METHODS AND COMPOSITIONS FOR THERAPEUTIC TREATMENT OF VIRAL OR VIRALLY-INDUCED INFECTIONS AND CONDITIONS, AND ANTI-VIRAL COMPOSITIONS AND THEIR PRODUCTION

Abstract

Methods and compositions for treating a viral infection, where the cells from the host to be treated are extracted from the host and modified with viral DNA, and then replaced back to the host to generate an immune response. The composition may be produced using fat cells that are mechanically or chemically transformed to a nanofat, and co-incubation of the nanofat with the adenovirus. The double-stranded DNA of the adenovirus codes for a specific antibody to be produced, and the transfer of genetic material from the virus to the nanofat stem cells takes place outside of the host where the host immune system cannot destroy it. The co-incubated cells are then administered to the host by introduction into the nasal passages or directive cavity, or through injection or other vehicle. The compositions provide an effective rapid response to an invasive pathogen, such as an adenovirus or bacteria, through passive immunity.

Claims

1. A method of delivering passive immunity against a pathogen to a mammalian host, comprising: a) extracting fat tissue of the mammalian host to be immunized; b) homogenizing the extracted fat tissue to extract the stem cell fraction (SVF) that contains the Adipose tissue-derived stem cells (ADSC); c) separating the fat fractions to obtain the nanofat containing the Adipose tissue-derived stem cells (ADSC); d) co-incubating the nanofat with the pathogen or pathogenic antigen to form an immunogenic composition; and e) administering to said mammalian host the immunogenic composition.

2. The method of claim 1, wherein said nanofat comprises the stromal vascular fraction (SVF) of the fat tissue extracted from the host and contains Adipose tissue-derived stem cells (ADSC).

3. The method of claim 2, including culturing the extracted stem cells prior to co-incubation with said gene for the antibody.

4. The method of claim 2, wherein the adenovirus is co-incubated with the ADSC (extracted stem cells) directly.

5. The method of claim 1, wherein the gene coding for the antibody to said pathogen is an adenovirus and wherein said adenovirus codes for the gene of the antibody.

6. The method of claim 5, wherein administering the immunogenic composition to said host includes spraying the immunologic composition into the mucosa of the digestive and/or respiratory tract of the host, or injecting the immunologic composition into the host via injecting into the skin, subcutaneous tissue, muscle or into a blood vessel.

7. The method of claim 5, wherein co-incubation is carried out by transfection of said nanofat with said adenovirus.

8. The method of claim 7, wherein said adenovirus is an antibody specific to the corona virus (COVID-19).

9. The method of claim 8, wherein said coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the coronavirus disease (COVID-19).

10. The method of claim 8, including repeating step e) at a plurality of spaced apart time intervals.

11. An immunologic composition for treating a viral infection by a virus in a host comprising: a modified host-generated stem cells transformed from nanofat having the viral DNA of the antibody directed against the viral infection to be treated, the modified host-generated stem cells being transformed to produce antibodies to the virus.

12. The composition of claim 11, wherein said modified host-generated stem cells comprise modified host-generated Adipose tissue-derived stem cells (ADSC) of the stromal vascular fraction (SVF) of fat cells from the host.

13. The immunologic composition of claim 11, wherein said viral DNA is an adenovirus DNA.

14. The immunologic composition of claim 11, wherein said adenovirus DNA is a gene that codes for the antibody against the adenovirus which is endangering the host.

15. The immunologic composition of claim 14, wherein said adenovirus DNA is a gene that codes for the antibody against the coronavirus which is endangering the host.

16. The immunologic composition of claim 15, wherein said coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the coronavirus disease (COVID-19).

