Polyinosinic acid-polycytidylic acid-based adjuvant

[0001] The invention generally relates to an adjuvant composition and a method for using it in increasing an immune response, more specifically, the invention relates to a composition, vaccines and a method for increasing antigen immunogenicity, and more particularly to polynucleotide adjuvant compositions and vaccines containing polynucleotide adjuvant compositions. The invention also relates to polynucleotide adjuvant compositions and vaccines for use in the claimed methods.

BACKGROUND OF THE INVENTION

1. Background Art [0002] The immune system may exhibit specific and non-specific immunity. In general, B and T lymphocytes, which have specific receptors for specific antigens on their cell surface, are a source of specific immunity. The immune system can respond to different antigens in two ways, by: 1) humoral immunity, which includes the stimulation of B lymphocytes and the production of antibodies or immunoglobulins, antigen presenting cells (APC) and helper T cells (Th1 and Th2), and 2) cellular immunity (CMI), which generally includes T lymphocytes including cytotoxic T lymphocytes (CTL), although other cells are also involved in forming CTL responses (e.g., Th1 and / or Th2 and APC).

[0003] Non-specific immunity includes various cells and mechanisms such as phagocytosis (absorption of foreign particles or antigens) by macrophages or granulocytes and, inter alia, natural killer (NK) cell activity. Nonspecific immunity is based on slightly less evolutionarily developed mechanisms and does not show acquired nature and specificity or memory, which are examples of specific immune response. The main differences between specific and non-specific immunity are based on the specificity of B and T cells. These cells usually reach their level of reactivity upon activation with specific antigens and have memory-capable mechanisms when exposed to this specific antigen in the future. As a result, vaccination (with specificity and memory) is an effective protocol for protection against harmful pathogens.

[0004] Adjuvants are generally compounds that when administered with an antigen (in mixed form or administered prior to administration of the antigen) enhance or modify the immune response to that particular antigen.

[0005] Exemplary adjuvants that have been used to enhance the immune response include aluminum compounds (all generally referred to as "alum"), water-oil emulsions (Freund's adjuvant (CFA) is an oil-in-water emulsion containing dried, thermally killed Mycobacterium tuberculosis), Saponin (isolated from the bark of Quillaja Saponoria, the active ingredient in an adjuvant known as Quile A), CpG ODN (synthetic oligodeoxynucleotide containing unmethylated CpG dinucleotides), MPL (derived from lipopolysaccharides from the body Salmonella minnesota Re595), liposomes (usually formed from biodegradable materials such as phospholipids) and biodegradable microspheric polymers (formed from various polymers such as PLA polymers such as PLA) and polyanhydrides). The adjuvant properties of these ingredients were evaluated after analyzing the pros and cons of each adjuvant.

[0006] The biggest problem with the use of adjuvants in human vaccines, and in particular in routine pediatric vaccines, is their toxicity and the adverse side effects of most adjuvant preparations. The use of modern technologies in vaccine development leads to the acquisition of purified subunits and synthetic antigens that have low immunogenicity. Developing new adjuvants to increase immunogenicity / efficiency and reduce side effects is one of the major challenges of vaccine research and development. Polynucleotide complexes have been studied for their various uses including their function as adjuvants. Double-stranded RNA (dsRNA) are very powerful biological modifying molecules that can exert significant effects on cells at nanomolar concentrations. The effects of dsRNA modulation cover a broad spectrum of activity at the molecular and cellular level.

[0007] At the molecular level, dsRNAs may have a biological effect such as interferon synthesis, protein kinase induction, enhancement of antigen histocompatibility, and inhibition of metabolism. At the cellular level, dsRNAs may have a biological effect such as pyrogenicity, mitogenicity, macrophage activation, activation of cellular immunity and induction of antiviral status. dsRNAs have promising potential through an immunomodulatory effect in therapies against microorganisms. US 4,124,702 discloses that double-stranded polynucleotides induced interferon in living animal cells. In US 3,906,092 it is disclosed that the antibody response to adjuvanted vaccines has been increased by incorporating the vaccine from a polynucleotide or polynucleotide complex. Houston et al. determined the PICLC complex (poly-L-lysine carboxy methyl cellulose complex of polyinosinic and polytitidylic acid) as a potential adjuvant by increasing the primary antibody response without the help of additional adjuvants. Houston et al., Infection and Immunity, 14: 318-9, 1976C. Mycoviral dsRNA was found to significantly increase

- 3 haemagglutination antibody response to sheep red blood cells (sRBC). Wright and Adler-Moore, Biochemical and Biophysical Research Communications, 131: 949-45, 1985.

[0008] However, PIC (polyinosinic and polytitidylic acid) shows significant toxicity when used in animals. For example, Phillips et al. Reported that significant toxicity was induced in dogs with sub-chronic PIC administration at a dose of 2.0mg / kg. Toxicity was characterized by reduced spontaneous activity, poor coordination, vomiting, anorexia, weight loss, hematological changes reflecting reduced hematopoiesis, increased alkaline phosphatase and transaminase activity, thymus degeneration, bone marrow destruction, dilatation of sinusoidal liver capillaries, and liver centrolobular cells collapse of liver structures and generalized arthritis. See Phillips et al., Toxicology and Applied Pharmacology, 18: 220-30, 1971.

[0009] PIC, one of the most commonly studied polynucleotide complexes, has not shown efficacy when used in monkeys and humans due to its instability in the body after its administration. Therefore, PIC has undergone many modifications to combat its weaknesses. For example, a complex of polyribosine and poly-biocytidilic acid with poly-L-lysine hydrobromide is about 5 to 15 times more resistant to pancreatic ribonuclease hydrolysis, like a PIC parent. Another example is the polyICLC dsRNA or PICLC for short, which has shown high efficiency as an antiviral or anti-cancer agent. PICLC is a synthetic dsRNA consisting of poly-ribosinic acid and poly-pyrocytidilic acid (PIC) fibers. Although PICLC is a promising immunomodulator that has remarkable potential in therapies against microorganisms and anti-cancer, it has been shown to cause serious side effects in humans, in particular when the drug is administered in repeated high doses. Some side effects that have been reported include: fever, hypotension, leukopenia, myalgia, thrombocytopenia and polyarthritis. The inherent toxicity problem must be eliminated to ensure the safe use of PICLC in humans. Moreover, the therapeutic efficacy of poly ICLC is limited by its in vivo stability.

[0010] An antiviral drug consisting of polyinosinic and polytitidylic acid (Poly I: C), kanamycin and calcium (Av-PICKCa) is used to treat viral infections. Av-PICKCa has been shown to be able to induce interferon and produce interleukin-2. Av-PICKCa administered alone as an antiviral drug stimulates a non-specific immune response, i.e. it stimulates a form of interferon that is not specific for any particular antigen. This antiviral response is significantly different from the specific antigen-specific immune response generated when the adjuvant is administered in combination with the antigen.

[0011] More importantly, the inventor has found that Av-PICKCa has adjuvant properties, i.e. it has the ability to elicit a specific specific immune response when administered with an antigen. The inventor also stated that Av-PICKCa was an effective adjuvant when used together with rabies antigens and

- 4 hemorrhagic fevers. Lin et al. Reported that Av-PICKCa can be used as an adjuvant (Lin, et al., A new immunostimulatory complex (PICKCa) in experimental rabies: antiviral and adjuvant effects, Arch Virol, 131: 307-19, 1993 and Chinese Patent No. 93105862.7). Chinese Patent No. 93105862.7 discloses the use of the general composition of Poly I: C, kanamycin and calcium (PICKCa) as a vaccine adjuvant for use in humans and mammals.

[0012] Lin et al. (1993) describe that PICKC's activity has been tested in experimental prophylaxis against rabies. PICKCa protected mice from peripheral infection in both strains of rabies: established and wild type. It also enhanced the protective activity of the experimental rabies vaccine injected before or after the occurrence of rabies infection. PICKCa enhanced both the specific and non-specific immune system responses including antibody production and cellular immune response as assessed by the production of interleukin-2.

[0013] Av-PICKCa samples are heterogeneous with respect to the size and weight of the molecules. Av-PICKCa has been described in the literature in terms of mean and range of sedimentation coefficient values, as measured by the Svedberg (S) sedimentation constant. The Av-PICKCa antiviral drug has in one embodiment a sedimentation constant ranging from 5S to 8S (Source A) see, Zhung JC, Research recollection of polyinosinic-polycytidylic acid (PIC). Work from the fifth conference on the use of interferon in clinical ventures and in theory, organized in China, Siam 1985, pp. 23-28. In other embodiments, Av-PICKCa has a sedimentation ratio of 4S to 12S with an average of 6S (Source B) or 5S to 12S with an average of 7S (Source C) or 8S to 10S (Source D), see Hu QG, Tianjin Av -PICKCa's laboratory research and clinical application, Fujian Medical Journal, 1983.12; (6): 28-30 and Hu QG Chinese Medical and Pharmaceutical Industry Journal, 1983 (9) 3134.

[0014] The sedimentation rate of this heterogeneous collection of molecules in Av-PICKCa can be converted to the corresponding molecular weight (mw in Dalton) using the transforming formula mw = 1100xS^2,2 (see Su BX et al; Introduction of Biochemical Technology, 1st edition, Zhongshan University, 1978, 356-357). The results of the conversion into Daltons are presented in the table below:

Table A: Av-PICKC characteristics

<td rowspan="2">AvPICKC source</td><td colspan="3">Sedimentation coefficient S</td><td colspan="3">Molecular mass in Dalton</td>

<td>Min.</td><td>Max.</td><td>Average</td><td>Min.</td><td>Max.</td><td>Average</td>

<td>AND</td><td> 5</td><td> 8</td><td> *</td><td> 38 000</td><td> 107 000</td><td> *</td>

<td>B</td><td> 4</td><td> 12</td><td> 6</td><td> 23 000</td><td> 260 000</td><td> 57 000</td>

<td>C</td><td> 5</td><td> 12</td><td> 7</td><td> 38000</td><td> 260 000</td><td> 79 000</td>

<td>D</td><td> 8</td><td> 10</td><td> *</td><td> 107 000</td><td> 174 000</td><td> *</td>

<td colspan="7">* data not cited in the references</td>

[0015] Original research on Av-PICKC as adjuvants was carried out by Lin et al. On samples containing molecules that exhibited Source A-like characteristics, i.e. their sedimentation coefficients were between 5S and 8S, which is equivalent to molecular weights ranging from 38,000 Dalton to 107,000 Dalton (see Lin et al., supra).

[0016] All forms of PICKCa were thought to be equally safe and effective considering that Av-PICKCa is significantly a form of PICKCa and further taking into account the history of use of Av-PICKCa as an antiviral drug. However, this was not a major problem. The research conducted by the inventor demonstrates that the effectiveness and toxicity of PICKCa, when used as an adjuvant in combination with an antigen, actually differ at different molecular weights. The inventor has indicated that AV-PICKCa does not provide sufficient optimal efficiency / safety for use as an adjuvant and under certain conditions PICKCa causes unacceptable negative side effects. Therefore, there remains a need to obtain an adjuvant that is more suitable for human use and which is safe and effective in providing the desired immunogenic effect. The invention addresses this need and provides other advantages that will be apparent with reference to the detailed description of this invention.

