OA21071A - Compositions and methods useful for the prevention and/or treatment of disease in mammals. - Google Patents

Abstract

Novel methods and pharmaceutical composition or medicaments are described for protecting a subject against, or treating a subject suffering from, disease characterised 5 by viral infection and/or diseases associated with immune system disorder and/or viral cancers, by raising the levels of Ksp37 in the blood plasma of the subject to a therapeutically effective concentration level. According to the invention, a therapeutically effective amount of one or more of; a clinically modified or genetically engineered Ksp37 protein and/or proteins having a molecular weight in the range 10 ranging from 24 kDa to 45 kDa, and/or a vector encoded with a KSP37 gene which will translate to a Ksp37 protein and/or proteins having a molecular weight in the range ranging from 24 kDa to 45 kDa, and/or a polar compound are administered to a subject.

Description

COMPOSITIONS AND METHODS USEFUL FOR THE PREVENTION AND/OR TREATMENT OF DISEASE IN MAMMALS

FIELD OF THE INVENTION

This invention relates to compositions and methods usefiil for the treatment of disease in mammals, characterised by viral infection and/or diseases associated with immune System disorders and/or viral cancers.

BACKGROUND TO THE INVENTION

The virus known as Human Immunodeficiency Virus (“HIV”), classified as a retrovirus, has affected the lives of millions of people across the world. The virus infects healthy individuals who progress to developîng Acquiredlmmunodeficiency Syndrome (“AIDS”) within a few years if no treatment is administered (Mann, J. et al. (2016) ‘The latest science from the IAS Towards an HIV Cure Symposium 16 - 17 July 2016, Durban, South Africa’, (July), pp. 235-241.).

HIV is a member of the lentivirus family of retroviruses (Teichez al., 1984, RNA Tumor Viruses, Weiss et al., eds., CSH-press, pp. 949-956). Retroviruses are small enveloped viruses that contain a single-stranded RNA genome, and replicate via a DNA intermediate produced by a viraily-encoded reverse transcriptase, an RNAdependent DNA polymerase (Varmus, H., 1988, Science 240:1427-1439).

Other retroviruses include, for example, oncogenic viruses such as human T-cell leukemia viruses (HTLV-1, -II, -III), and feline leukemia virus.

HIV Structure and Genome Organisation

Mature HIV virions are 100 nm -120 nm in diameter spherical structures consisting of a lipid bilayer membrane which encloses a dense truncated cone-shaped nucleocapsid (“core”). The core contains two 9.8kb long positive sense, single stranded, linear RNA molécules, molécules to initiate cDNA synthesis, cellular tRNA, Gag polyprotein, viral envelope (Env) protein and three enzymes: reverse transcriptase (RT), viral protease (PR), integrase (IN), and some other cellular factors (S Sierra, et al.,2005).

The HIV genome contains accessory and regulatory genes flanked by long terminal repeats (“LTR”), The viral genome has a total of nine genes which can be divided into three functional groups:

• structural genes, Gag, Pol, and Env;

• regulatory genes, Tat and Rev; and • accessory genes, Vpu, Vpr, Vif, and Nef (JM Costin, 2007).

The Gag gene codes for the core protein, Pol gene codes for RT, protease, integrase, and Env gene codes for the Envelope protein (gpl60). The Tat gene codes for the Tat protein and the Rev gene codes for the Rev protein. The Tat and Rev regulatory proteins function as RNA-binding proteins. In addition to RNA binding Tat proteins also act as activators of transcription ensuring that full length genomes of HIV are formed. Rev protein also helps in shift of gene expression of HIV from early to late phase. On the other hand, the accessory proteins coded for by the accessory genes, are multîfunctional. Nef or négative factor is involved in T-cell activation, downregulation of existing major histocompatibility complex (MHC) I, and CD4 on the cell surface by degranulation in lysosomes and also stimulate virion infectivity. Vpr acts as a nucleo-cytoplasmic transport factor which permits HIV to infect non-dividing cells. Vpu enhances release of virion through the development of an ion channel and also down-modulates expression of CD4 through ubiquitin-mediated dégradation. Réplication of HIV in lymphocytes, monocytes, and macrophages is regulated by Vif.

The envelope of the virion contains the transmembrane proteins, gpl20 and gp41, which project outwards from the virion in the form of spikes (up to 72 in number). Being a highly immunogenic protein, gpl20, which binds to the CD4 receptor, is a suitable target for majority of host antibodies. Most of these straîn-specific antibodies block the interaction of CD4 receptors with gpl20 protein by binding to these receptors. The matrix lying undemeath the lipid bilayer consists of Gag protein 17 (viral gag protein cleavage product), The core or capsid contains a coverîng of p24 protein (product of Gag gene), and a third Gag protein p7 (Lampejo Tel aZ.,2013).

HIV Life Cycle

Human immunodeficiency virus viral entry îs divided into basically three steps:

(1) binding;

(2) activation; and (3) fusion.

Major HIV-1 and HIV-2 receptors and co-receptors are CD4 and CCR5, CXCR4, respectively. The cycle starts with the récognition of HIV-enveloped trimeric complex, gpI20 and gp41 with CD4 receptor (58 kDa monomeric glycoproteîn) major co-receptor of MHC class II molécule, on cell surface. Upon binding of CD4 with gpl20, a conformational change occurs resulting in exposure of gpl20 domain where CCR5 chemokine co-receptors bind. So far, 17 chemokine receptor ligands are identified in this process (Fanales- Belasioei ûZ.,2010).

Following double binding of gpl20, a stable attachment complex formed which allows the N-terminal side of gpl20 peptide pénétration in plasma membrane. In gp41 protein, HR1 and HR2 sequences act together and form a hairpîn structure of gp41, which causes fusion of viral and cellular membranes (S Sierra et al.,2005).

After fusion viral core is released in cytoplasm, uncoating of viral capsid occurs mediated by MA, Nef, and Vif protein factors of virus (Lampejo T et al.,20\3).

By viral RT ribonucléase Hsite viral RNA is transcribed into DNA starting from primer binding site. After completion of transcription, ribonucléase H breaks the dsRN/DNA hybrid and by RT polymerization active site converted into dsDNA (Fanal es-Belasioe/ aZ.,2010). Proviral status is obtained by intégration of this dsDNA into host cell genome by integrase enzyme. The integrase protein produces sticky ends at 3 end of each DNA strand. Now modifiée! viral DNA is exported to nucléus through nuclear pore, dîrected by viral Vpr, and intégration function îs accomplîshed by this integrase (Sierra S).

For the viral genome to be expressed, the host genome intégration site should be in active State (Fanales-Belasio).

In the provirus state, the viral DNA may remain for several years in host genome and upon receiving activation signal express mRNA using host polymerase enzyme (Yousaf MZ étal,, 2011).

Latently infected T cells, macrophages, monocytes, and microglial cells are major réservoirs of HIV genome. In active cell State, transcription of HIV genome starts due to host RNA polymerase II and other transcription factors by binding with viral LTRs. Following transcription, translation results in basal amount of proteins (Tat, Rev and Nef), On adéquate production of Tat, further transcription is controlled by binding of Tat with TAR éléments on LTRs and other transcriptional cellular activators (Sierra S)·

In early stages, multiply spliced mRNA produces Rev, Tat, and Nef. On achieving adéquate amounts of Rev, non-spliced and longer mRNAs are produced, referred to polysome, resulting in the production of other viral proteins and genomic RNA. On the un-spliced RNA RRE, Rev response éléments are present where Rev binds and causes the safe transportation, without splicing, to cell cytoplasm for translation (Lampejo T).

REV also causes expression of enzymatic and structural proteins and regulatory proteins inhibition so plays a rôle in producing mature virion. In cytoplasm, ENV gene is translated into gpl60 glycosylated in ER resulted into mature gpl20 and gp!40 by HIV-1 protease (Fanales-Belasio).

During translation, ribosome-1 frame shift resulting in Gag pol proteins includes PR, RT, and IN. Nucléus of mature virions are formed by Gag and Pol gene proteins. From large 160 kDa precursor Gag and Pol proteins are formed cleaved by viral proteases into p24, p9, p7, pl7 Gag final products and Pol products. This cleavage is necessary for infectious viral particles ENV proteins, which after translation move toward membrane and is inserted into it. Gag and Gag—Pol polyprotein also move toward cell membrane and started to assemble mediated by Gag polyprotein. Full size genomic RNA, cellular tRNAlys-3-primer, enzymes, and ail cellular compounds become linked with immature viral core (Sierra S).

Budding of immature virus takes place through plasma membrane. It is necessary to hâve a reduced number of CD4 molécules on the cell surface when virus assembly and budding occurs. Nef, ENV, and Vpu are involved in this process. Nef in early stages médiate the endocytosis and mortification of MHC class I and II molécules. In later stages, Npu induces the dégradation of CD4 molécules. During budding, activation of protein protease takes place which auto-catalytically cleaves Gag and Gag—Pol polyprotein resulted in structural proteins and viral enzymes. Further interactions of individual proteins with capsid, nucleocapsid protein resulted in conic nucleocapsid, and MA remain associated to viral envelop (Sierra S).