17. A method of producing an immunologic composition for treating a viral infection in a mammalian host comprising: a) extracting fat tissue of the mammalian host to be immunized; b) homogenizing the extracted fat tissue to extract the stem cell fraction (SVF) that contains the Adipose tissue-derived stem cells (ADSC); c) separating the extracted fat tissue in step b) to obtain the nanofat fraction containing the Adipose tissue-derived stem cells (ADSC) by passing the fat tissue through a 500 micro mesh screen; d) co-incubating the nanofat fraction with the pathogen or pathogenic antigen to form an immunogenic composition that comprises antibodies against the virus; e) wherein said nanofat comprises the stromal vascular fraction (SVF) of the fat tissue extracted from the host and contains Adipose tissue-derived stem cells (ADSC); and f) wherein said viral infection to be treated is a virus that is attacking the host, or to which the host is susceptible.

18. The method of claim 17, wherein said viral infection to be treated is coronavirus (COVID19), and wherein said pathogen is coronavirus (COVID19) or pathogenic antigen is coronavirus (COVID19) antigen.

19. The method of claim 17, including an optional step of culturing the nanofat fraction passing through the 500 mesh screen in step c) that contains the Adipose tissue-derived stem cells (ADSC) in a cell-sustaining culture medium; and co-incubating the cultured nanofat fraction with the pathogen or pathogenic antigen to form an immunogenic composition that comprises antibodies to the virus.

20. The method of claim 1, wherein said mammalian host that receives the immunologic composition is asymptomatic or has no known prior exposure to the pathogen.

21. The method of claim 1, wherein said mammalian host that receives the immunologic composition is symptomatic or is confirmed to have an infection from the pathogen.

22. The method of claim 1, wherein homogenizing and/or separating the fat fractions to obtain the nanofat containing the Adipose tissue-derived stem cells (ADSC) comprises mechanical manipulation.

23. The method of claim 1, wherein homogenizing and/or separating the fat fractions to obtain the nanofat containing the Adipose tissue-derived stem cells (ADSC) comprises a chemical treatment.

Description

BRIEF DESCRIPTION OF THE DRAWING FIGURES

[0024] FIG. 1 is a flow diagram illustrating a schematic depiction of an exemplary implementation of a method for the treatment of a pathogen infection in a mammal according to the invention. Optional steps are depicted in broken lines, and each optional step may be implemented apart from any other optional step.

[0025] FIG. 2 is an exemplary illustration of a mechanism for producing antibodies from human stem cells.

DETAILED DESCRIPTION OF THE INVENTION

[0026] Immunologic compositions are provided to target specific pathogens, such as bacterial, viruses and other organisms. According to a preferred implementations, the method for producing the composition and for treating a mammalian subject involves transforming cells obtained from the mammalian host subject. According to the method, cells are extracted from a mammalian host. The preferred implementations obtain the cells from the same host that is to be treated for the pathogenic infection. The host cells preferably comprise cells that contain the stem cells, in particular Adipose tissue-derived stem cells (ADSC).

[0027] Preferred implementations of the method are carried out using Adipose tissue-derived stem cells from the adipose tissue or fat tissue of the mammalian host. The cells obtained are preferably the Adipose tissue-derived stem cells (ADSC) found in the stromal vascular fraction (SVF) which is obtained from processing the fat sample extracted from the host. The fat sample take from the host may be a fat tissue sample. In the process, cells within fat tissue are separated from the actual gloppy fat tissue itself (the fat tissue being the fat that is initially extracted from the host). Those cells are then concentrated to make the SVF, from which transformation of the stem cells through incubation with the adenovirus encoded for the gene of the desired antibody which upon being returned to the host results in the immunologic composition.

[0028] According to preferred implementations, the fat cells are transformed through a procedure where the larger fat cells are chemically or mechanically broken down or destroyed by manipulation of the cells to break the connections between fibrous components of the fat cells. The reduction of bulk of the fat extracted from the host results in the ultimate destruction of the larger fat cells. The bulk reduced extracted fat sample from the host is processed to transform it further by homogenization to obtain the fraction of the cells desired for production of the immunologic composition that may be administered to the mammalian host. According to some preferred embodiments, homogenization of the extracted fat sample may be carried out through a repeated transfer of the extracted fat between syringes having progressively smaller and smaller connectors. The fat is passed through the series of syringes.