LITERATURE [0017] The following references may be relevant:

• JP 1093540A2;

• US Patent 4,124,702 • US Patent 3,692,899 • US Patent 3,906,092 • US Patent 4,389,395 • US Patent 4,349,538 • US Patent 4,024,241 • US Patent 3,952,097 • Houston et al., Infection and Immunity, 14: 318-9, 1976C • Wright and Adler- Moore, Biochemical and Biophysical Research Communications, 131: 949-45, 1985 • Phillips et al., Toxicology and Applied Pharmacology, 18: 220-30, 1971 • Lin et al., A new immunostimulatory complex (PICKCa) in experimental rabies: antiviral and adjuvant effects, Arch Virol, 131: 307-19, 1993 • China Patent 93105862.7

- 6 • Zhung JC, Research recollection of polyinosinic-polycytidylic acid (PIC). The paper of fifth Chinese interferon conference in clinical application and theory, Siam 1985, pp23-28 • Hu QG, Tianjin Av-PICKCa's laboratory research and clinical application, Fujian Medical Journal, 1983.12; (6): 28-30 • Hu QG Chinese Medical and Pharmaceutical Industry Journal, 1983 (9) 3134.

• Su BX et al .; Introduction of Biochemical Technology, 1st Edition, Zhongshan University, 1978, 356-357 • Gupta RK et al., Adjuvants - a balance between toxicity and adjuvanticity, Vaccine, 11: 293-306, 1993 • Arnon, R. (Ed.) Synthetic Vaccines 1: 83-92, CRC Press, Inc., Boca Raton, Fla., 1987 • Sela, M., Science 166: 1365-1374 (1969) • US Patent No. 6,008,200 • Ellouz et al., Biochem. &amp; Biophy. Res. Comm., 59: 1317, 1974 • US Patent 4,094,971 • US Patent 4,101,536 • US Patent 4,153,684 • US Patent 4,235,771 • US Patent 4,323,559 • US Patent 4,327,085 • US Patent 4,185,089 • US Patent 4,082,736 • US Patent 4,369,178 • US Patent 4,314,998 • Patent US 4,082,735 • US Patent 4,186,194 • US Patent 6,468,558 • New Trends and Developments in Vaccines, edited by Voller et al., University Park Press, Baltimore, Md., USA, 1978 • Klein, J., et al., Immunology (2nd ), Blackwell Science Inc., Boston (1997) • Gupa RK and Siber GR, Adjuvants for human vaccines - current status, problems and future prospects, Vaccine, 13 (14): 1263-1276, 1995

- 7 • Richard T Kenney et al. Meeting Retort - 2nd meeting on novel adiuvants currently in / close to human clinical testing, Vaccine 20 2155-2163, 2002 • Laboratory Techniques in Rabies Edited by FX Meslin, MM Kaplan, H Koprowski, Edition 4th ISBN 92 4 1544

SUMMARY OF THE INVENTION [0018] The invention discloses a polynucleotide adjuvant composition and a polynucleotide adjuvant composition and a vaccine for use in the claimed methods. The invention also relates to an immunogenic composition comprising a polynucleotide adjuvant composition together with an antigen (e.g., as in the case of a vaccine). The adjuvant compositions of the invention exhibit certain physical properties (e.g. molecular weight, size, concentration and pH), which are addressed to the needs of creating an effective and safe adjuvant to elicit an increased immune response. The invention further contemplates adjuvant compositions for use in the process of eliciting an immune response to antigenic components.

[0019] In one embodiment, the invention relates to an adjuvant polynucleotide composition comprising polyribosine-polybybytytilic acid (PIC), an antibiotic and a positively charged ion, wherein the antibiotic is kanamycin and the positively charged ion is calcium. The invention also relates to an immunogenic composition comprising a polynucleotide adjuvant composition together with an antigen or vaccine.

[0020] The invention serves to broaden knowledge by defining a new composition that can be used safely and effectively as an adjuvant to enhance and / or modify the immune response in animals or humans. While previous disclosures show the use of the Av-PICKCa antiviral drug in use as an adjuvant, it has been observed that the PICKCa form induces only a limited specific immune response when administered with an antigen. Further, PICKCa was observed to cause unacceptable adverse side effects under certain conditions.

[0021] The invention is dedicated to these problems by providing an adjuvant composition, generally referred to as "PIKA", which can be administered with the greatest efficiency and safety as an adjuvant in animals, including humans.

[0022] PIKA is a composition containing polynucleotides, kanamycin and calcium ions that has been developed as an adjuvant. The invention includes compositions that exhibit unique product characteristics that make them most suitable for use as an adjuvant in an immunogenic composition for administration to animals and / or humans.

[0023] More specifically, the invention relates to a polynucleotide adjuvant composition comprising a polynucleotide, an antibiotic and a positively charged ion, wherein the polynucleotide may be poly-ribosine-polybybytytilic acid (PIC); the antibiotic is kanamycin and the ion is calcium.

[0024] More specifically, the invention relates to specifications, including molecular weights, concentrations and pH, of a composition comprising a polynucleotide, kanamycin and calcium ions that are addressed to the safety needs of an adjuvant that elicits the maximum level of desired immune response.

[0025] The invention also relates to an immunogenic composition comprising a polynucleotide adjuvant composition and an antigen or vaccine.

[0026] In certain embodiments, the invention is in the form of a kit comprising a polynucleotide adjuvant and an immunogenic component.

[0027] This disclosure relates to a method of enhancing the immune response to antigenic components by administering to the host an immunogenic composition. The host can be human or animal organisms. Administration can be by injection, i.e. intramuscularly, intraperitoneally, intravenously or subcutaneously, or by inhalation. In other embodiments, the immunogenic composition may be administered through the rectum, vagina, nose, oral, ophthalmic, topical, transdermal or intradermal.

[0028] Accordingly, the invention relates to an adjuvant and an immunogenic composition that can be used safely in humans and animals.

[0029] Accordingly, in one aspect, the invention features a polynucleotide adjuvant composition comprising polyribosine-polybybytytilic acid (PIC), kanamycin, and calcium ions, wherein the composition comprises molecules of heterogeneous adjuvant with a molecular weight ranging from about 66000 to 1200000 Daltons.

[0030] In related embodiments, the molecules in the adjuvant polynucleotide composition are heterogeneous in terms of molecular weight, where the molecular weight is in the range from 300,000 to 1,200,000 Dalton, or from about 66000 to 660000 Dalton, or from about 300000 to 660000 Dalton, or from about 300,000 to 2,000,000 Dalton, or from about 300,000 to 4,000,000 Dalton, or from about 500,000 to 1,000,000 Dalton, or from about 1,000,000 to 1,500,000 Dalton, or from about 1500,000 to 2,000,000 Dalton, or from about 2,000,000 to 2,500,000 Dalton, or from about 2,500,000 to 3,000,000 Dalton, or from about 3,000,000 to 3,500,000 Dalton, or from about 3,500,000 to 4,000,000 Dalton, or from about 4,000,000 to 4,500,000 Dalton, or from about 4,500,000 to 5,000,000 Dalton.

[0031] In related embodiments, the molecules in the polynucleotide adjuvant composition have an average molecular weight equal to or greater than 150,000 Dalton, or equal to or greater than 250,000 Dalton, or equal to or greater than 350,000 Dalton, or equal to or greater than 500,000 Dalton, or equal to or greater than 650,000 Dalton, or equal to or greater than 750,000 Dalton, or equal to or greater than 1,000,000 Dalton, or equal to or greater than 1,200,000 Dalton, or equal to or greater than 1,500,000 Dalton, or equal to or greater than 2,000,000 Dalton.

[0032] Accordingly, in one aspect, the invention is a polynucleotide adjuvant composition comprising polyribosine-polybybytytidilic acid (PIC), kanamycin and calcium ions, wherein the composition comprises adjuvant molecules heterogeneous due to their particle size, having Svedberg sedimentation coefficients (S) in the range about 6.43 S to 24.03S.

[0033] In related embodiments, the molecules in the adjuvant polynucleotide composition are heterogeneous in particle size, wherein the particle size is from about 12.8S to 24.035, or from about 6.43 to 18.31S, or from about 12.8 to 18.31S, or from about 12.8S to 30.31S, or from about 12.8S to 41.54S, or from about 13.5S, to 18.31S, or from about 13.5S to 24.03S, or from about 16.14 to 22.12S, or from about 22.12S to 26.6S, or from about 26.6S to 30.31S, or from about 30.31S to 33.55S, or from about 33.55S to 36.45S, or from about 36.45S to 39.1S, or from about 39.1S to 41.54S, or from about 41.54S to 43.83S, or from about 43.83S up to 45.95S.

[0034] In further related embodiments, the adjuvant polynucleotide composition has an average sedimentation rate (Svedberg) greater than 9, or greater than 12, or greater than 13.5 or greater than 15, or greater than 17 or greater than 18 or greater than 19 or greater than 20 or greater than 21 or greater than 22 or greater than 25 or greater than 30.

[0035] The calcium ion source may be provided by, for example, calcium chloride, calcium carbonate, calcium fluoride, calcium hydroxide, calcium phosphate or calcium sulfate.

[0036] In one aspect of particular interest, the invention relates to a polynucleotide adjuvant composition comprising polyribosine-polybybytytilic acid (PIC), kanamycin and calcium ion, wherein the composition comprises molecular weight heterogeneous adjuvant molecules having a molecular weight from about 66000 to 1200000 Dalton.

[0037] In related embodiments, the polyribosine-polyribybytidilic acid (PIC), kanamycin, and calcium molecules have a molecular weight of from about 300,000 to 1,200,000 Dalton, or from about 66000 to 660000 Dalton, or from about 300000 to 660000 Dalton, or from about 300,000 to 2 million Dalton, or from about 300,000 to 4,000,000 Dalton, or from about 500,000 to 1,000,000 Dalton, or from about 1,000,000 to 1,500,000 Daltons, or from about 1,500,000 to 2,000,000 Daltons, or from about 2,000,000 to 2,500,000 Dalton, or from about 2,500,000 to 3,000,000 Dalton, or from about 3,000,000 to 3,500,000 Dalton, or from about 3,500,000 to 4,000,000 Dalton, or from about 4,000,000 to 4,500,000 Dalton, or from about 4,500,000 to 5,000,000 Dalton.

[0038] In other related embodiments, polypyroinosine polybybydylidic acid (PIC), kanamycin, and calcium molecules of a heterogeneous molecular weight adjuvant have an average molecular weight equal to or greater than 150,000 Dalton, equal to or greater than 250,000 Dalton, or equal to or greater than 350,000 Dalton, or equal to or greater than 500,000 Dalton, or equal to or greater than 650,000 Dalton, or equal to or greater than 750,000 Dalton, or equal to or greater than 1,000,000 Dalton, or equal to or greater than 1,200,000 Dalton, or equal to or greater than 1,500,000 Dalton, or equal to or greater than 1,500,000 Dalton.

[0039] In one particularly interesting aspect, the invention relates to a polynucleotide adjuvant composition comprising polyribosine-polybybytytilic acid (PIC), kanamycin and calcium, wherein the composition comprises heterogeneous adjuvant molecules

- 11 by particle size, having a Svedberg sedimentation factor (S) in the range of from about 6.43S to 24.03S.