Current Treatment for HIV infection

The current treatment for HIV infection involves a combination of:

• Non-nucleosîde reverse transcriptase inhibitors;

• Nucleoside reverse transcriptase inhibitors; and • Protease inhibitors.

This combination of drugs îs commonly referred to as HÎghly Active Anti-Retroviral Treatment (“HAART”). HAART is provided to patients with the goal of slowing down the AIDS development, by inhibiting the viral DNA from being incorporated into the host DNA, and also by inhibiting the formation of viral DNA from viral RNA (ChupraditK et al., 2017 ‘Current peptide and protein candidates challenging HIV therapy beyond the vaccine Era’, Viruses, 9(10), pp. 1-14. doi: 10.3390/v9100281),

These are just a few of the drugs and their mechanisms of action that are given to patients that hâve tested positive for HIV.

To date, attempts at developing a vaccine against, or cure for, HIV infection hâve not proven successful.

The typical approach to vaccine development is to infect the body with parts of a virus and elicit an anti-body response. When the vaccinated host becomes infected with the real virus in future, the immune system will recognize the antigen ofthe virus which was contaîned in the vaccine, and an immédiate overwhelming immune response will be tnggered. While this vaccine development strategy works for some viruses, it does not work for the HI-Virus responsible for HIV infection and subséquent AIDS, because of the ability of the HI-Virus to mutate. As a resuit of the mutation ofthe HIVirus, détection of viral antigens is delayed, allowing prolifération of virus-infected cells.

In humans, HIV réplication occurs promînently in CD4⁺ T lymphocyte populations, and

HIV infection leads to a déplétion of this cell type and eventually to immune incompétence, opportunistic infections, neurologîcal dysfonctions, neoplastic growth, and ultîmately death.

South African Patent 1998/04649 entitled “Drug Delîvery Devices and Methods for Treatment of Viral and Microbial Infections and Wasting Syndromes”, teaches a drug delivery device for transdermal administration of a therapeutic agent, comprising a réservoir containing or having absorbed thereon a therapeutic composition comprising the polar compound A/N-dimethylformamide (“DMF”).

Individuals, the study of whom generated the teachings of ZA 1998/064649, despite being infected with HIV hâve maintained a CD4⁺ T-cell count above 350 for more than 20 years without being on antirétroviral treatment. These individuals are referred to herein as Long-term Non-Progressors (“LTNP”).

One such individual, had initially presented with HIV PCR detecting 63.298 HIV-1

RNA copies/ml of plasma in December 1996. A number of years after participation in the transdermal DMF study, a follow-up HIV PCR conducted in March 2006, detected <40 HIV-1 RNA copies/ml of plasma; and even more encouraging, in 2011 a further follow-up HIV PCR detected No HIV-1 RNA copies/ml of plasma and a CD4 Count of 920/pL.

The mode of action of the DMF was not fully understood, but further studies on this group of LTNP indivîduals revealed that their CD8⁺ cells produce elevated levels of a protein known as Ksp37.

The characteristics of the CD8⁺ cells that produce Ksp37 include:

• Phenotype type markers for these cells are: CD27, CD45RO, and CD57; and • The signal molécule for these cells has been confirmed to be ΜΙΡ-Ιβ (Bennett, Salter and Smith, 2018‘A New Class of Antirétroviral Enabling Innate Immunity by Protecting APOBEC3 from HIV Vif-Dependent Dégradation’, Trends in Molecular Medicine. Elsevier Ltd, 24(5), pp. 507-520. doi: 10.1016/j.molmed.2018.03.004.).

While Ksp37 is present in ail humans and many other animais, it is usually present in quantifies less than 400ng/mL.It is hypothesised that the levels of Ksp37 in the LTNP indivîduals may be a key reason why HIV is unable to progress in these indivîduals.

Characteristics of protein Ksp37

The KSP37 gene (also known as FGBP2) is commonly expressed by NK, CD8⁺ T, cd

T and CD4⁺ T cells and is composed of 223 amino acids(Ogawa et al., 2001‘A Novel

Sérum Protein That Is Selectively Produced by Cytofoxic Lymphocytes’, The Journal oflmmunology, 166(10), pp. 6404-6412. doi: 10.4049/jimmunol. 166.10.6404).

The protein known as Ksp37 or Killer-specific secretory protein of 37kDa, or Fibroblast Growth Factor-Binding Protein 2 (FGF-BP2) has been isolated, and sequenced as follows:

ORIGIN l ccctttaaagggtgactcgtcccacttgtgttctctctcctggtgcagagttgcaagcaa gtttatcagagtatcgccatgaagttcgtcccctgcctcctgctggtgaccttgtcctgc

121 ctggggactttgggtcaggccccgaggcaaaagcaaggaagcactggggaggaattccat

181 ttccagactggagggagagattcctgcactatgcgtcccagcagcttggggcaaggtgct

241 ggagaagtctggcttcgcgtcgactgccgcaacacagaccagacctactggtgtgagtac

301 agggggcagcccagcatgtgccaggcttttgctgctgaccccaaaccttactggaatcaa

361 gccctgcaggagctgaggcgccttcaccatgcgtgccagggggccccggtgcttaggcca

421 tccgtgtgcagggaggctggaccccaggcccatatgcagcaggtgacttccagcctcaag

481 ggcagcccagagcccaaccagcagcctgaggctgggacgccatctctgaggcccaaggcc

541 acagtgaaactcacagaagcaacacagctgggaaaggactcgatggaagagctgggaaaa

601 gccaaacccaccacccgacccacagccaaacctacccagcctggacccaggcccggaggg

661 aatgaggaagcaaagaagaaggcctgggaacattgttggaaacccttccaggccctgtgc

721 gcctttctcatcagcttcttccgagggtgacaggtgaaagacccctacagatctgacctc

781 tccctgacagacaaccatctctttttatattatgccgctttcaatccaacgttctcacac

841 tggaagaagagagtttctaatcagatgcaacggcccaaattcttgatctgcagcttctct

901 gaagtttggaaaagaaaccttcctttctggagtttgcagagttcagcaatatgataggga

961 acaggtgctgatgggcccaagagtgacaagcatacacaactacttattatctgtagaagt

1021 tttgctttgttgatctgagccttctatgaaagtttaaatatgtaacgcattcatgaattt

1081 ccagtgttcagtaaatagcagctatgtgtgtgcaaaataaaagaatgatttcagaaat

1Ό

The sequence is saved under the accession number AB021123, for BLAST identification. The protein has a 99% similarity to the Human Fibroblast Binding Protein 2(0gawa et al., 2001). As such the FGFBP2 is commonly used as a synonym for the Ki lier-spécifie secretory protein 37. The FGFBP2 gene is conserved in chimpanzee, Rhésus monkey, chîcken, zebrafish and frog. 137 Organisms hâve orthologs with human gene FGFBP2.

At the time of this invention no adéquate studies on the levels of Ksp37 in LTNPs, or on the différence in levels of Ksp37 between LTNPs, HIV négative individuals, HIV positive individuals on HAART and HIV positive individuals who had not yet begun treatment, had been conducted.

In light of the above, there is a need to identify and characterise the proteins or peptides secreted by activated CD8⁺T Lymphocytes of Long Term Non Progressors, to détermine the levels of Ksp37 produced by these Long Term Non Progressors, and to utilise these results in the development of a therapeutic vaccine for the treatment or prévention of viral infection and/or viral cancers.

Embodiments of the invention thus aim to address the issues îdentified above, at least to some extent.

SUMMARY OF THE INVENTION

The présent invention concerns compositions and methods usefui for the treatment of disease characterised by viral infection and/or diseases associated with immune System disorders and/or viral cancers in mammals, including humans.

The invention îs based on identifying the optimum concentration range within which a protein identified as Ksp37, having a molecular weight in the range ranging from 24kDa- 45 kDa, enhances the immune response of a subject against viral infection and/or diseases associated with immune system disorders and/or viral cancers, and utilising this identified concentration range of the protein for the treatment of viral infection and/or diseases associated with immune system disorders and/or viral cancers as well as in the préparation of medicines and médicaments for the treatment of viral infection and/or diseases associated with immune system disorders and/or viral cancers.

According to the invention, the optimum concentration range within which the Ksp37 protein enhances the immune response of the subject against viral infection and/or diseases associated with immune system disorders and/or viral cancers, is 400 ng Ksp37 per mL of blood plasma - 700ng Ksp37 per mL of blood plasma.

