Polyinosinic acid-polycytidylic acid-based adjuvant

IS 2 380 481 T3

DESCRIPTION

Immunogenic substances comprising an adjuvant based on polyinosinic acid and polycytidylic acid.

FIELD OF THE INVENTION

[0001] The invention generally relates to adjuvant compositions and methods, of their use to enhance an immune response, more in particular, to compounds, vaccines and methods for enhancing the immunogenicity of an antigen and, more particularly, to compositions and vaccines with polynucleotide adjuvants comprising the polynucleotide adjuvant compositions. The invention also relates to such polynucleotide adjuvant compositions and vaccines for use in methods as claimed.

BACKGROUND OF THE INVENTION

1. Description of Related Art

[0002] The immune system can display both specific and non-specific immunity. In general, B and T lymphocytes, which display specific receptors on their cell surface for a given antigen, produce specific immunity. The immune system can respond to different antigens in two ways: 1) humoral immunity, which includes the stimulation of B cells and the production of antibodies or immunoglobulins, antigen presenting cells (APC) and helper T cells (Th1 and Th2) and 2) cell-mediated immunity (IMC) which generally involves T cells including cytotoxic T lymphocytes (CTL), although other cells are also involved in generating a CTL response (e.g. g., Th1 and / or Th2 cells and APC).

[0003] Nonspecific immunity encompasses various cells and mechanisms such as phagocytosis (the action of engulfing foreign particles or antigens) by macrophages or granulocytes and the activity of natural cytotoxic (NK) cells, among others. Nonspecific immunity depends on evolutionarily less advanced mechanisms and does not display the acquired nature of specificity and memory, which are examples of characteristic features of a specific immune response. The key differences between specific and non-specific immunity are based on the specificity of B and T cells. These cells predominantly acquire their sensitivity upon activation with a specific antigen and have mechanisms to show memory in the event of future exposures to this antigen. specific. As a result, vaccination (which assumes specificity and memory) is an effective protocol for protection against harmful pathogens.

[0004] Generally, adjuvants are compounds, which when administered with an antigen (mixed with the antigen or administered prior to its administration) enhance or modify the immune response against this particular antigen.

[0005] Examples of adjuvants that have been used to enhance an immune response include aluminum compounds (all generally referred to as alum), oil-in-water emulsions (Freund's complete adjuvant (ACF) is an oil-in-water emulsion). water containing lyophilized heat-killed Mycobacterium tuberculosis organisms), saponin (isolated from the bark of Quillaja saponaria, the active component of the adjuvant known as Quile A), CpG ODP (synthetic oligodeoxynucleotide containing unmethylated CpG dinucleotides), MPL (derived from Salmonella minnesota Re595 lipopolysaccharide), liposomes (usually made from biodegradable materials such as phospholipids), and biodegradable polymer microspheres (made from various polymers such as PLA and polyanifoshyphazeides, polyanifoshyphazides ). The adjuvant properties of these compounds have been evaluated with each adjuvant that has shown advantages and disadvantages.

[0006] The main problem with the use of adjuvants for human vaccines, especially routine vaccines for children, is the toxicity and adverse side effects of most adjuvant formulations. The application of new technologies to vaccine development is leading to purified, subunit and synthetic antigens that tend to be poorly immunogenic. The development of new adjuvants, to improve immunogenicity / efficacy and reduce side effects, represents one of the main challenges in vaccine research and development. Various applications have been studied for polynucleotide complexes including their performance as adjuvants. Double-stranded RNAs (dsRNAs) are very powerful biological modifiers that can exert a profound influence on cells at nanomolar concentrations. The modulatory effects of dsRNAs include a broad spectrum of actions at the molecular and cellular levels.