[0040] In related embodiments, the molecules of the adjuvant polynucleotide composition are heterogeneous to the particle size, wherein the particle size is from about 12.85 to 24.03 S, or from about 6.43 to 18.31S, or from about 12.8 to 18.31S, or from about 12.8S to 30.3 1S, or from about 12.8S to 41.54S, or from about 13.5S to 18.31S, or from about 13.5S to 24.03S , or from about 16.14 to 22.12S, or from about 22.12S to 26.6S, or from about 26.6S to 30.31S, or from about 30.31S to 33.55S, or from about 33.55S to 36.45S, or from about 36.45S to 39.1S, or from about 39.1S to 41.54S, or from about 41.54S to 43.83S, or from about 43.83S up to 45.95S.

[0041] Further, in further related examples, poly-ribosine polybybyciditylic acid (PIC), kanamycin and calcium have an average sedimentation rate greater than 9, or greater than 12, or greater than 13.5, or greater than 15, or greater than 16, or greater than 17, or greater than 18, or greater than 19, or greater than 20, or greater than 21, or greater than 22, or greater than 25, or greater than 30.

[0042] In some embodiments, the invention relates to an adjuvant composition comprising poly-pyrosine-poly-pyrocytidilic acid (PIC), kanamycin and calcium, where the composition is preferred to exclude molecules, in particular to the extent that the excluded molecules do not have any significant immunogenic effect, wherein the excluded molecules have a molecular weight of about or less than 30,000 Dalton, about or less than 40,000 Dalton, about or less than 50,000 Dalton, about or below 60,000 Dalton, about or below 70,000 Dalton, about or below 80,000 Dalton, about or below 90,000 Dalton, about or below 100,000 Dalton, about or below 150,000 Dalton, about or below 200,000 Dalton, about or below 250,000 Dalton, about or less than 300,000 Dalton, approximately or below 350,000 Dalton, approximately or below 400,000 Dalton, approximately or below 450,000 Dalton, approximately or below 500,000 Dalton, approximately or below 600,000 Dalton, about or below 700,000 Dalton, about or below 800,000 Dalton, about or below 900,000 Dalton, about or below

And, 000,000 Dalton.

[0043] In some embodiments, the invention relates to a polynucleotide adjuvant composition comprising polyribosine-polybybytytilic acid (PIC), kanamycin and calcium, where it may be beneficial for the composition to exclude molecules, in particular to the extent that these excluded molecules do not cause any a significant immunogenic effect, wherein the excluded molecules have a particle size of about or less than 4.49S, about or less than 5.12S, about or less than 5.67S, about or below 6.16S, about or below 6.6S, about or below 7.02S, about or below 7.4S, about or below 7.77S, about or below 9.34S, about or below 10.64S, about or below

II, 78S, about or below 12.8S, about or below 13.73S, about or below 14.59S, about or below 15.39S, about or below 16.14S, about or below 17.54S, about or below 18 , 81S, about or less than 19.99S, about or less than 21.09S, about or less than 22.12S.

[0044] In one particularly interesting aspect, the invention relates to an immunogenic composition for enhancing the antigenicity of an antigenic component comprising a polynucleotide adjuvant composition.

[0045] In related embodiments, the immunogenic composition comprises a polynucleotide adjuvant and an antigen.

[0046] In related embodiments of the invention, the source of the antigen is a human antigen, animal but non-human animal antigen, plant antigen, bacterial antigen, fungal antigen, viral origin antigen, parasitic origin antigen or tumor antigen.

[0047] In related embodiments, the immunogenic composition includes a polynucleotide adjuvant composition and a rabies antigen.

[0048] In some embodiments, the antigens may be purified from a natural source, synthesized by solid phase synthesis, or may be obtained by recombinant genetics. The antigen may comprise a protein fragment containing one or more immunogenic regions of the molecule. Antigens can also be delivered in whole cells or microorganisms (e.g. whole virus particles) that can be alive, attenuated, limited or killed.

[0049] In other embodiments, the antigens include one or more agents from the group of infectious agents, plant antigens, cancer agents, allergenic agents and other human factors as well as for the development of autoimmune diseases. In other embodiments, the antigens include one or more infectious agents from any virus, bacteria, mycobacteria, fungus, and parasite.

[0050] The adjuvant polynucleotide composition of the invention can also be used to enhance the immune response directed against antigens produced by the use of DNA vaccines. The DNA sequences in these vaccines encoding the antigens may be either "naked" or may be contained in delivery systems, such as liposomes.

[0051] Furthermore, in other related embodiments, the rabies antigens are selected from rabies vaccines grown in culture of human diploid cells (HDCV), or inactivated, purified rabies vaccines grown in culture of hamster kidney cells (HKC-IPRV), or inactivated raw rabies vaccines grown in hamster kidney cell culture (HKC-ICRV), or purified rabies virus vaccines propagated in the Vero cell line (PVRV), or purified vaccines produced in chick embryo cell culture (PCEC), or purified vaccines produced on duck embryos (PDEV), or inactivated purified rabies antigens produced in hamster kidney cells

- 13 (HKC-IPRA) or inactivated raw rabies antigens produced in hamster kidney cells (HKC-ICRA).

[0052] In one particularly interesting aspect, the invention relates to an immunogenic composition for enhancing the antigenicity of an antigenic component consisting of a polynucleotide adjuvant composition that is capable of eliciting an antigen-specific cellular immune response.

[0053] In one particularly interesting aspect, the invention relates to an immunogenic composition for increasing the antigenicity of an antigenic component comprising a polynucleotide adjuvant composition that is capable of eliciting an antigen-specific cellular immune response (B).

[0054] In one particularly interesting aspect, the invention relates to an immunogenic composition for increasing the antigenicity of an antigenic component comprising a polynucleotide adjuvant composition that has the ability to elicit a mixed immune response of an antigen specific for T and B lymphocytes.

[0055] In one particularly interesting aspect, the invention relates to an immunogenic composition for increasing the antigenicity of a component comprising a polynucleotide adjuvant composition and inactivated purified rabies antigens produced in hamster kidney cells, where the presence of rabies antigens should achieve a minimum amount such as more than 1 International Unit (IU ).

International,

International,

International,

International, [0056] In related embodiments, the immunogenic composition consists of polynucleotide adjuvant compositions and inactivated purified rabies antigens produced in hamster kidney cells, where the presence of rabies antigens should reach a minimum amount such as more than 0.25 International Units, more than 0.5 International Units, more than 1.2 International Units, more than 1.4 International Units, more than 1.6 International Units, more than 1.8 International Units, more than 2.0 International Units, more than 2.2 International Units, more than 2.4 Units more than 2.6 International Units, more than 2, 8 Units more than 3.0 International Units, more than 3.2 Units more than 3.4 International Units, more than 3.6 Units more than 3.8 International Units, or is more than 4.0

International Units.

[0057] In one particularly interesting example, the invention relates to an immunogenic composition for increasing the antigenicity of a component comprising a polynucleotide adjuvant composition and inactivated purified rabies antigens from hamster kidney cells where the presence of the adjuvant and rabies antigens is in a ratio of 1 to

1.

[0058] In related embodiments, the immunogenic composition includes a polynucleotide adjuvant composition and inactivated purified rabies antigens from hamster kidney cells, wherein the presence of adjuvant and antigens

- 14 rabies is in a ratio of less than 1 to 10, than about 1 to 9, than about 1 to 8, than about 1 to 7, than about 1 to 5, than about 1 to 4, than about 1 to 3, than about 1 to 2, than about 2 to 1, than about 3 to 1, than about 4 to 1, than about 5 to 1, than about 6 to 1, than about 7 to 1, than about 8 to 1, than about 9 to 1 than about 10 to 1, greater than 10 to 1.

[0059] In one particularly interesting example, the invention relates to an adjuvant composition or immunogenic composition wherein the immunogenic composition or adjuvant composition comprises an immunogenic composition and is in solid or liquid form or in solution or suspension.

[0060] In one particularly interesting example, the invention relates to an adjuvant composition or immunogenic composition comprising an adjuvant composition, wherein the adjuvant composition or immunogenic composition has been lyophilized.

[0061] In related embodiments, the invention relates to a kit comprising an adjuvant composition and an antigenic component.

[0062] In one particularly interesting example, the invention relates to the use of a polynucleotide adjuvant composition for the preparation of a medicament for enhancing a host immune response.

[0063] In one particularly interesting example, the invention relates to a method of enhancing an immune response to an antigenic component, comprising administering to the host an immunogenic composition to enhance the antigenicity of the antigenic component comprising the polynucleotide adjuvant composition.

[0064] In related embodiments, the method of administering the immunogenic composition to the host may be selected from the group consisting of: parenteral injection, intramuscular injection, intraperitoneal injection, intravenous injection, subcutaneous injection, inhalation, rectal administration, vaginal administration, nasal administration , oral administration, ocular administration, topical, transdermal or intradermal administration.

[0065] The disclosure relates to a method of enhancing an immune response to an antigenic component, comprising administering to the host an immunogenic composition for increasing the antigenicity of the antigenic component comprising the polynucleotide adjuvant composition wherein the host is a human.

[0066] The disclosure relates to a method of enhancing an immune response to an antigenic component, comprising administering to the host an immunogenic composition for increasing the antigenicity of the antigenic component comprising the polynucleotide adjuvant composition wherein the host is an animal.

[0067] These and other features and advantages of the invention will be apparent after reading the further detailed description of preferred embodiments in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES [0068] Figure 1 shows the relative molecular weight for Av-PICKCa and PIKA samples.

Figure 2 shows that PIKA induces a dose dependent production of specific cytokines: interferon-gamma.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION [0069] The invention can be easily understood by reference to the following detailed descriptions of specific embodiments of the invention and the examples included herein.

[0070] Unless otherwise stated, all technical and scientific terms in this application have the meaning commonly known and understood by those skilled in the art to which the invention belongs. Although methods and materials similar or equivalent to those described in the application may also be used in the practice or testing of the invention, these preferred methods and materials have been described. All publications mentioned herein are incorporated by reference to describe and disclose methods and / or materials in connection with the publications cited.

[0071] It should be noted that the singular forms used and claimed herein include references to the plural, unless the context of the sentence clearly indicates otherwise. The term "text" includes the plural of such texts and reference to "segment" includes reference to one or more segments and their equivalents known to those skilled in the art, and so on. Furthermore, it is noted that the reservations may be recorded so as to exclude any optional elements. As a result, this statement is intended to serve as the basis for the exclusive use of terms such as "only", "only" and similar in combination with the citation of the claimed elements or as a "negative" limitation.

DEFINITIONS [0072] Before discussing the details of the invention, it is worth providing an understanding of the definitions of many terms used herein.

[0073] The term "adjuvant," as used herein, refers to any substance or mixture of substances that increases or differentiates the host's immune response to an antigenic component. In particular:

1. The term "PICKCa" generally refers to a mixture of poly I: C, kanamycin and calcium regardless of specific physical and immunogenic properties.