The invention therefore provides for a method of protecting a subject from disease characterised by viral infection and/or diseases associated with immune system disorder and viral cancers, and or treating a subject suffering from disease characterised by viral infection and/or diseases associated with immune system disorder and viral cancers by raising the levels of Ksp37 in the blood plasma of the subject to a therapeutically effective concentration level, wherein the therapeutically effective level of Ksp37 is between 400 ng/mL - 700ng/mL.

The levels of Ksp37 in a subject may be increased by one or more of the following routes:

a) by administering a médicament comprising a therapeutically effective amount of a clinically modified or genetically engineered Ksp37 protein and/or proteins having a molecular weight în the range ranging from 24kDa- 45kDa, to the subject; and/or

b) by stimulating the production of Ksp37 in the subject to a therapeutically effective level by administering a vector encoded with a KSP37 gene which will translate to a Ksp37 protein and/or proteins having a molecular weight in the range ranging from 24kDa - 45 kDa that is useful in fïghting viral infection and/or diseases associated with immune system disorders and/or viral cancers to the subject; and/or

c) by stimulating the production ofKsp37 in the subject to a therapeutically effective level by chemîcally treating the subject with a polar compound, to activate increased Ksp37 production.

The therapeutically effective level of Ksp37 is preferably a blood plasma concentration level of 400ng/mL - 700ng/mL, and a therapeutically effective amount of clinically modified or genetically engineered Ksp37 protein and/or proteins having a molecular weight in the range ranging from 24kDa to 45kDa is an amount that results in a Ksp37 blood plasma concentration level of 400 ng/mL - 700ng/mL.

The invention also provides a clinically modified or genetically engineered Ksp37 protein and/or proteins having a molecular weight in the range ranging from 24kDa to 45kDa, and/or a vector encoded with a KSP37 gene which will translate to a Ksp37 protein and/or proteins having a molecular weight in the range ranging from 24kDa to 45kDa for use in a method of protecting a subject from disease characterised by viral infection and/or diseases associated with immune system disorder and viral cancers.

The invention further provides for the use of a clinically modified or genetically engineered Ksp37 protein and/or proteins having a molecular weight in the range ranging from 24kDa to 45kDa, and/or a vector encoded with a KSP37 gene which will translate to a Ksp37 protein and/or proteins having a molecular weight in the range ranging from 24kDa to 45kDa, in the manufacture of a médicament for the treatment and/or protection of a subject from disease characterised by viral infection and/or diseases associated with immune system disorder and viral cancers, wherein the médicament increases the levels of Ksp37 protein in the subject to between 400 ng/mL and 700ng/mL.

The invention yet further provides a pharmaceutical composition for use in a method of protecting a subject from disease characterised by viral infection and/or diseases associated with immune System disorder and viral, comprising a therapeutically effective amount of one or more of; a clinically modified or genetically engineered Ksp37 protein and/or proteins having a molecular weight in the range ranging from >

24kDa to 45kDa, and/or a vector encoded with a KSP37 gene which will translate to a Ksp37 protein and/or proteins having a molecular weight in the range ranging from 24kDa to 45kDa, and/or a polar compound.

The therapeutically effective amount of Ksp37 protein or modified proteins, comprises an amount capable of increasing the levels of Ksp37 protein in the subject to between 400 ng/mL and 700ng/mL when administered one or more times over a suitable period and is between 0.001 pg/kg body weight of a mammal microgram and 20 pg/kg body weight of a mammal.

The pharmaceutical composition or médicament may additionally include a pharmaceutically accepted excipient, including but not limited to water, saline, phosphate buffered solution, ringer’s solution, dextrose solution, Hank’s solution,polyethyleneglycolcontainingphysiologîcalbalancedsaltsolutionandotheraqueousp hysiologically balancedsaltsolution as well as nonaqueousvehicles,suchasfixedoils,sesameseed oil,ethy lene oleatetriglycerides.

The pharmaceutical composition or médicament may also include acontrolledrelease compositionthatiscapableofslowlyreleasîng Ksp37intoamammaL

The pharmaceutical composition or médicament may be administered to a subject via an acceptable administration route, including nasal, oral, topical, inhalation, transdermal, rectal or parentéral administration.

Additional compounds capable of enhancing the ability of Ksp37 to protect a mammal from disease characterised by viral infection, may be included in the pharmaceutical

1C composition or médicament, the compounds including, but not limited to compounds capable of regulatîng cell mediated immune response, regulating T-helper cell activity, regulating degranulatîon of mast cells, protecting sensory nerve endîngs, regulating eosinophil and or blast cell activity, and/or preventing or relaxing smooth muscle contractions.

The Ksp37 protein may be extracted from blood componentsand/or tissue then purified, acetylised, genetically engineered, cloned and transferred back to a mammalian host as a therapeutîc and/or preventatîve vaccine against viral infection and/or diseases associated with immune system disorders and/or viral cancers.

The vector encoded with the KSP37 gene that is useful in fighting viral infection and/or diseases associated with immune system disorders and/or viral cancers, contains a nucleic acid sequence that translates to a protein identical to the naturally occurring Ksp37 protein.

An appropriate vector includes a pGEM-T Vector or a pCMV3-C-GFPSpark.

The hosts cells may include ail blood components and mammalian tissue cells related to the hosts immune system which is îdentified as the primary location for production ofKsp37.

The invention yet further provides a pharmaceutical composition comprising a therapeutically effective amount of a Polar compound wherein the therapeutically .6 effective amount of the polar compound is an amount sufficient to actîvate increased Ksp37 production to levels of between 400ng/mL and 700ng/mL in a subject.

The polar compound is preferably W,W-dimethylformamide (DMF).

A therapeutically effective dose of DMF for activating Ksp37 production may be a dose that results in a peak plasma level of about 2 mg/l - 200 mg/l, more preferably about 100 mg/l - 200 mg/l, still more preferably about 150 mg/l of DMF. Especially preferred is a peak plasma level of 100 mg/l - 150 mg/l or 150 mg/l - 200 mg/l of DMF.

The virus may be a retrovirus, and the viral cancer may include ovarian cancer, Leukaemia, Burkitt’s lymphoma, nasopharyngeal carcinoma, and some forms of Hodgkin’s disease.

The subject is a mammal.

The invention also provides a therapeutic and/or preventative vaccine against viral infection and/or diseases associated with immune system disorders and/or viral cancers, the vaccine comprising a therapeutically effective amount of a clînically modified or genetically engineered Ksp37 protein and/or proteins having a molecular weight in the range ranging from 24kDa to 45kDa, and/ora vector encoded with a KSP37 gene that is useful in fighting viral infection and/or diseases associated with immune system disorders and/or viral cancers.

¹?

DETAILED DESCRIPTION OF THE INVENTION

The following description is provided as an enabling teaching of the invention, is illustrative of principles associated with the invention and is not intended to limit the scope of the invention. Changes may be made to the embodiment/s depicted and described, while stili attaining results of the présent invention and/or without departing from the scope of the invention. Furthermore, it will be understood that some results or advantages of the present invention may be attained by selecting some of the features of the present invention without utilîsing other features. Accordingly, those skilled in the art will recognise that modifications and adaptations to the present invention may be possible and may even be désirable in certain circumstances, and may form part of the present invention.

1. Comparing the levels of Ksp37 amongst different sample groups

A sample of five (5) known LTNP îndividuals (LTNP1-LTNP5) as well as a random sample of fifteen volunteers were selected from the George Mukhari Academie Hospital. The fifteen random volunteers were further divided into the following classes:

- 5 volunteers who are HIV Positive and on HAART (HAART1-HAART5);

- 5 volunteers who hâve just been diagnosed with HIV and hâve not y et started with HAART (NAIVE1-NAIVE5); and

- 5 volunteers who are HIV négative (NEGI-NEG5).

The sample size of five (5) LTNP individuals was determined by the scarcity of known LTNP individuals.