[0007] At the molecular level, dsRNAs can show biological effects, such as interferon synthesis, protein kinase induction, enhancement of histocompatibility antigen, and inhibition of metabolism. And at the cellular level, dsRNA can show biological effects such as pyrogenicity, mitogenicity, macrophage activation, activation

ES 2 380 481 T3 of cell-measured immunity and induction of antiviral status. A promising potential for dsRNAs is their immunomodulatory effect in antimicrobial treatments. In US Patent 4,124,702 it is disclosed that double stranded polynucleotides induce the induction of interferon in living animal cells. In US Pat. No. 3,906,092 describes that the anti-body response to an adjuvant-type vaccine was increased by incorporating a polynucleotide or a complex of polynucleotides into the vaccine. Houston et al., Established that PICLC (complex of polyinosinic acid, polycytidylic acid and poly-L-lysinecarboxymethylcellulose) as a potent adjuvant increasing the primary anticorpal response without the help of an additional adjuvant. Houston et al., In Infection and Immunity, 14: 318-9, 1976C. The mycoviral dsRNA was found to significantly potentiate the hemagglutinating anticorpal response on lamb red blood cells (GRc). Wright and Adler-Moore, Biochemical and Biophysical Research Communications, 131: 949-45, 1985.

[0008] However, PIC (polyinosinic acid polycytidylic acid) shows acute toxicity when used in animals. For example, Phillips et al. reported that severe toxic manifestations were observed in dogs after subchronic administration of PIC at doses of 2.0 mg / kg. Toxicity was characterized by a spontaneous decrease in activity, poor coordination, vomiting, anorexia, weight loss, hematological changes reflecting decreased hematopoiesis, increased alkaline phosphatase and transaminase activities, thymus degeneration, bone marrow destruction. , dilation of the hepatic sinusoidal capillaries in the centrilobular areas, necrosis of the hepatic cells, collapse of the hepatic structures and a generalized arthritis. See, Phillips et al., Toxicology and Applied Pharmacology, 18: 220-30, 1971.

[0009] PIC, one of the most studied polynucleotide complexes, was not effective when used in mice and humans due to its instability in the body after administration. Therefore, PIC has been modified in many ways to overcome one deficiency or another. For example, a complex of polyribocytidylic polyriboinosinic acid with poly-L-lysine hydrobromide is approximately 5 to 15 times more resistant to hydrolysis by pancreatic ribonuclease than the parent PIC. Another example is dsRNA polyICLC, or PICLC for short, which was found to be highly effective as an antiviral or antitumor agent. PICLC is a synthetic dsRNA composed of chains of polyriboinosinic acid and polyribocytidylic acid (PIC). Although PICLC is a promising immunomodulator that has great potential in antimicrobial and antineoplastic therapies, it has been found to produce serious side effects in humans, especially when the drug is administered in multiple high doses. Some of the reported side effects are fever, hypotension, leukopenia, myalgia, thrombocytopenia, and polyarthralgia. The inherent problem of toxicity must be overcome to make PICLC safe for use in humans. Furthermore, the therapeutic efficacy of poly ICLC is limited by its stability in vivo.

[0010] An antiviral drug composed of polyinosinic acid-polycytidylic acid (Poly I: C), kanamycin and calcium (Av-PICKCa) is used to treat viral infections. Av-PICKCa has been shown to be capable of inducing the production of interferon and interleukin-2. Av-PICKCa given only as an antiviral drug stimulates a non-specific immune response, that is, it stimulates a form of interferon that is not specific for any particular antigen. This antiviral response is profoundly different from the antigen-specific immune response generated when an adjuvant is administered together with an antigen.

More importantly, the inventor of the present invention discovered that Av-PICKCa had the properties of an adjuvant, that is, the ability to induce a specific immune response when administered with an antigen. Additionally, the inventor further discovered that Av-PICKCa was an effective adjuvant when used in conjunction with rabies and hemorrhagic fever antigens. Lin et al. described 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 provides the use of the general composition of Poly I: C, kanamycin and calcium (PICKCa) as an adjuvant in a vaccine for human and mammalian application.

Lin et al. (1993) describe that PICKCa activity was tested in experimental rabies prophylaxis. PICKCa protected mice against peripheral infection with both fixed and wild rabies virus strains. It also enhanced the protective activity of an experimental rabies vaccine injected before or after infection with the rabies virus. PICKCa enhanced both nonspecific immune responses and specific immunity, including antibody production and cell-mediated immunity as assessed by interleukin-2 production.