2. The term "Av-PICKCa" refers to the form of PICKCa used commercially as an antiviral drug.

3. The term "PIKA" refers to a composition of the invention comprising poly I: C, an antibiotic (kanamycin) and a positively charged ion (calcium ion), where PIKA is characterized by physical properties (e.g., molecular weight, size and the like as described herein), that after administration PIKA shows features

Characteristic for an adjuvant with reduced side effects (e.g. reduced toxicity) relative to, for example PICKCa and greater potency (e.g. stimulates an increased immune response) relative to, for example, Av-PICKCa.

[0074] "PIC containing molecule" or "PIC containing component" refers, without limitation, to PIC, which may optionally be present in the complex or otherwise be combined with at least one or two antibiotics (e.g., kanamycin) and a positively charged ion ( e.g. calcium ion) present in the composition containing the PIC molecule.

[0075] "Heterogeneous" as used herein in the context of an adjuvant composition of the invention indicates components of the composition, e.g., PIC containing molecules, which are not uniform with respect to physical properties of molecular weight, size or both.

[0076] The term "animal" includes humans and all domesticated and wild mammals and birds, including without limitation cattle, horses, cows, pigs, sheep, goats, dogs, cats, rabbits, deer, mink, chickens, ducks, geese, turkeys , courses and the like.

[0077] The term "antibody" includes polyclonal and monoclonal antibodies, and an antigenic component that binds fragments of such antibodies including Fab, F (ab ') 2, Fd, Fv fragments, and single chain derivatives of these compounds. In addition, the term "antibody" naturally antibodies present, including, for example, chimeric, bifunctional and human antibodies, and related synthetic isoforms.

[0078] As used herein, the term "antigenic component" refers to any substance that can be recognized by the immune system (e.g., bound by an antibody or processed to elicit a cellular immune response) under certain conditions.

The term "antigen" refers to a substance, including vaccine compositions, where the vaccine itself contains the antigen component and may or may not contain an adjuvant other than PIKA, which, when administered appropriately (e.g. parenterally), includes an immune response, e.g. antibody formation, including antibodies that specifically bind to the antigen. Two characteristic features of antigens and their immunogenicity are their ability to induce an immune response in vivo and their antigenicity, i.e. their ability to be selectively recognized by antibodies that are derived from antigens.

[0080] The terms "cellular immunity" and "cellular immune response" are intended to refer to immune protection provided by lymphocytes, such as protection provided by T lymphocytes when they come in close contact with their target cells. The cellular immune response normally involves lymphocyte proliferation. The term "lymphocyte proliferation" should refer to the process of B, T-helper or cytotoxic T-lymphocyte (CTL) proliferation.

[0081] The term "effective amount of an antigenic component" refers to the amount of an antigenic component that, optionally in combination with an adjuvant, will cause the subject to produce a specific immune response to the antigenic component.

[0082] The term "enhanced immune response" or the like defines a situation in which the immune response is increased, improved or enhanced to favorably affect the host with respect to the previous status of the immune response, e.g., prior to administration of the immunogenic composition of the invention.

[0083] The terms "humoral immunity" and "humoral immune response" refer to a form of immunity in which antibody molecules are produced in response to antigenic stimulation.

[0084] The term "immune response" refers to any response to an antigenic component by the immune system of a vertebrate subject. Exemplary immune responses include, but are not limited to, cellular as well as local, and humoral systemic responses such as CTL responses, including antigen-specific induction of CD8 + CTL molecules, T helper cell responses include c T cell proliferative responses, and cytokinin release, and responses B cells including antibody response a.

[0085] The term "eliciting an immune response" is used generally to include the induction and / or potentiation of an immune response.

[0086] The term "eliciting an immune response" refers to an immune response that is stimulated, initiated or induced.

[0087] The term "enhancement of an immune response" refers to a pre-existing immune response that has been improved, strengthened, supplemented, amplified, enhanced, increased or elongated.

[0088] The term "poly I: C" or "PIC" refers to a composition comprising polyribosine and polyribytytilic acid nucleic acids, which may also be called polyinosine polycytididyl acids, respectively.

[0089] The term "immunogenic amount" refers to a dose of an antigenic component sufficient to elicit an immune response when administered in a composition of the invention, as compared to the immune response observed in the absence of polynucleotide adjuvant.

[0090] The term "immunoassay dose" refers to the dose of adjuvant required to increase the antibody dose and / or cellular immunity when administered with the antigenic component in the composition of the invention, which can be compared with the increase in antibody levels and / or cellular immunity observed in the absence of polynucleotide adjuvant. .

[0091] As used herein, the term "mixing" includes any method of combining the components of a composition, which means without limitation: mixing, dispensing, dissolving, emulsifying, coagulating, suspending or other physically combining the components of the composition.

[0092] The term "pharmaceutically acceptable salt" of an ingredient means a salt that is pharmaceutically acceptable and that has the desired pharmacological activity of the parent compound. Such salts include: (1) acid addition salts, formed from inorganic

- 18 acids as acid: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like or formed from organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanopropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3- (4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, glucoheptonic acid, 4,4'-methylene bis- (3-hydroxy-2-eno -1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid and the like; or (2) salts formed when the acid proton is present in the parent component or is replaced by a metal ion, e.g., an alkali metal ion, a beryllium ion, or an aluminum ion; or there is coordination with an organic base such as ethanolamine, diethanolamine, tretanolamine, tromethamine, N-methylglucamine and the like.

[0093] The term "treatment" describes any treatment of a disease in animals (vertebrates), in particular in humans, and includes (I) preventing the occurrence of a disease in a subject who may have a predisposition to this disease but has not yet been diagnosed as ill, (ii ) inhibiting the disease, i.e., arresting the development of the disease, or (iii) relieving the disease, i.e., causing regression of the disease.

[0094] The term "unit dosage form" as used herein refers to physical discrete units suitable as human and animal dosage units, each unit containing a predetermined quantity of ingredients of the invention calculated in an amount sufficient to produce the desired effect in combination with a pharmaceutically / physiologically acceptable solvent or carrier.

OVERVIEW OF THE INVENTION [0095] The invention relates to ingredients and methods used to enhance an immune response that may be humoral and / or cellular, in humans, animals or in cell cultures. Generally, the composition includes an immunogenic composition comprising an adjuvant. The presence of an adjuvant strengthens or modifies the immune response. In this way, the humoral and / or cellular immune response is more effective with the presence of an adjuvant. Furthermore, an adjuvant may affect the quality of the immune response by influencing the subclasses (isotypes) of immunoglobulins and cytokines produced.

[0096] The main characteristic of an adjuvant is the ability to stimulate the desired level and type of immune response without causing adverse side effects. There is currently a limited number of adjuvants approved for human use that have this combination of characteristics. Security Standards

- 19 for immunogenic substances and in particular for vaccines are defined in detail and very strictly enforced. Significantly limiting the development of an effective adjuvant is to develop a product that is strong enough to elicit an appropriate immune response and will not cause adverse side effects.

[0097] A preferred embodiment is a polynucleotide adjuvant, wherein the polynucleotide is polyinosine polycytidilic acid. PIC has been shown to be an effective adjuvant on its own, but also exhibits an unacceptable safety profile and is unstable in humans and primates. The present invention relates to a PIC composition combined with an antibiotic and a positively charged ion and enhances the desired characteristics of the adjuvant immunogenicity while increasing the safety profile and stability.

[0098] The invention is based on the disclosure that the physical and biological characteristics of the PIKA adjuvant composition affect the characteristics of the immune response and adverse side effects. Research has unexpectedly revealed that by adapting specific characteristics of a polynucleotide adjuvant, it becomes stronger or less potent and / or more or less toxic as described later in this document. Hence, by defining the composition of the adjuvant with its physical characteristics, it is possible to more accurately describe the features of the adjuvant composition that provide a favorable immune response and favorable safety / stability.

[0099] The adjuvant of the invention, which is described herein for convenience as a PIKA adjuvant, is fully defined by the combination of its chemical composition and the fundamental physical characteristics of the molecules that make up the adjuvant. The specific form of PIKA, which has significant overarching characteristics of immunogenicity while being safe for use in animals and humans, is best defined by one or more specific characteristics such as composition, molecular weight, particle size, concentration and pH.

[0100] PIKA generally contains a polynucleotide, an antibiotic, and a positively charged ion, where the polynucleotide is polybyrosine-polyribybytytidyl acid (PIC), the antibiotic is an aminoglycoside (kanamycin) and the positively charged ion is calcium.

[0101] "Aminoglycoside" antibiotics refer to antibiotics whose structure contains aminosugars attached to the aminocyclitol ring (hexose nucleus) by glycosidic linkages. Aminoglycoside antibiotics are obtained from various species of Streptomyces and Micromonospora or are synthetically produced. For example, kanamycin is an aminoglycoside antibiotic derived from the soil bacterium Steptomyces Kanamycetics, used to treat various infections, in particular those caused by Gram-negative bacteria.

[0102] The PIKA composition is obtained by mixing polyinosinic acid, polytitidyl acid, an antibiotic and a source of positively charged ions in a sodium chloride / phosphate buffer solution that has a pH between 6 and pH. Polyinosinic and polytitidylic acids are generally provided at a concentration of 0.1 to 10 mg / ml, preferably 0.5 to mg / ml and even more preferably 0.5 to 2.5 mg / ml. The value of the hyperchromic effect

Should be higher than 10%, preferably higher than 15% and even more preferably higher than 20%. The preparation of PIC and its mixture with kanamycin and calcium is preferably carried out under standard quality conditions consistent with the international processing of goods.

[0103] In certain embodiments of the invention, kanamycin in the polynucleotide adjuvant composition may be used together or replaced by one or more antibiotics selected from the group consisting of: tobramycin, anthracycline, butyrosine sulfate, gentamycin, hygromycin, amikacin, dibecacin, nycamamidine, nebramycin puromycin, streptomycin and streptozotocin. Antibiotic (e.g. kanamycin or the like) in the polynucleotide adjuvant composition of the invention is generally provided at a concentration of from about 10 to 100,000 units / ml, preferably from about 100 to 10,000 units / ml, and even more preferably from about 500 to 5000 units / ml.

[0104] The positively charged ion (calcium ion) may be provided in the composition of the invention in a concentration ranging from about 10 μmol to 10 mmol / ml, preferably from about 50 μmol to 5 mmol / ml, and even more preferably from about 100 μmol to 1 mmol / ml.

[0105] As noted above, the positively charged ion may be provided in the form of any suitable salt or organic complex, including, but not limited to, chloride, fluoride, hydroxide, phosphate, or sulfate salts. For example, where the positively charged ion is calcium, the ion may be in the form of calcium bicarbonate, calcium chloride, calcium fluoride, calcium hydroxide, calcium phosphate or calcium sulfate.

[0106] Where the positively charged ion of the adjuvant composition is calcium, it may exist in combination or be replaced by other positively charged ions, including, cadmium, lithium, magnesium, cerium, cesium, chromium, cobalt, deuterium, gallium, iodine , iron and zinc, where these ions can be in the form of inorganic salts or organic complexes.

[0107] The resulting composition is further transformed into PIKA through an additional manufacturing process that involves the isolation of molecules of defined size and / or molecular weight. Separation of polynucleotide molecules exhibiting specific characteristics is accomplished using filtration, chromatography, heat treatment, separation by centrifugation, electrophoresis and similar methods that are standard and known to those skilled in the art.