[0029] When the fat is destroyed through mechanical manipulation or a chemical treatment using these procedures, there is a fraction of very small cells in the sample. The smaller cell fraction is separated from the mechanically manipulated or chemically treated fat sample preferably by passing the mechanically manipulated or chemically treated fat sample through a screen that has a desired opening or mesh size to permit passage of the desired cell fraction that contains the desired Adipose tissue-derived stem cells, while leaving the other cells and cell components behind. According to preferred embodiments, a suitable screen mesh may be 500 micron. The fraction passing through the screen is collected, and contains the Adipose tissue-derived stem cells, and more particularly, the stromal vascular fraction (SVF) that includes the Adipose tissue-derived stem cells (ADSC). The Adipose tissue-derived stem cells obtained from the host fat and separated out preferably comprise progenitor or mesenchymal cells, which are capable of further development and replication. The procedures concentrate the progenitor cells in the sample.

[0030] According to preferred embodiments the virus is added directly to the nanofat (the fraction of the extracted fat tissue from the host that has been processed mechanically or chemically to break apart the fat cells and screened through a mesh screen, e.g., 500 micron screen). The co-incubation by the addition of the virus to the nanofat containing the stromal vascular fraction (SVF) that contains the Adipose tissue-derived stem cells (ADSC), transfers the viral DNA to the stem cells. The immunologic composition comprising the co-incubated host stem cells obtained from the host's processed pat tissue, is administered to the patient. The immunologic composition when placed in the patient's body, causes an immune response, and produces antigens targeting the virus.

[0031] The stem cell fraction obtained from the host, now containing the concentration of stromal vascular fraction (SVF) or Adipose tissue-derived stem cells (ADSC), is co-incubated with the pathogen. According to a preferred implementation, an adenovirus is the pathogen that is co-incubated with the stem cell fraction. Preferably, the co-incubated adenovirus is the target pathogen that the patient host (from whom the fat was extracted) is to be treated for, or immunized against, using the immunogenic composition. A suitable incubation procedure carried out to associate the adenovirus double stranded DNA and the stem cells of the stromal cell fraction containing the Adipose tissue-derived stem cells (ADSC) obtained from processing the extracted fat of the mammalian host.

[0032] Adenoviruses have double stranded DNA that codes for a specific antibody. Since the method involves the transfer of genetic material from the adenovirus to the extracted and processed host cells outside of the host body, the adenovirus is less likely to be destroyed by the body's immune system. The progenitor nature of the ADSC allows for the development of this cell population to differentiate into B cells. The adenovirus may be a particular adenovirus for which treatment of the infection in a mammalian host subject is desired. One example of a virus is the novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes the infection coronavirus disease (COVID-19).

[0033] The co-incubation is carried out, and the immunogenic composition produced.

[0034] According to preferred embodiments, the immunogenic composition includes transformed cells of the host. The transformed host cells are capable of producing antibodies to the specific pathogenic antigen that was co-incubated with the Adipose tissue-derived stem cells. According to a preferred example, the immunologic composition produces antibodies that function against the adenovirus using the host patient body's immune system, when the immunologic composition is administered to the patient host.

[0035] The co-incubation of the virus and Adipose tissue-derived stem cells isolated from the fat (nanofat) promotes the development of the antibodies needed to attack the viral infection. The co-incubation is carried out on the extracted and processed host cells, without interference from the host's other cells and immune system response.

[0036] According to preferred embodiments, once the co-incubation has been carried out, the composition may be used as an immunologic composition by administering it to the host human or animal. According to preferred embodiments, the procedure may be carried out in a single host visit, to provide immediate passive immunity to the person or animal being treated.

[0037] The cell culture of the nanofat extracted cells may be co-incubated using a suitable procedure. For example, the co-incubation may be carried out by transfection or transduction.