Blood samples were drawn from these 20 individuals.

a. Cell Isolation

Isolation of total RNA from whole blood was done using an RNA extraction kit (Thermo Fischer Tempus Spin RNA Isolation Kit), according to the manufacturer’s instructions. Cells were separated from whole blood by centrifugation at 250 x g for 5 minute/s (min/s). Next the cell pellets were washed twice with ice cold PBS, pH7.4, resuspended in 600 μΐ lysis buffer supplemented with β-mercaptoethanol, followed by vortex mixing for 10 second/s (sec/s).360pl of éthanol were added to cell lysate, and mixed by aspiration. The 700 μΐ lysate was then transferred onto the RNA purification column inserted in a collection tube. The column contents were subjected to centrifugation for at 12 000 x g for 1 minute. The flow-through was discarded. The purification column was washed with wash buffers 1 and 2, followed by centrifugation at 12 000 x g for 1 minute, with the flow-through discarded, RNA was eluted with 50 μΐ nuclease-free stérile deionized water, followed by centrifugation at 12 000 x g for 1 minute. The elute RNA was stored at -70°C.

b. Conversion of RNA to cDNA

Extracted RNA was reverse transcribed to cDNA using the cDNA synthesis kit (Thermo Scientific Superscript VILO cDNA Synthesis Kit), according to the manufacturer’s instructions. The cDNA reaction mixture contained 10 μΐ template RNA, 2 μΐ oligo d(T) primer, 12 μΐ nuclease-free deionized water, 4 μΐ 5x reaction buffer, 1 μΐ RibobLockRNase inhibitor and 1 μΐ MuLV reverse transcriptase. The resulting cDNA was stored at -70 G until used.

c. Amplification and quantification of the résultant cDNA

The cDNA was amplified using polymerase chain reaction kit (Kapa Bio Systems, USA)according to the manufacturées instructions. The PCR reaction mixture contained 2μ1 forward5’ -CTTCCGAGGGTGAC AGGTGA-3 ’ and reverse 5 TCCAGTGTGAGAACGTTGGATTG-3’ primers (0.4μΜ each), 5μ1 of template cDNA, 16 μΐ of nuclease free deionized water and 25 μ! of 2x Ready Mîx. The PCR reaction consisted of 90s dénaturation at 95 C, primer annealing for 30 s at 59 G and élongation for 1 min at 72 G for 7 min. PCR products were analyzed on 2 percent (%) agarose gel electrophoresis at 75 Volts (V) for at least 60mins at room température. The gel was viewed, and band intensifies estimated using Chemo (Bio-Rad). Protein polymorphism was then determined using restriction fragment length polymorphism.

d. Cytokines quantification in different groups

The quantities of selected cytokines (IFN-y, IL-5, GM-CSF, TNF-aIL-2, IL-13, IL41L-10, IL12p70) were determined amongst the different groups using direct, quantitative measurement of cytokine proteins in single human CD8 lymphocytes from fresh peripheral blood of healthy donors (Saxena et al., 2018Ultrasensitive

Quantification of Cytokine Proteins in Single lymphocytes from Human Blood following ex-vivo stimulationFront. Immunof, 9:2462. doi:

10.3389/fimmu.2018.02462.eCollection2018).

Results

Real time PCR analysis showed Ksp37 levels as indicated in the table below:

Sample	Ksp37 value (ng/mL)
LTNP1	507.24
LTNP2	473.23
LTNP3	530.99
LTNP4	571.53
LTNP5	655.88
HAART1	173.98
HAART2	152.70
HAART3	158.31
HAART4	158.90
HAART5	176.70
NAÏVE 1	375.34
NAIVE2	none
NAIVE3	348.40
NAÏVE4	236.28
NAIVE5	433.81

NEG1	243.49
NEG2	274.41
NEG3	249.38
NEG4	none
NEG5	none

Table 1: Ksp37 Values

The real-time PCR results confirm the HPLC peaks observed by the fewer cycles for détection of Ksp37 in LTNP blood sérum (average 26 cycles) when compared to the sérum of other study groups as reflected in the graph below:

Graph 1: Real time PCR Results

The results indicate that the blood sérum of Long-term Non-Progressor Group had significantly higher levels of the protein Ksp37.

The protein was also seen to be associated with higher levels of other cytokines and proteins namely IL-12p70, IFN-γ and IL-4 as illustrated in the following table 2.Levels are measured in ng/mL

Sample	IFN-γ . -	IL-5	GM-CSF	TNF- a	IL-2	IL-13	IL-4	IL-10	IL12p 70
LTNP1	4,5 ’	3,5	-i. ,,'W·	15	4	6,5	3,5	1,5	4
LTNP2	. 2^·	2		8,5	4	3	^2'	5,5	2,5
LTNP3	2	3	-1 'L	45,5	4	2,5	1,75	4	2,5
LTNP4	2,5	3,25	1,5	85	8,5	3	2,5	0	2,5
LTNP5	3,5	2,5	1 U / r -	105	7,75	1	1	-10	2
HAART1	0 -----	0,5		12	1	0	0,5	0	0
HAART2	0	2,5	-3,5 ' .	26	4	2	2,5	6	1
HAART3	-1	0,5	-4,5 . •j^C	16	1	1	0,5	-1	0
HAART4	'2··.—.—···-	0,5		6	0	2	OÆ:	1	1
HAART5	6	1,5	-2,5	10	0,5	2	0,5	2,5	2
NAÏVE 1	-1	-0,5	-6,5 A	18	0	1	0,5	-9	0
NAIVE2	0:	2,5	-2,5	6	0	I	-0,5	-2	I
NAIVE3		0,5		10	0	1	-0,5 .....	1	0 f l:
NAÏVE4	1	1,5	-4,5' ?	3	1	1	-0,5 $	1	I
NAIVE5	0 w	1,0	-4,0 Λ r- -'.·	6	0	1	0	1	0
NEG1		0,5		5	0	0	-0,5	-2	0
NEG2	1 l	0,5	-2,5' A.r.	8	1	1	0,5	1	0

NEG3	0	1,5	-5,5^.,^	6	0	2	0,5	4	1,5
NEG4	1 :	2,5	-4,5 _ T	19	0,5	3	0,5	0,5	0
NEG5	1	1,5	-5,5	11	1	1	0,5	0	1

Table 2: Cytokine Results

While it should be borne in mind that the samples used were from cultured cells and the values tabled above are therefore not représentative of cytokines values that would be expected from samples taken directly from a human host, the values do, however, guide the observations as to the pathways of immunity associated with high levels of Ksp37.

The production of IFN-γ and IP-12p70 is partly regulated by a positive feedback loop, with IFN-γ and GM-CSF promoting IL-12p70 production and IL-12p70 in turn stimulating IFN-γ and GM-CSF sécrétion. IL-12p70 and IFN-γ promote ThI différentiation, favouring cell-mediated immunity and inhibiting Th2 responses.

When considering the above IFN-y results, the average value in LTNPs is considerably higher than the values from the other study groups. IFN-γ is a cytokine critical to both innate and adaptive immunity, and functions as the primary activator of macrophages, in addition to stimulating natural killer cells and neutrophils.

IFN-γ has been identified as a correlator of better disease prognosis in HIV infection, and is positively associated with CD8+ T cell and activated NK cell counts(Lôpez

Mer al., 201 l‘The expansion ability but not the quality of HIV-specific CD8 + T cells

U is associated with protective human leucocyte antigen class I alleles in long-term nonprogressors’, Immunology, 134(3), pp. 305-313. doî: 10.111 I/j.13652567.2011.03490.x.).

Simîlarly, when considering the IL-12p70 values, the average value in LTNPs is considerably higher than the values from the other study groups. IL-12p70 stimulâtes growth and function of T cells, production of interferon-gamma (IFN-γ) and tumour necrosis factor-alpha (TNF-α) from T cells and natural killer (NK) cells, and reduces IL-4-mediated suppression of IFN-γ.

In SlV-infected macaques, IL-12p70 treatment during acute infection was associated with decreased viral loads, increased CD8⁺ NK and T cells, reduced naïve CD4+ T cells expressing homing markers, rétention of HIV-specific CTL and prolongea survival.

The above results also point to higher levels of IL-4 in the LTNP group when compared to other study groups. IL-4 has many biological rôles, including the stimulation of activated B-cell and T-cell prolifération, and the différentiation of B cells into plasma cells. It is a key regulator in humoral and adaptive immunity. IL-4 induces B-cell class switching to IgE, and up-regulates MHC class II production. IL4 and IL-12p70 hâve a complementary rôle. The main function of the IL-4 is to stimulate the adaptive immune system and CD8⁺ cytotoxic cells. Whole IL-12p70 prevents the suppression of IL-4.

A recent study also indîcated that IL-10 can significantly inhibit HIV-1 réplication in monocytes/macrophages. The inhîbitory effects of IL-10 on HIV-1 production în monocytes/macrophages are the resuit of IL-10- induced inhibition of the synthesis of other cytokines, such as tumour necrosis factor alpha and IL-6, capable of upregulating HIV-1 expression în these cells.

2. Modes of Action of Ksp37

While research into the modes of action of Ksp37 îs ongoing, increased levels of Ksp37 appear to inhibit the progression of HIV into AIDS by a number of different mechanisms. These include:

a. CD8+ considération

One possible mechanism by which increased levels of Ksp37 inhibit the progression of HIV to AIDS is linked to the site of production of Ksp37, the HIV GAG-specific cells. The GAG-specific CD8⁺ cells are characterised by the production levels of CD107a IFN-γ MIP-Ιβ IL-2 TNF-α (T, H. C. D. et al. (2006) ‘IMMUNOBIOLOGY HIV nonprogressors preferentially maintam highly functîonal’, Blood, 107(12), pp. 4781-4789. doi: 10.1182/bîood-2005-12-4818).