[0108] In certain examples of the invention, the polynucleotide adjuvant composition is then defined by its physical properties - molecular weight. In the course of the research, it was surprisingly found that there is a positive correlation between the molecular weight and the effectiveness of the adjuvant polynucleotide composition. The observed level of potency of the immunogenic composition containing the polynucleotide adjuvant composition, including the ability to induce the production of immunoglobulins and cytokines, increases with increasing weight of the polynucloethide adjuvant composition. The molecular weight of the polynucleotide adjuvant can be determined by agarose gel electrophoresis as described in Example 1.

[0109] As shown in the examples in the following section, the inventor of the invention has noticed that vaccine compositions containing PIKA adjuvant with different molecular weights showed a direct correlation between the molecular weight and the protective power of specific antigens (Example 2). Similarly, the inventor has found that there is a direct correlation between the molecular weight of the PIKA adjuvant composition and the ability to induce interferon-gamma production when administered to a host in combination with rabies antigen (see Example 3).

[0110] The inventor further identified that during clinical trials conducted in humans in 1996 in China using a rabies vaccine with an adjuvant containing PICKCa of particularly high molecular weights, the resulting composition unexpectedly showed an unacceptable level of negative side effects. The results of clinical trials conducted in 1996 that were not previously published are presented in Example 4. Molecular weight studies are presented in Examples 5 and 6. Clinical studies were conducted under the jurisdiction of the Chinese Food and Drug Administration. This would prevent the adjuvant from being administered to people in a controlled clinical trial environment if such side effects were anticipated based on existing knowledge of the subject.

[0111] The inventor has found that PIKA adjuvant compositions of the invention in preclinical studies with molecular weights up to 1.0 x 10⁶ and vaccine compositions comprising PIKA adjuvant compositions with molecular weights up to 5.5 x 10⁵ showed a wide safety margin in specific toxicity tests (see Example 7). PIKA with a maximum molecular weight of 1.2 x10⁶ was successfully used in preclinical studies (see Example 3). Further research by the inventor demonstrates the safety of PIKA when used in combination with the vaccine antigen component (see Example 8).

[0112] The results of the next experiments conducted by the inventor in China in 2002 also show that the use of PIKA relates to a safe and effective adjuvant for human use. The results of this experiment not previously published are presented in Example 9.

[0113] Based on the above observations, a preferred embodiment of PIKA includes molecules having physical molecular weight and / or size properties that are beneficial for increasing power and efficiency when providing an adequate level of safety so that they do not cause any negative side effects. Molecules present in Av-PICKCa with lower molecular weight ranges may be effective as antiviral compositions, but are significantly less effective than PIKA molecular compositions when used with an adjuvant in an immunogenic composition. Furthermore, PIKA has been shown to have a safety profile that is better than PICKC.

[0114] One aspect of the invention is therefore the PIKA molecular weight of the composition of the invention.

[0115] The ingenious PIKA composition generally comprises a collection or population of molecules, where the molecules have physical properties, e.g., molecular weight and / or size, that provide the desired effect in eliciting an immune response while, alleviating or avoiding adverse side effects (such as those associated with the PICKC administration). Generally, PIKA molecules are heterogeneous due to their molecular weights and / or sizes.

[0116] As generally used herein, and unless otherwise stated, PIKA, the adjuvant composition of the invention contains PIC acids that may be present in a complex with an antibiotic (kanamycin) and a positively charged ion (calcium ion). The molecules in the PIKA molecule are heterogeneous in molecular weight (e.g. as determined by Dalton) or size (e.g. as determined by sedimentation factor).

[0117] Where the range is used to refer to the characteristics of heterogeneous PIKA molecules (e.g., molecular weight or size), reference to such range here indicates the respective upper and lower molecular weight limits or PIKA particle size in the composition, but does not indicate that the composition comprises a PIKA molecule that has a molecular weight or size that is representative of all molecular weights or sizes within this range. Thus, for example, a molecular weight of about 66,000 to 1,200,000 Dalton indicates that PIKA of about 66,000 Dalton and about 1,200,000 Dalton are included in the composition, but there is no requirement that PIKA of 88,000 Dalton is included in the composition (although may indeed be present there).

[0118] Where the physical characteristics of the PIKA molecules in the ingenious composition are defined in the molecular weight range, the PIKA molecules are heterogeneous with respect to molecular weights, where the molecular weights are in the range from about 300,000 to 660000 Dalton, from about 300000 to 1200,000 Dalton, from about 66000 to 660000 Dalton, or from about 66000 to 1200000 Dalton.

[0119] The invention also discloses compositions comprising heterogeneous PIKA molecules based on molecular weight, wherein the molecular weight ranges from about 300,000 to 2,000,000 Dalton, from about 300,000 to 4,000,000 Dalton, from about 500,000 to 1,000,000 Dalton, from about about 1,000,000 to 1,500,000 Dalton, from about 1,500,000 to 2,000,000 Dalton, from about 2,000,000 to 2,500,000 Dalton, from about 2,500,000 to 3,000,000 Dalton, from about 3,000,000 to 3,500,000 Dalton, from about 3,500,000 to 4,000,000 Dalton, from about 4,000,000 to 4,500,000 Dalton, or from about 4,500,000 to 5,000,000 Dalton. PIKA molecules having molecular weights within the upper and lower limits of these ranges as well as within these ranges are present in the composition.

[0120] Where the physical properties of PIKA molecules of the invention are defined by average molecular weight, PIKA molecules may have an average molecular weight equal to or greater than 150,000 Dalton, equal to or greater than 250,000 Dalton, equal to or greater than 350,000 Dalton, equal to or greater than 500,000 Dalton, equal to or greater than 650,000 Dalton, equal to or greater than 750,000 Dalton, equal to or greater than

- 23 1,000,000 Dalton, equal to or greater than 1,200,000 Dalton, equal to or greater than

1500000 Dalton, or equal to or greater than 2 million Dalton.

[0121] Where the physical properties of PIKA molecules in an ingenious composition are defined by a sedimentation factor, which is a measure of molecular weight and size (particle size), PIKA may have a sedimentation factor greater than 9, or greater than 12, or greater than 13.5, or greater than 15, or greater than 16, or greater than 17, or greater than 18, or greater than 19, or greater than 20, or greater than 21, or greater than 22, or greater than 25, or greater than 30.

[0122] In some embodiments, the invention relates to a polynucleotide adjuvant composition comprising polyribosine-polybybytytilic acid (PIC), kanamycin and calcium, wherein the composition excludes detectable amounts of molecules that have a molecular weight of about or less than 30,000 Dalton, about or less than 40,000 Dalton, about or less than 50,000 Dalton, about or less than 60,000 Dalton, about or less than 70,000. Dalton, approximately or below 80,000 Dalton, approximately or below 90,000 Dalton, approximately or below 100,000 Dalton, approximately or below 150,000 Dalton, approximately or below 200,000 Dalton, approximately or below 250,000 Dalton, approximately or below 300,000 Dalton, approximately or below 350,000 Dalton, about or below 400,000 Dalton, about or below 450,000 Dalton, about or below 500,000 Dalton, about or below 600,000 Dalton, about or below 700,000 Dalton, about or below 800,000 Dalton, about or less than 900,000 Dalton, or about or less than 1,000,000 Dalton. In this embodiment, the exclusion of molecules with such low molecular weights is particularly important due to the fact that these excluded molecules do not have any significant immunogenic effect.

[0123] The inventor has revealed that PIKA containing molecules with molecular weights up to 1.0 x 10⁶Dalton, is safe for use in animals in specific toxicity tests (see Example 7). PIKA containing molecules with molecular weights up to 1.2 x 10⁶Dalton is safely used in preclinical studies (see Example 3). PIKA has also demonstrated safe use in an immunogenic composition (see Example 8). This PIKA compound composition has advantages in the form of efficacy. PIKA containing molecules with molecular weights up to 6.6 x 10⁵ The Dalton also elicits an effective immune response in a wider range of safety when used for animals and humans. By increasing the molecular weight of the smallest particles present up to 6.6 x 10⁵ Dalton and preferably up to 3.0 x 10⁵ Dalton improves the effectiveness of an adjuvant without violating safety standards.

[0124] It has also been disclosed that the concentration of the polynucleotide adjuvant composition can affect the molecular weight of the molecules contained in the composition. The molecular weight of PICKCa has been shown to increase with increasing concentration of the adjuvant composition (see Example

5). The inventor has observed that the increasing concentration of polynucleotide adjuvant can lead to coalescence (aggregation) of PICKCa molecules, which will result in high molecular weight molecules. This process has been shown to be irreversible. Therefore, the following dilution of the polynucleotide adjuvant composition in

A suitable medium does not result in the reduction of the molecular weight of the adjuvant molecules. As shown in Example 6, when the concentrated large molecular form of the polynucleotide adjuvant composition is bound to rabies antigen, the result is a composition that maintains its high molecular weight range. The tuberculosis vaccine thus obtained showed negative side effects in human clinical trials (see Example 4).

[0125] The PIKA composition of the invention may be provided in any physiologically acceptable buffer, but phosphate buffers are preferred. Other acceptable buffers such as acetate, TRIS, bicarbonate, carbonate or the like can be used as substitutes for phosphate buffers.

[0126] The pH of the aqueous component will preferably be between 4.0 and 10.0 although it is preferred to adjust the pH of the system to be in the range of 6 to 8.5, where the pH does not significantly reduce the stability of the other components of the composition and is in no other physiologically inappropriate way. In some embodiments, the aqueous portion of the immunogenic composition is buffered with saline. When these compositions are dedicated to parenteral administration, it is preferred that these solutions be prepared in such a way that their tonicity, i.e. osmolality, is the same as that of normal physiological fluids, to prevent swelling after administration or rapid absorption of the composition due to varying concentrations ions between the composition and physiological fluids.

[0127] The quality of the buffered saline used in these compositions will be that amount needed to bring the value of the composition into one. That is, the amount of buffered saline needed to create 100% will be mixed with the other ingredients to make up the volume to the specified volume.

[0128] In some specific embodiments, the antigens may be purified from a natural source, synthesized by solid phase synthesis, or may be obtained by recombinant genetics. The antigen may contain a protein fragment containing one or two regions of immunogenicity of the molecule. Antigens can also be delivered in whole cells or through microorganisms (e.g. whole virus particles) that can be alive, attenuated, truncated or killed.

[0129] In other embodiments, the antigens include one or more agents derived from infectious agents, plant antigen, cancer, allergenic agents and other human antigens, such as those for causing autoimmune diseases. In other embodiments, the antigens contain one or more infectious agents derived from any virus, bacteria, mycobacteria, fungus, and parasite.

[0130] The polynucleotide adjuvant composition of the invention can also be used to enhance the immune response against antigens produced by the use of DNA vaccines. The DNA sequences in these vaccines encoding the antigens may be either "naked" or may be included in a delivery system, such as liposomes.

[0131] In certain embodiments, the polynucleotide adjuvant composition can be used in combination with vaccines. It doesn't matter if the vaccine contains adjuvants or not. Vaccine classes are included in the group: against infectious, anti-cancer, anti-allergic, anti-autoimmune diseases and immuno-contraception.

[0132] The invention also describes the use of the polynucleotide adjuvant of the invention in combination with any suitable rabies antigen.