[0038] According to some alternate embodiments, the nanofat extracted cells may be cultured for a period of time, e.g., a few to several days, prior to introduction of the adenovirus (viral DNA), or in the case of alternate embodiments, another pathogen.

[0039] According to some alternate embodiments, the nanofat cells that contain the SVF fraction (containing the Adipose tissue-derived stem cells (ADSC)) are cultured in a suitable culture media. The extracted nanofat cell fraction may be washed with a suitable washing agent, such as for example saline, e.g., a phosphate buffered saline, (e.g., Dulbecco's Phosphate Buffered Saline (DPBS), which may be commercially obtained (Invitrogen, Dun Laoghaire, Ireland]). The cell fraction may be centrifuged, and the centrifuged cells transferred to a container, such as a flask, along with a suitable culture medium that is designed to sustain the cells. For example, a suitable medium may comprise a human or animal sera, and a suitable cell growth supplement also may be added. Preferably, a human derived serum is utilized, as it may be advantageous over animal sera, and in addition, countries may restrict the use of animal serum during the production or treatment process. According to some preferred embodiments, human serum from type AB donors which is lacking in antibodies towards the A and B blood-type antigens, may be more preferred as immunoreactivity is minimized against the human cells in culture. In addition, male donor serum may be preferred over female in order to lower risks of major histocompatibility class (MHC) antibodies being present. (Women having a past pregnancy may have developed these antibodies to MHC antigens carried on the father's cells or the developing fetus.). Some examples of cell culture growth supplements include GroPro™ Cell Culture Growth Supplement (ZenBio, Inc.) and Human Platelet Lysate (HPL), which are non-animal derived cell culture growth supplements obtained from human platelets. HPL contains abundant growth factors and cytokines necessary for cell growth and proliferation. According to some embodiments, platelet lysate may be used as a replacement for traditional Fetal Bovine (FBS) supplemented cell culture medium. The aforementioned cell culture media are provided as examples, and other suitable culture media may be used. In some alternate applications, where a culture is carried out, the cells may be animal derived cells and may be fortified with growth factors or other supplements suitable for the animal host cells (e.g., when treating an animal).

[0040] During the cell culture period, the culture media may be changed and/or augmented as needed.

[0041] According to some alternate embodiments, the adenovirus is co-incubated to the cells of the cell culture. The cell culture preferably is compatible with the viral material antigen, such as the adenovirus, to permit the stromal cells to receive the viral DNA. The progenitor nature of the ADSC allows for the development of the cultured cell population comprising the extracted nanofat from the host to differentiate into B cells. During the co-incubation period, the gene is introduced into the cell population and incorporated into the cells. Once the gene is incorporated, the cell can produce antibodies that confer passive immunity to the host against a specific antigen which resides on the surface of a virus. The transfected nanofat cells of the host stromal vascular fraction (SVF) comprise an immunologic composition is then transferred to the host, which may be done by administering the composition (comprising the co-incubated cells) to the host via injection, spraying or other delivery means. According to some preferred implementations, the immunologic composition is administered to the host at preferred locations which include as a spray into the mucosa of the digestive and/or respiratory tract, or an injection into a site on the body of the host. The presence of the administered immunologic composition in the host neutralizes the virus and protects the host from infection.

[0042] Although the preferred methods are used without the need for culturing the cells and/or the use of a cell culture, according to some alternate embodiments a culture may be generated. In these embodiments, the stem cell fraction or stromal vascular fraction according to some embodiments may be cultured in a suitable culture media, which may also include nutrients and/or growth factors to permit the cells to stabilize and strengthen prior to co-incubation. A human derived culture media (versus animal derived media) is utilized according to preferred embodiments. According to some alternate implementations, the viral DNA may be introduced into a eukaryotic stromal vascular fraction (SVF) that contains the Adipose tissue-derived stem cells (ADSC) (the nanofat extracted cells) through chemical and physical methods in a physician's office or in a laboratory. According to some alternate embodiments, one or more additional agents may be used to promote the co-incubation, e.g., via transfection. For example, chemicals like calcium phosphate and diethylaminoehtyl (DEAE)-dextra may be used. Other agents which are commercially available and sold as transfection reagents may be used. The agents neutralize or may even impart an overall positive charge on DNA molecules so that the viral DNA molecule can more easily cross the negatively charged cell membrane of the stem cells. Alternatively, other methods such as physical methods, e.g., electroporation or microinjection which pokes holes in the cell membrane so DNA can be introduced directly into the cell may be used according to some embodiments. Microinjection requires the use of a fine needle to deliver nucleic acids to individual cells. Electroporation on the other hand uses electrical pulses to create transient pores in the cell membrane that genetic material can pass through.

