the monitoring of “HIV Viral Load” throughout the treatment period. Moreover, it also illustrates the screening profile of the patients' viral load after 6 months from which patients stopped the treatment. Viral load is reported as copies of HIV in one cubic millimeter of blood.
All patients regardless of their lifestyle, race, age and sex showed that their case of HIV/AIDS to be improved and 0 cases became worse. According to the criteria of curative effect the 17 cases are effective and 0 cases were ineffective in this study.
No case in the study showed side effects in general, except that namely 1 case felt diarrhea and tired for a short period. All of the other 16 cases continued to take the medicine in the prescribed quantity.
This therapeutic composition was very effective to suppress HIV replication as well as to stimulate its eradication from the blood stream. During the treatment process, all patients had positive response with no side effects, except that namely 1 case felt diarrhea and tired for a short period. However, the symptoms of the patients were noticeably improved after two weeks of the treatment including alleviation of weakness, depression, and stegnosis. The abdominal region pain and uncomfortable feeling also disappeared. Those patients who lost weight had about 10 Kg increase of body weight after two months of treatment. In the treatment, most if not all of the symptoms of the cases showed improvement after taking the HIV-immunized camel's milk. This was also associated with immune function improvement as well as increased in the number of the CD4+ T cells. The HIV-immunized camel's milk for all cases was found to be effective in the eradication of the HIV from the patients' blood streams. This was confirmed after four months after patients stopped taking the medication and were screened by Real Time PCR.
The therapeutic composition was therefore found to be highly effective to quickly decrease HIV viral load, and has the potential to eradicate HIV infection. The therapeutic composition also has the potential to stimulate and increase WBC significantly.
Two similar and independent investigations were performed where two groups of patients were given either (i) the medicament milk from camels intubated with the herb alone (without DNA immunization), or (ii) the medicament milk from camels that had been DNA immunized, but had not been fed the herbal composition. Both of these groups demonstrated reduction of viral load after treatment; however, neither of these experimental groups demonstrated full eradication of the HIV infection, as the virus was detected four months from the time that treatment was stopped (Table 6 & Table 7).
It is to be understood that the present invention is not limited to the embodiments described above, but encompasses any and all embodiments within the scope of the following claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 16/7084 | Oct 2016 | ZA | national |
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| 20050129780 | Holcomb-Halstead | Jun 2005 | A1 |
| 20110104163 | Dimitrov | May 2011 | A1 |
| 20130028968 | Huang | Jan 2013 | A1 |
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| Number | Date | Country | |
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| 20180105582 A1 | Apr 2018 | US |