[0133] In certain embodiments, the rabies antigens may be inactivated raw rabies antigens, such as inactivated crude rabies vaccines propagated in the culture of hamster kidney cells (HKC-ICRA) or inactivated purified rabies kidney cells HIPC (HKC) ).

[0134] In certain embodiments, the adjuvant polynucleotide composition can be used in a rabies vaccine. Suitable rabies vaccines are commercially available or under development including inactivated, subunits, recombinant and peptide vaccines such as rabies virus vaccines grown in culture of human diploid cells (HDCV), or inactivated, purified rabies vaccines grown in culture of hamster kidney cells ( HKC-IPRV) or inactivated raw rabies vaccines propagated in the culture of hamster kidney cells (HKC-ICRV), or purified vaccines with rabies virus grown in the Vero cell line (PVRV), or purified vaccines produced in the culture of chick chick embryo cells (PCEC), or purified vaccines produced on duck embryos (PDEV). However, not all rabies vaccines elicit a cellular immune response that is important before and after immunization. When an adjuvant polynucleotide composition (e.g., PIKA) is administered with a rabies vaccine, the induced immune response includes; non-specific response (e.g. enhanced macrophage function), humoral response (e.g. increased production of specific antibodies), and cellular response (e.g. cytokine production including interferon and interleukin-2).

[0135] In specific embodiments, the invention relates to a kit comprising a polynucleotide adjuvant and an antigenic component.

[0136] An immunogenic composition containing PIKA is capable of eliciting an immune response in two ways: i) a humoral response that involves stimulation of B lymphocytes and the production of antibodies or immunoglobulins (other cells are also involved in generating an antibody response, e.g. antigen presenting cells (APCs, including macrophages), helper T cells (Th1 and Th2) and ii) cellular immunity, which generally includes T lymphocytes containing cytotoxic T cells (CTL), although other cells are also involved in the CTL response process (e.g. Th1 and / or Th2 and APC). Ways to achieve an immune response

Humoral and / or cellular individuals are known in the art (see Examples 10, 11, 12 and 13).

[0137] Further, the polynucleotide adjuvant composition can affect the quality of the immune response by influencing the subclasses (isotypes) of produced immunoglobulins (IgG1, IgG2, IgG3 and IgG4 for human IgG molecules; IgG1, IgG2a, IgG2b and IgG3 for human IgG molecules) as also on their affinity.

[0138] The response regulated by Th1 cells in mice will induce IgG1, IgG2a, IgG2b and, to a lesser extent, IgG3 and will also favor the cellular immune response to the antigen. If the IgG response to an antigen is regulated by Th2-type cells, it will mainly enhance IgGI and IgA production.

[0139] NIH potency tests using the PIKA adjuvant composition and inactivated, purified rabies antigens grown in hamster kidney cell culture have surprisingly demonstrated that the potency of the immunogenicity of the composition requires minimal presence of rabies antigens (see Example 14). The potency of the composition increases significantly with respect to the presence of additional rabies antigens in an excess of 1 IU antigen. Thus, it was observed that the rate of increase in potency of the composition is highest at values ​​of about 1.5IU to 2.5 IU of foreign rabies antigen in the composition. The NIH test is described in the book: Laboratory Techniques in Rabies, Edited by FX Meslin, MM Kaplan H Koprowski, 4th Edition, ISBN 92 4 1544 1.

[0140] Tests using the PIKA adjuvant composition and inactivated, purified rabies antigens grown in hamster kidney cell culture showed that the potency of the immunogenicity of the composition increased with the amount of adjuvant present when it exceeded the amount of antigen present. The potency increased when the ratio of PIKA to rabies antigens grown in hamster kidney cell culture was increased with a favorable ratio greater than 3: 1 (see Example 15).

[0141] The invention describes methods of using the polynucleotide adjuvant of the invention with an antigen to, for example, elicit a specific humoral antigenic response and / or a specific cellular (e.g. T lymphocytes) response to a subject. An induced immune response may be an antigen response in a subject unaware of the experiment being carried out, or may be used to enhance an existing immune response (e.g., as a boost effect).

[0142] In certain embodiments, the PIKA adjuvant composition and immunogenic composition comprising the PIKA adjuvant and the antigenic component can be lyophilized to obtain long-term stability and storage in solid form. Freeze drying is known to those skilled in the art. Reconstitution of the immunogenic composition containing PIKA and the antigen component showed increased levels of efficacy (see Example 16).

[0143] The immunogenic composition may be prepared as an injectable, liquid solution, suspension or emulsion. Preparation of preparations with the desired immunogenic composition is generally described in the work New Trends and Developments in Vaccines,

27 edited by Voller et al., University of Park Press, Baltimore, Md., USA, 1978. The immunogenic composition of the invention can be used in such forms as capsules, liquid solutions, emulsions, suspensions or elixirs for oral administration or sterile liquid forms such as solutions, emulsions or suspensions. Any inert carrier such as saline or phosphate buffered saline or any type of carrier in which the ingredients used in the method of the invention have suitable solubility properties for use in the methods of the invention is preferred.

[0144] The immunogenic composition of the invention may be administered to a subject using a variety of methods known in the art. In specific embodiments, the immunogenic composition may be delivered parenterally, by injection, such as intramuscular, intraperitoneal, intravenous or subcutaneous, or by inhalation. In other embodiments, the immunogenic composition may be delivered rectally, vaginally, intranasally, orally, ophthalmically, topically, transdermally, intradermally. When the administration method is injection, the capsule-surrounded antigenic components can remain at the injection site for up to two weeks, providing an antigen storage that will allow sustained release or release in vivo doses. Such a delivery system may allow the use of single-injection preparations to be produced for antigenic components that would otherwise require multiple repetitive injections to elicit an immune response.

[0145] For parenteral administration in the form of an aqueous solution, for example, the solution should be appropriately buffered if required and the liquid solvent should first be brought to an isotonic state with a suitable saline or glucose. These particular aqueous solutions are particularly suitable for intravenous and intraperitoneal administration. In this regard, sterile aqueous media that can be used will be known to those skilled in the art in light of the disclosure. Exemplary injection media that can be used in the present invention include a buffer with or without dispersing and / or preserving agents, edible oil, mineral oil, cod liver oil, squalene, mono-, di- or triglycerides and their additives.

[0146] The exact amount of such required compositions will vary depending on the subject, depending on the species, age, weight and general condition of the subject of the study, the condition of the diseases, infections or diseases that are being treated or prevented, the particular compound used, the method of its administration and the like. The appropriate dose can be determined by those skilled in the art only from the routine testing of the techniques described herein. After initial administration, the test subjects may receive one or more immunization enhancements appropriately spaced.

[0147] The above disclosure generally describes the present invention. The following examples will assist in understanding the invention. These examples are described by way of illustration only and are not intended to limit the scope of the invention. Changes in the form of substance and

- 28 substitution changes to equivalent are considered as possibilities and may indicate their usefulness. The terms used are descriptive and not intended to limit the invention.

EXAMPLES

EXAMPLE 1. DETERMINATION OF THE PIKA MOLECULAR WEIGHT AND Av-PICKCA [0148] This example shows how the molecular weight was determined for PIKA adjuvants compared to Av-PICKCa.

[0149] Agarose gel electrophoresis is a technique known to those skilled in the art and therefore only some of the elements used in this invention are described herein. The agarose gel used in the invention had an agarose concentration of 1.5%. The molecular markers were DNA ladders with a length of 100 bp from 100 bp to 1000 bp corresponding to the molecular weight range from 6.6 out of 10⁴ up to 6.6 x 10⁵ Daltons. 4μ1 samples with a concentration of 1 mg / ml were applied. Figure 1 shows a representative image of the result of agarose gel sample analysis after applying the knowledge of this paragraph. Five (5) different tested batches showed a broad distribution of their molecular weights. The upper limits of their molecular weights ranged from 2.3 x 10⁵ Dalton for Av-PICKC up to 5.28 x 10⁵Dalton for PIKA.

EXAMPLE 2. IMMUNOLOGICAL PERFORMANCE OF PIK IN COMPARISON WITH

AV-PICKCA [0150] This example shows the difference between the Av-PICKC power with a maximum molecular weight of 230,000 Dalton and between PIKA with a maximum molecular weight of up to 528,000 Dalton.

[0151] Three series of PIKA adjuvant with different molecular weights and one series with molecules with a molecular weight corresponding to the Av-PIKA molecule were mixed with inactivated purified rabies antigens produced in hamster kidney cells (HKC-IPRA). The resulting compositions were subjected to the NIH efficiency test.

[0152] The NIH test is a rigorous and extensive comparative study between rabies vaccines tested , and standard rabies vaccines. Vaccinated mice are infected with a strain of live rabies viruses, then their survival is measured. Different dilutions of rabies vaccine are given to different groups of mice. Comparison of survival between groups of mice exposed to experimental and standard vaccines determines the potency of the experimental vaccine (Laboratory Techniques in Rabies, Edited by FX Meslin, MM Kaplan H Koprowski, 4th Edition, ISBN 92 4 1544 1).

[0153] The effectiveness of each combined vaccine has been normalized with respect to the standard, non-combination vaccines, where the yield of the non-combination vaccine has been described as 1, and the relative performance has been described as the multiple with which the compound yield increased over the yield of the non-combined vaccine. Table 1

- 29 summarizes the results. As can be seen from Table 1, the higher the molecular weight of the adjuvant

PICKCa is the higher the performance of a growing dose of rabies vaccine.

Table 1. Effect of molecular weight on the potency of rabies vaccine.

<td>^Typ adjuvant</td><td>Antigen</td><td>Sample number</td><td>Upper limit of the molecular weight of an adjuvant</td><td>ED50</td><td>Power (IU / ml)</td>

<td>PIQUE</td><td>HKC-IPRA</td><td> 20000304</td><td>5,28x10⁵</td><td> 2,10</td><td> 5,00</td>

<td>PIQUE</td><td>HKC-IPRA</td><td> 20000907</td><td>4,62x10⁵</td><td> 2,00</td><td> 3,98</td>

<td>PIQUE</td><td>HKC-IPRA</td><td> 990202</td><td>3,96x10⁵</td><td> 1,98</td><td> 3,80</td>

<td>Av-PICKCa</td><td>HKC-IPRA</td><td> 000703</td><td>2,30x10⁵</td><td> 1,88</td><td> 3,00</td>

<td></td><td>HKC-IPRA</td><td>Vaccine control</td><td></td><td> 1,40</td><td> 1,00</td>

EXAMPLE 3. COMPARISON OF INTERFERON PRODUCTION BETWEEN PIK AND

Ay-PICKCA [0154] This example demonstrates the difference in ability to induce interferon production between Av-PICKC samples with a maximum molecular weight of 230,000 Dalton and PIKA samples with a maximum molecular weight of up to 1200,000 Dalton.

[0155] Two PIKA series with upper molecular weight limits of 1.2 x 10⁶ Dalton and 4.6x10⁵ Dalton have been compared with a series of Av-PICKC molecules with an upper molecular weight limit of 2.3x10⁵ Daltons.