[0043] According to some embodiments, a co-culture of the nanofat fraction and the virus may be generated by placing the nanofat fraction containing the patient's stromal cells and virus in the same tissue culture vessel (e.g., a plate, tube, or flask) without any barrier to impede contact between either the patient stromal cells and the virus or the medium in which they are cultured. In another embodiment, the nanofat cells and virus may be placed in the same tissue culture vessel but with a barrier that impedes contact between the them but does not impede transfer of the medium and any compounds smaller than the pore size of the barrier in which they are grown. Thus, the first and second cells can be exposed to the same tissue culture medium and/or any agents secreted by the nanofat cells or the virus (or the component agent that may contain the virus).

Proposed Example 1

[0044] A mammalian patient having symptoms of, or who is confirmed to have a virus is treated for the infection. Cells comprising fat cells are extracted from the patient, by extracting a sample of fat tissue from the patient. In this example, areas proposed from which the fat cells are extracted include a suitable location, such as the buttock, cheek or breast of the patient. The tissue and cells therein are manipulated mechanically or chemically treated to disrupt and break apart the fibers of the fat cells. In the case of mechanical manipulation, according to a preferred implementation, the cells are mechanically manipulated by passing the extracted fat sample through a series of syringes having narrowing connectors, where the syringes are narrowing to impart forces on the extracted sample. The resultant substance containing the transformed fat cells is separated to obtain the desired nanofat fraction by passing the mechanically manipulated or chemically treated fat sample through a 500 micron mesh screen. The filtrate portion containing the Adipose tissue-derived stem cells in the nanofat is co-incubated with the virus to incorporate the viral DNA into the cells (Adipose tissue-derived stem cells (ADSC)) of the filtrate. The composition of the co-incubated fat cells and viral DNA are incubated at suitable temperature conditions for a suitable time. An immunologic composition is formed that includes Adipose tissue-derived stem cells that are capable of producing antibodies to the virus.

Proposed Example 2

[0045] A patient was treated using the method set forth in Proposed Example 1, and the virus co-incubated is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the coronavirus disease (COVID-19), and the immunologic composition treats the patient for coronavirus disease (COVID-19).

Proposed Example 3

[0046] The patient treated in accordance with Proposed Examples 1, 2 or 3, was given a second administration of the immunologic composition after a period of time from the administration of the first treatment with the immunologic composition.

[0047] In accordance with the treatments provided by the methods and compositions disclosed herein, when the immunologic composition is administered to the patient, such as the mammalian host, the B lymphocytes (B cells) produce antibodies that can attach to the specific antigen of the pathogen being targeted and stimulate the process of destroying the antigen. When the antibodies attach to the specific antigen targeted by the method and composition it makes it easier for the host patient's immune cells to destroy the antigen.

[0048] Referring to FIG. 2, a depiction of an exemplary diagram showing a stem cell, a progenitor cell, that may differentiate to form a B cell. The B cell is shown producing the antibodies that can confer immunity to the host against the specific antigen residing on the surface of the targeted virus. In FIG. 2, the gene or DNA of the virus may be incorporated so that the antibodies produced target the specific virus. FIG. 2 shows the formation of white blood cells. The differentiation of multipotent progenitors is represented, and there are two main lines. The lymphoid line is shown giving rise to T-lymphocytes and B-lymphocytes and natural killer cells. The viral co-incubation is represented and the antibodies produced by the B lymphocytes are also depicted.