The production of these components are believed to negate the immunosuppressive abilities of HIV. Ksp37 is responsable for increasing the life span of these HIV spécifie CD8⁺ cells. Ksp37 is hypothesised to control the immunosuppressive effects of HIV by also controlling the release of components such as perforin, TNF-α, and IL2 3 from the CD8⁺ cells. The initial response to HIV infection in LTNP individuals îs the same as seen in other study groups. Once the HIV spécifie CD8⁺ cells hâve been activated in the LTNP individuals, a few outcomes are observed. The levels of TNF-a rise but decrease as the levels of Ksp37 increase, as shown in Table 3 below:

	Initial Ksp37 levels before HIV infection	Ksp37 levels 4 hrs after HIV infection.	Initial TNF- α levels	TNF-a levels after 4 hrs
LTNPs	441 ± 235 ng/ml	800 ±529 ng/ml	9 ±2 pg/mL	3 ± 1 pg/mL
Progressors	380 ± 200 ng/ml	246 ± 127 ng/ml	7 ± 2pg/mL	15 ±6 pg/mL

Table 3: Levels of TNF-α and Ksp37

The main rôle of TNF-α in the body îs linked to inflammation în the human body. The 10 decreased levels of TNF-α decreases inflammation at the site of activation, thus lowers response from the CD4⁺ cells. This leaves the virus exposed in the blood stream and unable to infect the CD4⁺ host cells. This allows for the HIV spécifie CD8 cells to directly attack the virus. Viruses are typically elîminated by virusspecific CD8⁺ T cells, which recognize processed viral proteins that are presented as a 15 complex with an HLA class I molécule at the surface of an infected cell. Récognition through the T cell receptor (TCR) initiâtes a cascade of activation events, ultimately

2“ leading to the release of granzymes and perforin and klllïng of the infected cell, which can occur before infectious progeny virions are produced (R. Brad Jones et al., 2016).

High IL-2 levels lead to a decrease in overall génération of early memory T cells by both decreasing central memory T cells and augmenting effectors (T. Kaartinenet al., 2017). There is, therefore, an inverse relationship between the levels of IL-2 and the génération and/or lifespan of CD8⁺ T cells. Ksp37-expressing CD4 and CD8 T cells lack the abîlity to produce IL-2. (Ogawa et al., 2001). Therefore, high levels of Ksp37 are associated with lower levels of IL-2 production at activation. The levels of Ksp37 are therefore inversely proportional to the levels of IL-2 .

Therefore, when Ksp37 levels are high, IL-2 is low, and CD8⁺ memory cell génération increases with a longer lifespan. This supports a greater ability to kill the virus. The initial activation of the immune system is as a resuit of IL-2, and once IL-2 is removed this results in massiveinfected cell death. As HIV-specific CD8+ cells lack the ability to produce IL-2 (Ogawa et al., 2001), these are not heavîly affected by IL-2 levels.

As noted, the sample study utilised cells grown inside the lab, and outside of the hosts immune system, as a resuit the IL-2 levels recorded are not fully représentative of what would be normally found in LTNP hosts.

The action of polar agents on oncogenic expression is hypothesized to be the induction of cancer cells to become more benign. It would seem reasonable that to couvert a maiignant cell to a benign type, there should be sortie modulation of gene expression causing malîgnancy in the first place.

A study done by Ogawa et al in 2001 suggest that Ksp37 may be involved in an essential process of cytotoxic lymphocyte-mediated immunity in patients with Epstein-Barr virus and that Ksp37 may also hâve clinical value as a new type of sérum indicator for monitoring cytotoxic lymphocytes in vivo. EBV is associated with Burkitt’s lymphoma, nasopharyngeal carcinoma, and some forms of Hodgkin’s disease. EBV can readily infect and alter the genetîc code of human B cells, and may prédisposé immunosuppressed patients to maiignant tumeurs.

b. Ksp37 as a Vif Inhibitor

Ksp37 is also believed to act as a Vif inhibitor.

The body’s natural immune response to retroviruses is based on the function of the APOBEC3 proteins. Human APOBEC3 (A3) proteins are cellular cytidine deaminases that potently restrict the réplication of retroviruses by hypermutating viral cDNA and/or inhibiting reverse transcription. There are seven members of this family includîng A3A, B, C, D, E, F, G, and H, ali encoded in a tandem array on human chromosome 22. A3F and A3G are the most potent inhibitors of HIV-1, but only in the absence of the virus-encoded protein, Vif (Shingo K, et al., 2011).

The cytidine deaminase APOBEC3G (A3 G) exerts a multifaceted antiviral effect against HIV-1 infection. Firstly, A3 G was shown to be able to terminate HIV infection by deaminating the cytosine residues to uracil in the minus strand of the viral DNA during reverse transcription (Sadler H et al., 2O1O‘APQBEC3G Contributes to H1V-1 Variation through Sublethal Mutagenesis’, Journal of Virology, 84(14), pp. 7396-7404. doi: 10.1128/jvi.00056-l 0.). A number of studies hâve also indicated that A3G inhibits HIV-1 reverse transcription by a non-editingmediated mechanism.

HIV Vif antagonizes the human antiviral protein APOBEC3G by hijacking the human Elongin B/C (EloBC)-cullin-SOCS box (ECS)-type E3 ubiquitin ligase, resulting in the polyubiquitinatîon of APOBEC3G and subsequently its proteasomal degradation(Matsui Yer al., 2016‘Core binding factor β protects HIV, type 1 accessory protein viral infectivity factor from MDM2-mediated dégradation’. Journal of Biological Chemistry, 291(48), pp. 24892-24899. doi: 10.1074/jbc.Ml 16.734673.).

HIV Vif protein has a similar function to the p23 antigen found on the Epstein BanVirus (ËBV”) (among other functions, in their respective interactions with the Hsp90/70 chaperone proteins). The antigen p23 is an Isoform of the Vif HIV protein. A protein isoform, or protein variant, is a member of a set of highly similar proteins that originate from a single gene or gene family and are the resuit of genetic différences. Protein Isoforms tend to hâve the same or similar biological functions. Ogawa et al. found that in the presence of EBV there was a greater production of Ksp37. This indicates the antiviral properties of Ksp37.

It is believed that the mechanism of Ksp37 in supporting the function of APOBEC3G proteins in fighting EBV is similar to the mechanism of Ksp37 in supporting the function ofAPOBEC3G proteins in fighting HIV, given the slmilarity of the HIV Vif and EBV p23 antigen.

Therefore, the mechanism of action for Ksp37 may give it a sub-classification of a Vif inhibitor, inhibiting the formation of the E3 ligase complex. The possible inhibition of the Vif protein would allow the A3G protein to disrupt the translation of viral DNA.

3. Concentration Levels at which Ksp37 is most effective

The results in Tables l to 3 above indicate that Ksp37 is functional in blood sérum concentration levels of between 400ng/mL and 700ng/mL.

Ksp37 is present in normal healthy individuals at concentrations lower than 400ng/mL. At this level it is not effective in inhibiting the immune disabling réplication of the HIV virus and possibly other retroviruses. Conversely, concentration levels of Ksp37 higher than 750ng/mL may lead to autoimmune disorders, including asthma and Down Syndrome, due to the positive effect Ksp37 has on cytokines such as IL-5 and TNF-a which are associated with the increased activity of the immune system.

In light of this, the invention provides for a method of protecting a subject from disease characterised by viral infection and/or diseases associated with immune system disorder and viral cancers, by increasing the levels of Ksp37 in the subject to between 400ng/mL and 700ng/ml.

3-1

The levels of Ksp37 in a subject may be increased by one or more of the following routes:

(a) by administering a therapeutically effective amount of a clinically modified or genetically engineered Ksp37 protein and/or proteins having a molecular weight in the range ranging from 24kDa to 45kDa, to the subject;

(b) by stimulating the production of Ksp37 in the subject to a concentration level of 400 ng/mL - 700ng/mL by administering a vector encoded with KSP37 gene which will translate to a Ksp37 protein to the subject; or (c) by stimulating the production of Ksp37 in the subject to a concentration level of 400-700ng/mL by treating the subject with a polar compound, to activate increased Ksp37 production.

Subjects may include ali mammals, and are not restricted to humans only.

4. Administering a clinically modified or genetically engineered Ksp37 protein to a subject

In one embodiment of the invention, a Ksp37proteinandorproteinsmtherangerangingfrom24kDato45 kDa whichhasbeenclinicallymodified/clonedorgeneticallyengineered, and/or a formulation comprising a Ksp37proteinandorproteînsintherangerangingfrom24kDato45 kDa whichhasbeenclînicallymodified/clonedorgeneticallyengïneered, may be administeredto a subject to treat or prevent disease characterised by a viral infection and/ ordiseases associated with immune system disorder and viral cancers.

The subject is a mammal.

The proteinisselectedfromthegroupofKspproteÎnsandail it species,wheresaidproteinisamammalianprotein.

The proteinishasamolecularweightofapproximately 37 kDa with 223 amino acid chains, contains an N-terminal signal sequence, ashort C-terminal hydrophobie région,and a potential Ο-Glycosylation site and a cysteine sidechain.