[0156] The PIKA and Av-PICKCa compositions were mixed with inactivated purified rabies antigens produced in hamster kidney cells (HKC-IPRA). The compositions were injected subcutaneously in mice. After two hours, the presence of interferon in each mouse was assessed. The general procedure for measuring the amount of interferon is known to those skilled in the art. Briefly, in a 96-well plate, each well was inoculated with L929 cells at 0.15 ml / well or approximately 30,000 cells. After three (3) days, when the cells reached confluence, wells were added to serum samples (0.1ml / well), where the sera were diluted 1:20 to 1: 640. Three wells were for each diluted sample. The wells were incubated overnight at 37 ° C. Serum samples were washed away. Vesicular stomatitis VSV virus particles were used to detect interferon production. Table 2 shows the production of interferon induced by the mixtures. As can be seen from Table 2, the higher the molecular weight of PIKA samples, the better the excited production of interferon.

- Table 2. Relationship between molecular weight and interferon production.

<td>^Typ adjuvant</td><td>Serial number</td><td>Upper mass limit in Dalton</td><td>Serial number HKC-IPRA</td><td>Ratio PIKA: HKCIPRA</td><td>Dose produced interferon</td>

<td>PIQUE</td><td> 20010601</td><td>1.20x10⁶</td><td> 20001205</td><td> 4:1</td><td> 868,6</td>

<td>PIQUE</td><td> 200009-7</td><td>4.62x10⁵</td><td> 20001205</td><td> 4:1</td><td> 530,6</td>

<td>Av-PICKCa</td><td> 200009-7</td><td>2.30x10⁵</td><td> 20001205</td><td> 4:1</td><td> 46,4</td>

EXAMPLE 4. CLINICAL STUDY ON A VACCINE FOR PEOPLE 1996 (Z

TOXICITY AS A SIDE EFFECT) [0157] This example demonstrates that a PICKC adjuvant combined with a vaccine generates an unacceptable level of side effects when administered to humans.

[0158] The aim of the study was to assess the safety and immune response of a rabies vaccine containing 11.95 mg / ml PICKC adjuvant and with a molecular weight of 69 700 (note: molecular weight is not equivalent to Dalton in this case, see example 5) and inactivated raw rabies antigens produced in hamster kidney cells (HKC-ICRA). The results and conclusions of the above clinical trial have not been published before.

[0159] 40 patients participating in the study were divided into two groups of 20 people. Each group received five (5) doses of 2ml administered intramuscularly on Day 1, Day 3, Day 7 and Day 30. One group received rabies antigens with PICKCa adjuvant and the other group received rabies antigens with alum adjuvant.

[0160] From a safety perspective, body temperature, local and systemic symptoms were observed after 24 hours, 28 hours and 72 hours after each injection. The following observations were made:

Table 3. Adverse side effects after HKC-ICRA injection with Alum or

PICKCa

<td>Effect side</td><td>Group</td><td>number volunteers</td><td>Number of people, u that have negative side effects</td>

<td rowspan="2">Local</td><td>PICKCa plus HKC-ICRA</td><td> 20</td><td> 6</td>

<td>Alum plus HKC-ICRAx5</td><td> 20</td><td> 2</td>

<td rowspan="2">system</td><td>PICKCa plus HKC-ICRA</td><td> 20</td><td> 4</td>

<td>Alum plus HKC-ICRAx5</td><td> 20</td><td> 0</td>

[0161] Systemic negative side effects have included: fever (1), rash (2), joint pain (2), lymph nodes (1), throat edema (1). Local side effects included:

skin redness at the injection site (6) [0162] The following research by the inventor attributed the observed side effects to the particle size of the molecules in the adjuvant (see Examples 5 and 6).

EXAMPLE 5. RELATIONSHIP BETWEEN PICKC CONCENTRATION AND WEIGHT

MOLECULAR PICKCa [0163] This example demonstrates that the increasing concentration of PICKCa adjuvant results in increased molecular weight compositions.

[0164] PICKCa can be prepared in various concentrations. It has been hypothesized that PICKCa as a polymer complex would exist in various forms when prepared in different concentrations. For this purpose, the laser light scattering method was used. The scattering of laser light has been widely used to determine the average molecular weight (Mw) and radius of inertia (Rg). The devices are commercially available and the process is known to those skilled in the art. Table 4 shows that the observed molecular weight of PICKCa determined by laser light scattering corresponded to its intensity.

Table 4. Observed molecular weight by laser light scattering.

<td>PICKCa concentration (mg / ml)</td><td>Average molar mass</td>

<td> 11,95</td><td>6.97 x 10⁴</td>

<td> 2,00</td><td>7.30 x 10³</td>

<td> 1,00</td><td>2.00 x 10³</td>

EXAMPLE 6. RELATIONSHIP BETWEEN PICKC CONCENTRATION BEFORE

MIXING AND MOLECULAR MASS OF THE VACCINE [0165] This example illustrates the relationship between the increased molecular weight of PICKC's adjuvant and the resulting molecular weight of a composition that contains PICKC's adjuvant and inactivated raw rabies antigens produced in hamster kidney cells.

[0166] It was also suspected that pre-combination PICKCa concentration could affect antigens in vaccines. PICKCa samples were combined with inactivated raw rabies antigens produced in hamster kidney cells. The method of laser light scattering was used for the analysis. The scattering of laser light has been widely used to determine the average molecular weight (Mw) and radius of inertia (Rg). The equipment used is commercially available and all processes are known to those skilled in the art. Table 5 shows that the increase in PICKCa concentrations before mixing caused an increase in Mw of rabies vaccines.

- 32 Table 5. Relationship between concentration

PICKCa before mixing and Mw rabies vaccines

<td>PICKCa concentration (mg / ml)</td><td>Average molar mass</td><td>Radius of inertia</td>

<td> 11,95</td><td>29,6x10⁴</td><td>17,2x10²</td>

<td> 4,00</td><td>22,2x10⁴</td><td>15,0x10²</td>

<td> 2,00</td><td>13,8x10⁴</td><td>11,8x10²</td>

<td> 1,00</td><td>5,60x10⁴</td><td>7,55x10²</td>

<td> 1,00</td><td>5,29x10⁴</td><td>6,50x10²</td>

EXAMPLE 7. PIKA TOXICITY TEST [0167] This example demonstrates the safety characteristics of PIKA adjuvant, including molecular weight reduction.

[0168] The toxicity test was carried out in accordance with the regulations of the Chinese National Drug Standards (WS1-XG-050-2000). Briefly, five (5) mice with body weights in the range of 18-22 grams were injected intravenously with 0.5nm / mouse sodium chloride solution containing 0.3ml PIKA adjuvant with an upper molecular weight limit from about 525,000 to about 1,000,000 Dalton. Injected mice were observed for 7 days and weighed at the end of the observation. Table 6 summarizes the results that showed that the molecular weight of PIKA adjuvant can be as much as 1.0x10⁶ Dalton without causing visible toxicity.

Table 6. PIKA adjuvant toxicity test

<td>Serial number</td><td>upper border weight cząsteczko input (Daltons)</td><td>Pre-test mouse body weight (g)</td><td>The dose of injection into a vein caudal</td><td>Mouse status at the end of the study</td><td>Potestowe research weight the body mice (G)</td><td>Comments</td>

<td> 20000304</td><td>5,25x10⁵</td><td> 18-19</td><td>0.5ml / mouse</td><td>Healthy</td><td> 23-26</td><td>Satisfying</td>

<td> 20010103</td><td>5,20x10⁵</td><td> 18-19</td><td>0.5ml / mouse</td><td>Healthy</td><td> 22-25</td><td>Satisfying</td>

<td> 20010816</td><td>5,20x10⁵</td><td> 18-19</td><td>0.5ml / mouse</td><td>Healthy</td><td> 23-25</td><td>Satisfying</td>

<td> 20010511</td><td>1,00x10⁶</td><td> 18-20</td><td>0.5ml / mouse</td><td>Healthy</td><td> 24-26</td><td>Satisfying</td>

- 33 EXAMPLE 8: PIKA IN COMPOSITION TOXICITY STUDIES

VACCINES [0169] The purpose of this experiment is to validate the safety of PIKA adjuvant.

[0170] PIKA adjuvant (molecular weight 66000 Dalton to 660000 Dalton) was mixed with inactivated purified rabies antigens produced in hamster kidney cells (HKC-IPRA) in a PIKA: HKC-IPRA ratio of

4:1.

[0171] The PIKA and HKC-IPRA vaccine composition was compared with commercially available inactivated, purified rabies vaccines (IPRV) that contained alum adjuvant.

[0172] Mice were given five (5) doses of the vaccine composition on day 0, day 3, day 7, day 14 and day 28. The dose administered was equivalent to 300 times the dose administered to an adult human at the normal dosage for immunizing humans against rabies.

[0173] The results of toxicity observations are presented in Table 7 below:

Table 7: Safety observation after administration of the Tuberculosis Vaccine

<td>preparations</td><td>Effect</td><td>Day 0</td><td>Day 3</td><td>Day 7</td><td>Day 14</td><td>Day 28</td>

<td>HKC-IPRA plus PIKA</td><td>allergy</td><td> 0/20</td><td> 0/20</td><td> 0/20</td><td> 0/20</td><td> 2/20</td>

<td>HKC-IPRA plus PIKA</td><td>death</td><td> 0/20</td><td> 0/20</td><td> 0/20</td><td> 0/20</td><td> 0/20</td>

<td>IPRV (including Alum)</td><td>allergy</td><td> 0/20</td><td> 0/20</td><td> 0/20</td><td> 5/20</td><td> ?</td>

<td>IPRV (including Alum)</td><td>death</td><td> 0/20</td><td> 0/20</td><td> 0/20</td><td> 2/20</td><td> 7/20</td>

Key: (observed occurrence) / (integer) [0174] Conclusion: the PIKA / HKC-IPRA combination is more secure than commercially available IPRV.

EXAMPLE 9. SAFE USE OF PIKA ADIUVAN IN PEOPLE [0175] In 2002, five volunteers (5) were immunized with the PIKA composition (molecular weight 66000 to 660000 Dalton) and inactivated purified rabies antigens produced in hamster kidney cells (HKC-IPRA). Volunteers were given the vaccine composition on day 0, day 3, day 7, day 14 and day 30.

[0176] No local or systemic side effects were observed after any vaccination in any patient.

[0177] Vaccine potency was measured using a standard NIH test and the results are shown in Table 8, below:

- Table 8: Observed Potency of the Rabies Vaccine

<td>Day</td><td>ED50</td><td>IU / ml neutralizing antibody</td>

<td> 0</td><td> 0</td><td> 0</td>

<td> 14</td><td> &gt; 1,9</td><td> &gt; 1,84</td>

<td> 45</td><td> 2,35</td><td> 5,17</td>

[0178] The results indicate that the PIKA and HKC-IPRA vaccine composition induces a specific immune response and induces the production of protective neutralizing antibodies.

EXAMPLE 10. TEST AFTER EXPOSURE TO VIRUS (CELLULAR RESISTANCE) [0179] The test after exposure to the virus is the definitive proof that the vaccine has the ability to eliminate pathogens from the injection host. This is evidence of a vaccine-induced cellular immune response.