[0013] The Av-PICKCa samples were heterogeneous with respect to the size and weight of the molecules. AvPICKCa is described in the literature in terms of a mean value or range of the value of the sedimentation coefficient measured in Svedbergs, S. The antiviral drug Av-PICKCa exists in an embodiment from 5S to 8S (source A); see Zhung JC, Research collection of polyinosinic-polycytidyl (PIC). The role of the fifth

Chinese Conference on Clinical Application and Theory of Interferon, Siam 1985, pp. 23-28. In other embodiments,

IS 2 380 481 T3

Av-PICKCa exists with a sedimentation coefficient of 4S to 12S with a mean of 6S (source B), 5S to 12S with a mean 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 coefficient of this heterogeneous collection of molecules in Av-PICKCa can be converted into an equivalent molecular weight (Mw in Daltons) using the conversion formula pm = 1,100 x S^2.2(see Su BX et al; Introduction of Biochemical Technology, 1^to Edition, Zhongshan University, 1978, 356357). The results of the conversion in Daltons are presented in the following table:

Table A: Av-PICKCa characteristics

<td>Source of Av-PICKCa</td><td colspan="3">Sedimentation coefficient S</td><td colspan="3">Daltons molecular weight</td>

<td></td><td>Min.</td><td>Max.</td><td>Half</td><td>Min.</td><td>Max.</td><td>Half</td>

<td>TO</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> 38.000</td><td> 260.000</td><td> 79.000</td>

(continuation)

<td>Source of Av-PICKCa</td><td colspan="3">Sedimentation coefficient S</td><td colspan="3">Daltons molecular weight</td>

<td></td><td>Min.</td><td>Max.</td><td>Half</td><td>Min.</td><td>Max.</td><td>Half</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 reference</td>

The original investigation on Av-PICKCa as an adjuvant by Lin et al., Was with a sample that had molecules with similar characteristics to source A, that is, a sedimentation coefficient of 5S to 8S, which is a molecular weight equivalent of molecules ranging from 38,000 Daltons to 107,000 Daltons. (see Lin et al., supra).

[0016] All forms of PICKCa were considered to be equally safe and effective given that Av-PICKCa is essentially a form of PICKCa and further given the historical use of Av-PICKCa as an antiviral drug. However, this has proven not to be the case. Research by the inventor shows that the efficacy and toxicity of PICKCa when used as an adjuvant in combination with an antigen currently varies with different molecular weights. The invention finds that Av-PICKCa does not provide the optimal efficacy / safety profile for use as an adjuvant and that PICKCa does not induce unacceptable adverse side effects under certain conditions. Thus, there remains a need for an adjuvant that is more suitable for use in humans and that is safe and effective in providing the desired immunogenic effect. The current invention addresses this need and provides other advantages that will become apparent with reference to the detailed description.

BIBLIOGRAPHY

[0017] The following references may be of interest:

• Document JP 10935440A2;

• US Patent No. 4,124,702 • US Patent No. 3,692,899 • US Patent No. 3,906,092 • US Patent No. 4,389,395 • Patent US Patent No. 4,349,538 • US Patent No. 4,024,241 • US Patent No. No. 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 • Chinese patent 93105862.7 • Zhung JC , Research collection of polyinosinic-polycytidylic acid (PIC). The paper of fifth Chinese interferon conference in clinical application and theory, Siam 1985, pp. 23-28 • Hu QG, Tianjin Av-PICKCa's laboratory research and clinical application, Fujian Medical Journal, 1983.12; (6): 28-30

ES 2 380 481 T3 • Hu QG Chinese Medical and Pharmaceutical Industry Journal, 1983 (9) 3134.