The protein may be extracted from blood componentsand/or tissue then purified, acetylised, genetically engineered, cioned and transferred back to a mammalian host as a therapeutic and/or preventative vaccine for protection against or treatment of viral infections and/ ordiseases associated with immune system disorder and viral cancers.

One or more recombinant molécules can be used to produce the Ksp37 protein ex vivo. In one embodiment, an encoded product is produced by expressing a nucleic acid molécule under conditions effective to produce the protein.

A preferred method to produce an encoded protein învolves transfecting a host cell with one or more recombinant molécules having a nucleic acid sequence encoding a

Ksp37 protein, to form a recombinant cell. Suitable cells for transfection are any cells that can be transfected. Host cells can either be transfected cells or cells that are aiready transformed with at ieast one nucleic acid molécule.

Host cells useful in the present invention can be any cell capable of producing a Ksp37 protein, including bacterial, fungal, mammal and insect cell.

Transfectionofanucleicacidmoleculeintoahost cell canbeaccomplishedbyanymethodbywhich anucleicacidmoleculecanbeinsertedintoacell.Transfectiontechniquesincludebut are notlimitedto,transfection,electrophoretic_}microinjection,lipofection,adsorption andprotoplasmfusion.IntheuseofrecombinantDNAtechnology,expressioncanbeimprovedbyt ransfectednucleicacidmoleculeswithinahostceli,theefficiencywithwhichnucleicacid moleculesencodinga Ksp37canbetranscribed,theefficiency with which theresultanttranscriptsaretranslatedandefficiencyofpost translational modification recombinant techniques useful for increasing expression of nucleicacidmolecules.

Ex vivo production of Ksp37protein includes,but is notlimitedto,operativelylinkingnucleicacidnioleculestohighcopynumberplasmids,integrationofthe nucleicacid moleculesinto oneor morehostcellschromosomes,additionofvectorstabilitysequencesto plasmids,subsectioningormodification of transcription control signais (promotors,operators,enhancers) substitutions onnodifications of translational control signais (e.g. ribosome binding sites, Shere-Dalogans Signals),modificationsof nucleic acid molécules to correspond to the codum usage ofthe host cells anddeletionof sequencesthat destabilisetranscrîpts..The activityof an expressedrecombinantKsp37proteinmaybeimprovedbyffagmenting,modifyingorderivatising nucleicacidmoleculesencodingsuchaprotein.

Deliveringaformulation,includingaKsp37protein to a target cell in a mammal.

ATarget Site refers to a site în a mammal to which onedesîres to deliver a therapeutic formulation. For example,a target site can be a lymphocyte, stemcells,allbloodcomponents,andotherdeliveryvehiclesincluding,butnotlimitedto,natural lipid-containing delivery vehicles, including cellsand cellularmembranes; and artificïallipidcontainingdeliveryvehiclesincludingliposoniesandmicelles.

The deliveryvehiclecanbemodified by known techniquestotargetaspecificsiteinamammal,therebytargetingandmakinguseofanucieicacid moleculeatthatsite.

Suitablemodifications include manipulatingthe chemical formula of the lipid position of the delivery vehicleand / or introducinginto the vehicle a compound capable pf specificallytargetinga delivery vehicletoapreferredsite.forexample,apreferredcelltype.Speciallytargetingrefers tocausingadeliveryvehicle to bind to particular cell by the interaction of the compoundin the vehicle to a molécule onthesurfaceofthecell.Suitabletargetingcompoundsincludeiigandscapableofselectivelybind inganother molécule at a particular site. Example of such ligands includesantibodies, antigens,receptors and receptor ligands.

Manipuiatingthe Chemical formula of tire lipid position of thedeliveîyvehiclecanmodulatetheextracellularorintracellulartargetingofthedeliveryvehicl e.Forexampie, a Chemical can be added to the lîpid fonnula of a liposome that alters the charge of thelipid bi layer of the liposome so that the liposome fuses with particular cells having a particularchargecharacteristics.

Excipients

A formulationcomprising the Ksp37 proteinto beadmînisteredto a subjectcanalsoincludeother componentssuch as a phannaceutically acceptable excipient. For example,formulationsofthepresentinventioncanbefonnulatedinaexcipientthatthesubjectcan tolerate, examples of such excipients include water, saline, phosphate buffered solution,rîngerssolutîon, dextrosesolution,

Hank’ss0liition,polyethyleneglycoicontamingphysiologicalba!ancedsaltso!utionandothera queousphysiologically balancedsaltsolution.Nonaqueousvehicles,suchasfixedoils,sesameseed oil, and ethyleneoleatetriglyceridescanalsobeused.

Otherusefulformulationsincludesuspensionscontainingviscosityenhancingagentssuchassod iumcarboxymethylcellulose, sorbitol, or dextrin. Excipients can also contain minor amounts ofadditives,suchsubstancesthat enhanceisotonicityandchemicalstabilityorbuffers.Examples ofbuffers include phosphate buffer, bicarbonate buffer, très buffer, while examples of preservativesincludeTrimeresal,m-oro-cresol,formalinandbenzyIalcohol.

Standard formulait oncanbeeîtherliquids or injectable or solids which can be taken up in a suitable liquid as a suspension or solutionfor injection. Thus, in a nonliquidformulation,the excipientcan comprise of dextryls, Humanserumalbumin,preservativesetc.to whichsterilewaterorsalinecanbeaddedpriorto administration.

The Ksp37 protein may beadministeredbyatleastonerouteelected from the group consistîng of oral, nasal, topical, by inhalation, transdermal, rectal and parentéral (subcutaneous/ întramuscular) administration.

Controlled Release

A formulation including the Ksp37 protein or modified proteinto beadministeredto a mammal may include a controlled release composition that îs capable of slowly releasing Ksp37 into a mammal. As used herein a controlled release composition comprises a Ksp37 protein or in a controlled release vehicle.

Suitable controlled release vehicles include but are not limited to biocompatible polymers, other polymeric matrix capsules, micro capsules, micro particles, Bolus préparations, Osmotic pumps, diffusion devices, liposomes, lipospheres, dry powders and transdermal delivery Systems. Other controlled release of the invention includes liquids that, upon administration to a mammal form a solid or gel in-situ.

3?

Additional Compounds

Additional compounds capable of enhancing the ability of Ksp37 to protect a mammal from disease characterised by viral infection may be administered sequentially or simultaneously. Such compounds include compounds capable of regulatîng cell mediated immune response, regulatîng T-helper cell activity, regulatîng de granulation of mast cells, protecting sensory nerve endîngs, regulatîng eosinophii and or blast cell activity, and/or preventing or relaxing smooth muscle contractions. Such compounds will further induce microvascular permeability or modulating Thl and or Th2 cell subset différentiation.

The choice of compound to be administered in conjunction with Ksp37 protein can be made by one of skill in the art based on various characteristics of the mammal. In particular a mammafs genetic background, health history, physical signs, use of rescue médication and blood gases, and blood analysis.

Dose

A therapeutic dose of Ksp37 protein or modified proteins administered to a mammal, comprises a dose capable of protecting a mammal from and/or treating a disease characterised by infections and/or a Th-1 type immune response, when administered one or more times over a suitable period.

Alternatively, a therapeutic dose of Ksp37 protein or modified proteins comprises a

Q ³ £ dose that improves the immune system of a mammal. Further alternat!vely, a therapeutic dose of Ksp37 protein or modîfied protein comprises a dose that reduces viral infections and/or încreases Th 1-type cytokines.

A preferred single dose pf Ksp37 protein or modîfied proteins, which is hypothesised to produce a therapeutic or preventative resuit has been identified to be of between 0.001 pg/kg body weight of a mammal microgram and 20 pg/kg body weight of a mammal.

. Method of stimulating expression of KSP37 in a ceii

Gene therapy is a new therapeutic modal ity under considération for the treatment of various inherited and acquired disorders. It works on the premise of manipulating gene expression towards a therapeutic end. Recent advances in bîotechnology hâve stimuiated the development of in vivo gene therapy approaches based on the direct delivery ofthe therapeutic gene to the cells in vivo. Gene therapy aims to introduce a normal copy of the gene in question to restore, increase, or to modify the function of a protein.

A Nucleic acid molécule encoding a Ksp37 protein can be obtaîned from its natural source, either as an entire (complété) gene or a portion thereof. Altematively, a nucleic acid molécule can be produced using recombinantDNAtechnology(PolymeraseChainReactionamplificationcloning)orchemical synthesis.

Nucleicacidmoleculesincludenaturalnucleicacid moleculesandhomologuesthereof includingbut not limîtedto, naturalailelicvariants andmodifiednucleic acid molécules in which nucléotides hâve been înserted, deleted substituted and/orinvertedin such a manner that such inodifïcationsdo not substantially interfère with nucleic acid abilitytoencodeKsp37 proteinusefulinthemethodof thepresentinvention.