Proposed Example 4

[0049] Patients who do not have immunity to the virus and who have not been exposed could similarly be treated and be given Passive Immunity. There by allowing them to interact with others without the concern for “contracting” the virus as passive immunity exists.

[0050] In Table 1 below are common viral vectors that may be useful for co-incubation with the adipose tissue-derived stem cells according to the present method and compositions.

TABLE-US-00001 TABLE 1 Characteristics of the commonly-used viral vectors. Viral system Adenovirus (Ad5) AAV Retrovirus Lentivirus HSV-1 Baculovirus Genome material dsDNA ssDNA RNA RNA dsDNA dsDNA Genome size  36 kb 8.5 kb 7-11 kb 8 kb  150 kb 80-180 kb Enveloped No No Yes Yes Yes Yes Biosafety level BSL-2 BSL-1 BSL-1/2 BSL-2/3 BSL-2 BSL-1 Insert size 8-36 kb   5 kb   8 kb 9 kb 30-40 kb No limit known Max titer 1 × 10.sup.13 1 × 10.sup.11 1 × 10.sup.9 1 × 10.sup.9 1 × 10.sup.9 2 × 10.sup.8 (particles/ml) Tropism Broad, low Broad, low Broad Broad Neurons Some for blood for blood (pan or (pan or mammalian cells cells pseudo-typed) pseudo-typed) cells Infectivity Dividing and Dividing and Dividing Dividing and Dividing and Dividing and non-dividing non-dividing cells non-dividing non-dividing non-dividing cells cells cells cells cells Transgene Transient Transient Stable Stable Transient Transient expression or stable or stable Vector genome Episomal Episomal Integrated Integrated Episomal Episomal or form (>90%), site- integrated specific integration (<10%) Inflammatory High (low Low Low Low High High potential for “HC- AdVs) Advantages High titers; Safe Persistent Persistent Large Large cargo extremely transgene gene transfer gene transfer packaging sizes; high efficient delivery; in dividing in most tissues capacity; level of gene transduction non-inflammatory; cells strong tropism expression of most cell non-pathogenic for neuronal types and cells tissues Drawbacks Capsid Small Only Integration Inflammatory; Limited mediates a packaging transduces might induce no expression mammalian potent capacity; dividing cells; oncogenesis during latent host range inflammatory requiring integration in some infection; response helper AdV might induce applications transient gene (eliminated for replication oncogenesis expression in in HC-AdVs) and difficult in some non-neuronal to produce applications cells pure viral stocks

[0051] The preferred embodiments of the invention have been described in connection with fat cells from adipose tissue. However, according to some other embodiments, the host cells may comprise progenitor cells that are found in brain, bone marrow, blood vessels, skeletal muscle, skin, teeth, heart, gut, liver, and other (although not all) organs and tissues. However, the utilization of the fat cells is preferred.

[0052] Therapeutic immunologic compositions are produced as described herein, and preferably are generated from the host patient's own cells, and in particular the extracted fat sample. The processing and transformation of the fat sample to the nanofat containing the stromal vascular fraction (SVF) that contains the progenitor stem cells (adipose tissue-derived stem cells) provides a substrate fraction that is co-incubated with the virus. Once the co-incubation process has been carried out, the cells contain the DNA of the virus. This immunologic composition comprises host based cells that have been transformed through a mechanical manipulation process or chemical treatment to destroy the fat cells and dissociate fibrous bonds and that contain the DNA coding for the antibodies to the viral antigen. The immunologic compositions may be provided to target specific pathogens, to provide a rapid treatment for the patient host.

[0053] The methods and compositions may be applied to mammals that include animals. The method has utility not only for treatment of infections in a human host, but also may be useful to treat infections in farm animals, such as swine or bovine. The methods herein include methods for treating an infection in a mammalian host, as well as methods for producing an immunologic composition.