• Su BX et al; Introduction of Biochemical Technology, 1^to 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 No. 4,094,971 • US Patent No. 4,101,536 • US Patent No. 4,153,684 • US Patent .UU. No. 4,235,771 • US Patent No. 4,323,559 • US Patent No. 4,327,085 • US Patent No. 4,185,089 • US Patent No. 4,082,736 • US Patent No. 4,369,178 • US Patent No. 4,314,998 • US Patent No. 4,082,735 • US Patent No. 4,186. 194 • US Patent No. 6,468,558 • New Trends and Developments in Vaccines, edited by Voller et al., University Park Press, Baltimore, Md., USA. 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 • Richard T Kenney et al. Meeting Retort - 2nd meeting on novel adjuvants currently in / close to human clinical testing, Vaccine 20 2155-2163, 2002 • Laboratory Techniques in Rabies Edited by FX Meslin, MM Kaplan, H Koprowski 4th Edition ISBN 92 4 1544

SUMMARY OF THE INVENTION

[0018] The present invention provides a polynucleotide adjuvant composition and a polynucleotide adjuvant composition and vaccines for use in methods as claimed. The present invention also provides an immunogenic composition comprising the polynucleotide adjuvant composition together with an antigen (eg, as in a vaccine). The adjuvant compositions of the invention have particular physical properties (e.g. g., molecular weight, size, concentration, and pH) that address the need for an effective and safe adjuvant to induce an enhanced immune response. The present invention further contemplates these adjuvant compositions for use in eliciting an immune response to an antigenic compound.

In one embodiment, the invention provides a polynucleotide adjuvant composition comprising polyriboinosinic-polyribocytidylic acid (PIC), an antibiotic and a positive ion, where the antibiotic is kanamycin and the positive ion is calcium. The present invention also provides an immunogenic composition comprising the polynucleotide adjuvant composition together with an antigen or a vaccine.

[0020] The present invention serves to advance the body of 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 an animal or human host. While previous descriptions showed the application of the antiviral drug Av-PICKCa for use as an adjuvant, it was observed that this form of PICKCa elicited only a limited specific immune response when administered with an antigen. Additionally, PICKCa was found to trigger unacceptable adverse side effects under certain conditions.

[0021] The present invention addresses these problems by providing an adjuvant composition, generally referred to herein as "PIKA" that can be more effectively and safely administered as an adjuvant in animals, including humans.

[0022] PIKA is a composition comprising a polynucleotide, kanamycin and a calcium ion that has been specifically developed as an adjuvant. Included in the invention are compositions that have unique product attributes that make them more suitable for use as an adjuvant in an immunogenic composition for administration to animals and / or humans.

IS 2 380 481 T3

More specifically, the present invention provides a polynucleotide adjuvant composition comprising a polynucleotide, an antibiotic and a positive ion, where the polynucleotide can be polyriboinosinic acid-polyribocytidylic acid (PIC), the antibiotic is kanamycin and the positive ion is calcium.

More specifically, the present invention provides the specification, including molecular weight, concentration, and pH of the composition comprising a polynucleotide, kanamycin, and a calcium ion that addresses the need for a safe adjuvant that triggers the immune response. desired maximum.

[0025] The present invention also provides an immunogenic composition comprising the polynucleotide adjuvant composition and an antigen or a vaccine.

In certain embodiments, the present invention is in the form of a kit comprising the polynucleotide adjuvant and an immunogenic compound.

[0027] The disclosure provides a method of enhancing immune responses to an antigenic compound by administering the immunogenic composition to a host. The host can be a human or an animal. Administration can be by injection, such as intramuscular, intraperitoneal, intravenous or subcutaneous injection, or by inhalation. In other embodiments, the immunogenic composition can be administered rectally, vaginally, nasal, orally, ophthalmic, topical, transdermal, or intradermal.

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

Consequently, in one aspect the invention presents a polynucleotide adjuvant composition comprising a polyriboinosinic-polyribocytidylic acid (PIC), kanamycin and a calcium ion, where the composition contains heterogeneous adjuvant molecules in terms of molecular weight, with a molecular weight of between about 66,000 to 1,200,000 Daltons.