[0180] In the test after exposure to the virus, the mice were infected with a wild strain of rabies virus and then inoculated with: inactivated purified rabies antigens from hamster kidney cells (HKC-IPRA) in combination with PIKA adjuvant (molecular weight ranges from 1.65 x 10⁵ up to 1.2 x 10⁶ Dalton), or HKC-IPRA in combination with aluminum hydroxide adjuvant (alum), commercially available purified rabies vaccines grown in the Vero cell line (PVRV) or phosphate buffered saline (PBS). The results ultimately show that PIKA adjuvant led to increased survival, see Table 9.

Table 9. Test after exposure to the virus

<td colspan="3">9.1 Mortality after treatment</td>

<td>group</td><td>Predefined 80% lethal dose</td><td>Pre-determined 50% lethal dose</td>

<td>PIKA plus HKC-IPRA</td><td> 2/20</td><td> 0/20</td>

<td>Alum plus HKC-IPRA</td><td> 10/20</td><td> 9/20</td>

<td>PVRV</td><td> 16/20</td><td> 3/20</td>

<td>Control (PBS)</td><td> 15/20</td><td> 14/20</td>

<td colspan="3">9.2 Survival after treatment</td>

<td>group</td><td>Predefined 80% lethal dose</td><td>Pre-determined 50% lethal dose</td>

<td>PIKA plus HKC-IPRA</td><td> 90.00%</td><td> 100.00%</td>

<td>Alum plus HKC-IPRA</td><td> 50.00%</td><td> 55.00%</td>

<td>PVRV</td><td> 20.00%</td><td> 85.00%</td>

<td>Control (PBS)</td><td> 25.00%</td><td> 30.00%</td>

[0181] Infected rabies virus, administered by subcutaneous injection, mice were given the vaccine after 6 hours, 1 day, 2 days and 3 days after injection.

EXAMPLE 11. PRODUCTION OF GAMMA INTERFERON IN SWÓJA, TO

ANTIGEN, CELLULAR IMMUNOLIOGICAL RESPONSE [0182] Production of interferon-gamma is an indicator of cellular immunity activity.

[0183] In this experiment, blood samples were taken from two test patients and two people as controls. Volunteers were vaccinated with PIKA rabies vaccines that contained PIKA (molecular weight ranges from

66000 up to 660000 Daltons), and vaccines containing inactivated purified rabies antigen from hamster kidney cells (HKC-IPRA) 2.5 years before blood sampling.

[0184] The results in Figure 2 show a significant difference in the level of interferon-gamma produced by two patients compared to the subjects that were treated as controls.

[0185] Isolated monocytes from blood samples were incubated with the same HKC-IPRA that was used in the original test. After three days of incubation, the non-cell supernatants were harvested and their level of interferon-gamma was measured using an ELISPOT specific cytokine assay. A dose-dependent effect was observed.

[0186] The conclusions of the above observation are as follows:

• intravenous vaccines that contain the PIKA adjuvant of the invention have the ability to induce interferon-gamma production and to induce a cellular immune response through implication • the interferon-gamma response is specific (i.e., the response is directed to rabies antigens as opposed to non-specific reaction). If the interferon-gamma response were nonspecific, there would be no differentiation in the level of interferonugamma production in the blood of vaccinated patients, as the concentration of antigen stimulant would increase.

EXAMPLE 12: PIKA PERFORMANCE TEST [0187] The purpose of this experiment is to demonstrate that PIKA has the ability to induce the production of interferon-gamma and interleukin 12 (IL-12).

[0188] Splenocyte samples from normal healthy mice were incubated for three days in the presence of PIKA (molecular weight in the range 66000 to

660000) in a clean environment. At the end of the period, the level of cytokines in the supernatant was tested by IL-12 (p40) specific ELISA and interferon-gamma molecules.

The results of the experiment are shown in Table 10 below:

Table 36: In Vitro Test for Cytokine Production

<td>PIKA μg / ml</td><td>IFN-gamma pg / ml</td><td>IL-12P40 pg / ml</td>

<td> 0</td><td> 3</td><td> 0</td>

<td> 0,4</td><td> 23</td><td> 91</td>

<td> 2</td><td> 22</td><td> 98</td>

<td> 10</td><td> 30</td><td> 134</td>

<td> 50</td><td> 179</td><td> 186</td>

<td> 100</td><td> 559</td><td>N / D</td>

<td> 250</td><td> 1340</td><td>N / D</td>

[0189] The conclusion of the experiment is as follows: PIKa induces a dose-dependent production of interferon-gamma and IL-12 cytokines and as such induces a cellular immune response.

[0190] In a further experiment, four mice (4) were administered 500 Pg / ml PIKA (molecular weight in the range from 66000 to 660000 Dalton) by intraperitoneal injection. A phosphate buffer solution was used as a negative control. Five hours after injection, a blood sample was taken and serum prepared. Serum cytokine levels were measured by IL-12 (p40) and interferonugamma specific ELISAs. The results of the experiment are presented in Table 11 below:

Table 11: In vivo cytokine production assay.

<td>Group</td><td>IFN-gamma pg / ml</td><td>IL-12P40 pg / ml</td>

<td>PBS</td><td> 4</td><td> 2</td>

<td>PIQUE</td><td> 410</td><td> 40</td>

[0191] The conclusion of the above experiment is as follows: PIKA is effective in stimulating a cellular immune response.

EXAMPLE 13: USE OF INACTIVE PIK

PURIFIED RABIES ANTIGENE PLACED IN THE LINE

VERO CELL [0192] The purpose of this experiment is to evaluate the effectiveness of PIKA in combination with a purified vaccine with Vero rabies virus (PVRV).

[0193] PIKA with a molecular weight in the range 66000 to 660,000 Dalton was mixed with PVRV to form a rabies vaccine. The NIH test was used to assess the potency of the resulting vaccine composition. The results are shown in Table 12 below:

- Table 12: NIH test results for PIKA and purified vaccines with rabies virus Vero.

<td>Composition vaccine</td><td>Antigen</td><td>adjuvant</td><td>The power of composition (IU / ml)</td>

<td>PVRV</td><td>0,02IU / ml</td><td>n / a</td><td> 0,46</td>

<td>PVRV plus PIKA</td><td>0,02IU / ml</td><td>PIQUE</td><td> 3,68</td>

[0194] The conclusion of this experiment is: PIKA enhances the potency of the purified vaccine with rabies virus Vero.

EXAMPLE 14. DOSE OF ANTIGEN [0195] This experiment sets out the requirements for having a minimum dose of inactivated purified rabies hamster kidney antigens (HKC-IPRA) present in the composition together with PIKA adjuvant (molecular weight in the range of 66000 to 660,000 Dalton) needed for a significant increase in the level of the specific immune response. Elevated levels of rabies antigens have been added to PIKA 0.1mg solid adjuvant. Power was measured using a standard NIH test for Measuring Rabies Vaccine Power. After a predictable initial increase in power, a clear, dramatic increase in power was observed before the power reached low levels, as expected after the addition of the antigen (see Table 13).

Table 13: Vaccine potency with increased doses of inactivated purified rabies antigen from hamster kidney cells.

<td>HKC-IPRA (IU)</td><td>HKC-IPRA plus 0.1mg PIKA (IU)</td><td>Marginal increase in power</td>

<td> 0,25</td><td> 0,42</td><td> -</td>

<td> 0,51</td><td> 1,43</td><td> 3,88</td>

<td> 1,01</td><td> 2,73</td><td> 2,60</td>

<td> 1,40</td><td> 6,07</td><td> 8,56</td>

<td> 2,07</td><td> 19,78</td><td> 20,46</td>

<td> 2,91</td><td> 21,38</td><td> 1,90</td>

[0196] The marginal potency increase is the potency increase of the HKC-IPRA / PIKA vaccine observed after the addition of one IU of the current HKC-IPRA.

[0197] The conclusion drawn from this experiment is that minimal antigen presence is necessary before a significant immune response is elicited. Moreover,

- 38 the occurrence of antigen exceeding the initiation point causes only a marginal increase in power recovery.

EXAMPLE 15. RELATIONS OF ANTIGEN TO ADIUVANT [0198] This experiment demonstrates the optimal mixture of inactivated purified rabies antigens from hamster kidney cells (HKC-IPRA) and PIKA adjuvant (molecular weight in the range of 66000 to 660000).

[0199] Different amounts of antigen were mixed with different amounts of adjuvant in PBS to provide a constant volume. The potency of the resulting vaccines was determined using the NIH potency test. The results are presented in Table 14.

[0200] Consideration of the set of test results indicates the optimal combination of PIKA vaccine: antigen in a ratio of at least 3 to 1.

Table 14: Potency of rabies vaccines at different antigen to adjuvant ratios

<td>samples</td><td>HKC-IPRA (Ml)</td><td>PIKA (ml)</td><td>PBS (ml)</td><td>Ratio (PIKA HK C-IPRA)</td><td>ED50</td><td>Power (IU / ml)</td>

<td>AND</td><td> 0,20</td><td> 0,80</td><td> -</td><td>4.0 to 1</td><td> 2,65</td><td> 7,84</td>

<td>B</td><td> 0,20</td><td> 0,70</td><td> 0,10</td><td>3.5 to 1</td><td> 2,46</td><td> 5,20</td>

<td>C</td><td> 0,20</td><td> 0,60</td><td> 0,20</td><td>3.0 to 1</td><td> 2,40</td><td> 4,22</td>

<td>D</td><td> 0,20</td><td></td><td> 0,80</td><td>0 to 1</td><td> 1,85</td><td> 1,43</td>

<td>standard</td><td></td><td></td><td>on</td><td>on</td><td> 2,52</td><td> 6,70</td>

EXAMPLE 16. STORAGE OF PIK AND PIK IN CONNECTION WITH

VACCINE AGAINST RABIES IN THE FORM OF A LYOPHILISATE [0201] This example demonstrates that PIKA is stable in the form of a lyophilisate.

[0202] Freeze-drying technology has been used to store rabies vaccines for up to three years. The inventor sought the answer to the question whether storage in the form of PIKA lyophilisate (molecular weight in the range 66000 to 660000) and rabies vaccines containing PIKA would be beneficial. The following compositions were used to test the storage methods in the form of a lyophilisate: i) non-frozen PIKA added to dissolved lyophilisates of inactivated purified rabies antigens produced in hamster kidney cells (HKC-IPRA), ii) dissolved lyophilisates of the PIKA plus HKC-IPRA composition, iii) dissolved lyophilisates of commercially available rabies vaccines (without the addition of PIKA) and iv ) non-frozen commercially available rabies vaccines. Table 15 shows that the rabies vaccine lyophilisates containing PIKA and PIKA were ideal for long-term storage of rabies vaccines.

- Table 15: effect of storage in the form of lyophilisates on the potency of rabies vaccines

<td>A sample</td><td>ED50</td><td>relative power</td><td>IU / ml</td>

<td>i) rabies vaccine lyophilisate dissolved in PIKA</td><td> 2,89</td><td> 2,34</td><td> 15,71</td>

<td>ii) rabies vaccine containing PIKA, dissolved in PBS</td><td> 3,00</td><td> 3,00</td><td> 20,23</td>

<td>III) lyophilisate of a commercial rabies vaccine containing PIKA, dissolved in PBS</td><td> 1,85</td><td> 0,21</td><td> 1,43</td>

<td>iv) unfrozen standard rabies vaccine</td><td> 2,52</td><td> 1,00</td><td> 6,70</td>

Prepared and verified

Dorota Rzążewska Patent attorney