In one embodiment, a nucleic acid molécule encoding a Ksp37 protein that is useful in fighting viral immune system related infections such as HIV and viral cancers, is a nucleic acid sequence that translates to a protein that îs identîcal to the naturally occurrîng Ksp37 protein.

An isolated, or biological pure nucleic acid molécule, is a nucleic acid molécule that has been removed from its natural milieu.

A nucleic acid molécule encoding a Ksp37 protein can be produced using any of a number of methods known to those skilled in the art, includîng recombinant DNA techniques, such as site directed mutagenesis, Chemical treatment of nucleic acîd molécules with polar compounds to induce mutation, restriction enzymes cleavage of a nucleic acid fragment, irrigation of nucleic acid fragments, polymerase chain réaction (PCR) and or mutagenesis of select régions of nucleic acid sequence, synthesis of Oligonucleotide mixtures and ligation of mixture groups to build a mixture of nucleic acid molécules and combinations thereof. Nucleic acid molécule envelopes can be selected from a mixture ofmodified nucleic acid by screening ofthe function encoded by the nucleic acid.

A nucleic acid molécule used for encodîng a K.sp37 protein that îs useful în the method of present invention can be operatively linked to one or more transcription control sequences to form a recombinant molécule. The phrase “operatively linked” refers to linking nucleic acid molécule to a transcription controlled sequence in a manner such that the molécule is able to be expressed when transfected, transduced or transformed into a whole cell. Transcription control sequences are sequences which control the initiation, élongation and termination of transcription. Of particular importance are controlled transcription initiation, promotor, enhancer, operator and repressor sequences.

Suitable transcription control sequences include any transcription control sequence that can transcript in a recombinant cell useful for the expression of the Ksp37 protein, and or useful to administer în a mammal in the method of the present invention, Preferred transcription control sequences include those that function in mammalian, bacterial, or insect cells.

Transcription controlledsequence of the present invention can also include naturally occurrîngtranscrîptionscontrolled sequences naturally associated with gene encodîng a KSP 37 protein usefülina methodof thepresentinvention.

Recombinant molécules of the present invention, which can be either DNA or RNA, can also contain additional regulatory sequences, such as translation regulatory sequences, origins or réplications, and other regulatory sequences that are compatible with the recombinant cell. In one embodiment, a recombinant molécule of the present invention contains secretory signais (signal segment nucleic acid sequences) to enable an expressed Ksp37 protein to be secreted from a cell that produces the protein.

Suitable signal segments include, but are not limited to, signal segments naturally associated with any of the here afore menti oned Ksp37 proteins and ail associated species and nucléotides of Ksp37 proteins.

The rate limiting technologies of gene therapy are the gene delivery vehicles, known as vectors, used to accomplish gene transfer. Vectors can also be used to increase gene production of a spécifie protein.

Suitable Vector

Examples of phanna grade acceptable vectors which are particularly useful for the administration of nucleic acid molécules encoding Ksp37 protein are:

• pGEM-T Vector • pCMV3-C-GFPSpark

These are some of the vectors that are currently used on the market as vectors for KSP37 sequence.

An expression vector, otherwise known as an expression construct, is usually a plasmîd or virus designed for gene expression in cells. The vector is used to introduce a spécifie gene into a target cell, and can commandeer the cell's mechanism for protein synthesis to produce the protein encoded by the gene.

1

The gene of interest in this case is KSP37, which is normally produced by CD8⁺ cells part ofthe adaptive immunity in human hosts (Lôpez et ai, 2011).

The pCMV3-C-DDK (Flag) is an expression vector that is used for the expression of KSP37 in mammalian cells. The vector should contain spécifie segments to allow expression, these include a promoter, the correct translation initiation sequence such as a ribosomal binding site and start codon, a termination codon, and a transcription termination sequence. After the expression of the gene product, ît may be necessary to purify the expressed protein; however, separating the protein of interest from the great majority of proteins of the host cell can be a protracted process. To make this purification process easîer, a purification tag may be added to the cloned gene.

Vectors are transfected into the cells and the DNA may be integrated into the genome 20 by homologous recombination in the case of stable transfection, or the cells may be transiently transfected. Examples of mammalian expression vectors include the adénoviral vectors, the pSV and the pCMV sériés of plasmid vectors, vaccînia and retroviral vectors, as well as baculovirus. The promoters for cytomégalovirus (CMV) and SV40 are commonly used in mammalian expression vectors to drive gene 25 expression.

4^

In particular, pGEM-T vector, KSP37/FGFBP2 cDNA ORF CLONE supplied by SÎnoBiological has been identified as a suitable cloning vector of the full-length clone DNA of human fîbroblast growth factor binding protein 2,2 units to 10 units, for use in gene therapy against rétro viral infections, viral cancers and prions in mammals.

Excipients/Delivery Vehicles

According to the present invention, a nucleic acid molécule encoding a Ksp37 protein may be administered with a pharmaceutically accepted excipient. A pharmaceutically accepted excipient can include but is not limited to a natural lipid containing substrate, an oil, an ester, glycol, a virus, a métal partîcle or cationic molécule.

A pharmaceutical acceptable excipient which is capable of targetîng is herein referred to as a delivery vehicle. Pharmaceutical acceptable excipients of the present invention are capable of delivering a formulation, including a Ksp37 protein and/or nucleic acid molécule encoding a Ksp37 protein, to a target cell in a mammal. A Target Site refers to a site in a mammal to which one desires to deliver a fherapeufic formulation. For example, a target site can be a lymphocyte, stem cells, ail blood components.

Delivery vehicles include, but are not limited to, natural lipid-containing delivery vehicles, including cells and cellular membranes, artificial lipid-containing delivery vehicles, including liposomes and micelles.

A delivery vehicle of the present invention can be modified to target a spécifie site in a mammal, thereby targeting and making use of a nucleic acid molécule at that site. Suitable modifications include manipulating the Chemical formula of the lipid position of the delivery vehicle and/or introducing into the vehicle a compound capable of specifically targeting a delivery vehicle to a preferred site, for example, a preferred cell type.

Specially targeting refers to causing a delivery vehicle to bind to a particular cell by the interaction of the compound in the vehicle with a molécule on the surface of the cell. Suitable targeting compounds include ligands capable of selectively binding another molécule at a particular site. Examples of such ligands include antibodies, antigens, receptors, and receptor ligands.

Manipulating the Chemical formula of the lipid position of the delivery vehicle can modulate the extra cellular or intracellular targeting of the delivery vehicle. For example, a Chemical can be added to the lipid formula of a liposome that alters the charge of the lipid bi layer of the liposome so that the liposome fuses with particular cells having particular charge characterîstics.

Administration of the Vector

The vector is administered to the subject via an acceptable administration route, including nasal, oral, topical, inhalation, transdermal or parentéral administration

Additional Compounds

Additional compounds capable of enhancîng the ability of Ksp37 to protect a mammal from disease characterised by viral infection may be adminîstered sequentially or simultaneously. Such compounds include compounds capable of regulating cell mediated immune response, regulating T-helper cell activity, regulating degranulation of mast cells, protecting sensory nerve endîngs, regulating eosinophil and or blast cell activity, and/or preventing or relaxing smooth muscle contractions. Such compounds will further induce microvascular permeability or modulating Thl and or Th2 cell subset différentiation.

The choice of compound to be adminîstered in conjunction with a nucleic acid molécule encoding a Ksp37 protein can be made by one of skill in the art based on various characteristics of the mammal. In particular a mammal’s genetic background, health history, physical signs, use of rescue médication and blood gases, and blood analysis.

.ChemicaI Treatment with Polar Compounds to Activate Ksp37 Production

As previously disclosed, the polar compounds dimethylformamide (DMF) and DMSO hâve been shown to be powerful inhibitors of HIV production. Treatment of patients infected with HIV-1 with a transdermal patch containing dimethylformamide (DMF) showed promîsing results.

The action of polar agents on oncogenic expression is hypothesized to be the induction of cancer cells to become more benign. It would seem reasonable that to couvert a malignant cell to a benign type, there should be some modulation of gene expression causing malignancy in the first place. In the HL-60 human promyelocytic leukaemia cells, Diemthylsulfoxide [DMSO] reduced the expression of c-myoncogenes by 80 to 90% [5].

It is implicated that chemîcal inducers of cell différentiation play an important rôle in viral réplication by affecting cellular mechanisms of the host cell.

Polar compounds, including DMF may be used in the clînical management of viral infection and diseases through a cascade of events that starts with the activation of CD8 cells, which în turn activate production of Ksp37.

Polar compounds such as dimethylformamide [DMF] may be used as an activator of KSP37 protein.

DMF is generally used as a polar solvent and is readily absorbed through the skin, by inhalation, and upon oral ingestion. DMF is rapîdly metabolized, mainly in the liver, and excrétion occurs principally in the urine.

Transdermal and suppository delivery of Chemicals is a common method of altering hormonal properties of a patient.

DMF may be administered to a patient via transdermal application, using any suitable drug delivery device, for example by applying one or more dermal patches, or a suppository for rectal application to activate Ksp37 production. Treatment with a dermal patch to activate Ksp3 7 production would comprise application of the patch to

4?

the skin for a period of about 8 hours once a week, while treatment by rectal application of DMF to activate Ksp37 production would ideally comprise use of a suppository once a week.

A therapeutically effective dose of DMF for activating Ksp37 production be a dose that results in a peak plasma level of about 2 mg/l - 200 mg/l, preferably about 100 mg/l - 200 mg/I, still more preferably about 150 mg/l of DMF, Especially preferred is a peak plasma level of 100 mg/l -150 mg/l or 150 mg/l - 200 mg/l of DMF.

For transdermal administration of a polar compound, the rate of absorption is determined by the skin of the subject. Upon exposure to the human skin, liquid DMF is absorbed at a steady-state rate of approximately 9.4 mg/cm²/hour (see Mraz and Nohova, 1992, Occup. Env. Health 64:85-92).

Accordingly, the desired rate of absorption may be achieved by controlling the surface area of the skin exposed to the drug, as by determinîng the area of each patch and the number of patches applied to the skin. For example, two patches of diameter 9 cm will expose a total skin surface area of 127 cm² to the polar compound; for DMF, this will resuit in an absorption rate of about 1.2 g of DMF per hour.

An initial dose of about 15 mg/kg of DMT is especially preferred.

It is anticîpated that long term treatment of about 2 years with DMF would be required, before the production of the Ksp37 protein is suffïciently genetically modified to the therapeutic range required for at least 20 years.

The invention therefore identifies the optimum concentration range within which Ksp37, having a molecular weight în the range ranging from 24kDa to 45kDa, enhances the immune response of a subject against viral infection and/or diseases 5 associated with immune system disorders and/or viral cancers, and provides for the préparation of medicines and médicaments for the treatment of viral infection and/or diseases associated with immune system disorders and/or viral cancers and a method of treatment and/or protection against viral infection and/or diseases associated with immune system disorders and/or viral cancers.

AH publications cited herein are incorporated by reference in their entirety. Citation or discussion of a reference herein shall not be construed as an admission that such is prior art to the present invention.

Claims (28)

1, A pharmaceutical substance comprising a therapeutically effective amount of one or more of a clinically modified or genetically engineered Ksp37 protein and/or proteins having a molecular weight in the range ranging from 24 kDa to 45 kDa; and/or a vector encoded with a KSP37 gene which will translate to a Ksp37 protein; and/or proteins having a molecular weight in the range ranging from 24 kDa to 45 kDa; and/or a polar compound, for use in enhancing the immune response of a subject against viral infection and/or diseases associated with immune system disorders and/or viral cancers.

2. The pharmaceutical substance as claimed in claim 1, wherein the substanceis formulated to raise the Ksp37 blood plasma concentration level of the subject to between 400 ng/mL and 700 ng/mL when administered to the subject one or more times over a suitable period.

3. The pharmaceutical substance as claimed either of claims 1 or 2, wherein the substance includes a pharmaceutically accepted excipient.

4. The pharmaceutical substance as claimed in any one of claims 1 to 3, wherein the vector encoded with a KSP37 gene that is useful in fighting viral infection and/or diseases associated with immune system disorders and/or viral cancers, contains a nucleic acid sequence that translates to a protein identical to the naturally occurring Ksp37 protein.

5. The pharmaceutical substance as claimed in any one of the preceding claims.

wherein the vector encoded with the KSP37 gene which will translate to a Ksp37 protein includes a pGEM-T vector or a pCMV3-C-GFPSpark.

6. The pharmaceutical substance as claimed in claim 5, wherein the vector is pGEM-T vector, KSP37/FGFBP2 cDNA ORF CLONE (SinoBiological).

7. The pharmaceutical substance as claimed in any one of the preceding claims, wherein the polar compound is N, A-dimethylformamide (DMF).

8. The pharmaceutical substance as claimed in claim 7, wherein theamount of DMF for activating Ksp37 production is a dose that results in a peak plasma level of 2 mg/l - 200 mg/l, more preferably 100 mg/l - 200 mg/l, and most preferably 100 mg/l - 150 mg/l or 150 mg/l - 200 mg/l of DMF.

9. The pharmaceutical substance as claimed in any one of the preceding claims for use in protecting a subject from disease characterised by viral infection and/or diseases associated with immune system disorder and/or viral cancers, and/or treating a subject suffering from disease characterised by viral infection and/or diseases associated with immune System disorder and/or viral cancers.

10. Use of a therapeutically effective amount of the pharmaceutical substance of any one of claims 1 to 8 in the manufacture of a médicament for protecting a subject from disease characterised by viral infection and/or diseases associated with immune system disorder and/or viral cancers, and/or treating a subject suffering from disease characterised by viral infection and/or diseases associated with immune system disorder and/or viral cancers.

11. The use as claimed in claim 10, wherein the medicamentis formulated for administration in an amount that raises the Ksp37 blood plasma concentration level of the subject to between 400 ng/mL and 700 ng/mL when administered one or more times over a suitable period.

12. The use as claimed in either of daims 10 or 11, wherein the clinically modified or genetically engineered Ksp37 is formulated for administering în a dose between 0.001 pg/kg body weight of a mammal microgram and 20 pg/kg body weight of a mammal.

13. The use as claimed in any one of daims 10 to 12, wherein the médicament includes acontrolledrelease compositionthatiscapableofslowlyrdeasing the clinically modified or genetically engineered Ksp37intoamammal.

14. The use as claimed în any one of daims 10 to 13, wherein theclinically modified or genetically engineered Ksp37 protein is extracted from blood componentsand/or tissue, then purified, acetylised, genetically engineered, cloned and transferred back to a mammalian host as a therapeutîc and/or preventative vaccine against viral infection and/or diseases associated with immune system disorders and/or viral cancers.

15. The use as claimed ïn any one of daims 10 to 14, wherein the médicament is formulated for administration to the subject via an acceptable administration route, includîng nasal, oral, topical, inhalation, transdermal, rectal or parenteral administration.

16. The use as claimed in any one of claims 10 to 15, wherein the virus is a retrovirus, specifically HIV.

17. The use as claimed in any one of claims 10 to 16, wherein the viral cancer is ovarian cancer or leukaemia.

18. The use as claimed in anyone of claims 10 to 17, wherein the subject is a mammal.

19. A method of protecting a subject from disease characterised by viral infection and/or diseases associated with immune system disorder and viral cancers, and/or a method of treating a subject suffering from disease characterised by viral infection and/or diseases associated with immune system disorder and/or viral cancers, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical substance of any one of claims 1 to 8 to increase the blood plasma levels of Ksp37 protein in the subject to between 400 ng/mL and 700 ng/mL.

20. The method as claimed in claim 19, wherein the therapeutically effective amount of clinically modified or genetically engineered Ksp37 protein and/or proteins having a molecular weight in the range ranging from 24 kDa to 45 kDa, is between 0.001 pg/kg body weight of a mammal microgram and 20 pg/kg body weight of a mammal.

21. The method as claimed in either of claims 19 or 20, wherein the pharmaceutical substance is administered to the subject via an acceptable administration route, including nasal, oral, topical, inhalation, transdermal, rectal or parentéral administration.

22. The method as claimed in any one of claims 19 to 21, wherein the Ksp37 protein is extracted from blood componentsand/or tissue, then purified, acetylised, genetically engineered, cloned and transferred back to a mammalian host as a therapeutic and/or preventative vaccine against viral infection and/or diseases associated with immune system disorders and/or viral cancers.

23. The method as claimed in any one of claims 19 to 21, wherein the therapeutically effective amount of DMF for activating Ksp37 production is a dose that results in a peak plasma level of 2 mg/l - 200 mg/l, preferably 100 mg/l - 200 mg/l, and most preferably 100 mg/I -150 mg/l or 150 mg/I - 200 mg/l ofDMF.

24. The method as claimed in claim 23, wherein the DMF is administered transdermal.

25. The method as claimed in claim 19, wherein the virus is a retrovirus, specifically HIV.

5 >

26. The method as claimed in claim 19, wherein the viral cancer is ovarian cancer or leukaemia.

27. The method as claimed inclaim 19, wherein the subject is a mammaL

28. A therapeutic and/or preventative vaccine against viral infection and/or diseases associated with immune system disorders and/or viral cancers, the vaccine comprising a clinically modified or genetically engineered Ksp37 protein and/or proteins having a molecular weight in the range rangîng from 24 kDa to 45 kDa, and/or a vector encoded with a KSP37 gene which will translate to a Ksp37 protein and/or proteins having a molecular weight in the range ranging from 24 kDa to 45 kDa.