[0030] In related embodiments, the polynucleotide adjuvant composition molecules in the composition are of heterogeneous molecular weight, where the molecular weight is from about 300,000 to 1,200,000 Daltons, or from about 66,000 to 660,000 Daltons, or from about 300,000 to 660,000 Daltons, or approximately 300,000 to 2,000,000 Daltons, or approximately 300,000 to 4,000,000 Daltons, or approximately 500,000 to 1,000,000 Daltons, or approximately 1,000,000 to 1,500,000 Daltons, or approximately 1,500,000 to 2,000,000 Daltons, or approximately 2,000,000 to 2,500,000 Daltons, or approximately 2,500,000 to 3,000,000 Daltons, or approximately 3,000,000 to 3,500,000 Daltons, or approximately 3,500,000 to 4,000,000 Daltons, or approximately 4,000,000 to 4,500,000 Daltons, or approximately 4,500,000 to 5,000,000 Daltons.

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

Consequently, in one aspect the invention presents a polynucleotide adjuvant composition comprising a polyriboinosinic-polyribocytidylic acid (PIC), kanamycin and a calcium ion, where the composition contains adjuvant molecules of heterogeneous molecular size that have a coefficient of Svedbergs (S) sedimentation from approximately 6.43S to 24.03S.

[0033] In related embodiments, the polynucleotide adjuvant composition molecules in the composition have a heterogeneous molecular size, where the molecular size is from about 12.8S to 24.03S, or from about 6.43S to 18.31S, or from about 12.8S to 18.31S, or from about 12.8S to 30.315S, 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 16.14S 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 at 39.1S, or from about 39.1S to 41.54S, or from about 41.54S to 43.83S, or from about 43.83S to 45.95S.

IS 2 380 481 T3

In other related embodiments, the polynucleotide adjuvant composition has a mean sedimentation coefficient (Svedbergs) 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 over 18, or over 19, or over 20, or over 21, or over 22, or over 25 or over 30.

[0035] The source of calcium ions can be, for example, calcium chloride, calcium carbonate, calcium fluoride, calcium hydroxide, calcium phosphates or calcium sulfate.

[0036] In an aspect of special interest, the invention provides a polynucleotide adjuvant composition comprising a polyriboinosinic-polyribocytidylic acid (PIC), kanamycin and a calcium ion, where the composition includes adjuvant molecules of heterogeneous molecular weight having a weight molecular weight of between approximately 66,000 to 1,200,000 Daltons.

[0037] In related embodiments, polyriboinosinic-polyribocytidylic acid (PIC), kanamycin, and calcium molecules have a molecular weight of about 300,000 to 1,200,000 Daltons, or about 66,000 to 660,000 Daltons, or about 300,000 to 660,000 Daltons. , or approximately 300,000 to 2,000,000 Daltons, or approximately 300,000 to 4,000,000 Daltons, or approximately 500,000 to 1,000,000 Daltons, or approximately 1,000,000 to 1,500,000 Daltons, or approximately 1,500,000 to

2,000,000 Daltons, or approximately 2,000,000 to 2,500,000 Daltons, or approximately 2,500,000th

3,000,000 Daltons, or approximately 3,000,000 to 3,500,000 Daltons, or approximately 3,500,000th

4,000,000 Daltons, or approximately 4,000,000 to 4,500,000 Daltons or approximately 4,500,000a

5,000,000 Daltons.

[0038] In other related embodiments, polyriboinosinic-polyribocytidylic acid (PIC), kanamycin, and calcium molecules of the heterogeneous molecular weight adjuvant having an average molecular weight equal to or greater than 150,000 Daltons, or equal to or greater than 250,000 Daltons, or equal to or greater than 350,000 Daltons, or equal to or greater than 500,000 Daltons, or equal to or greater than 650,000 Daltons, or equal to or greater than 750,000 Daltons, or equal to or greater than 1,000,000 Daltons, or equal to or greater than 1,200,000 Daltons, or equal to or greater than 1,500,000 Daltons or equal to or greater than 1,500,000 Daltons.

[0039] In an aspect of special interest, the invention provides a polynucleotide adjuvant composition comprising a polyriboinosinic-polyribocytidylic acid (PIC), kanamycin and a calcium ion, where the composition includes adjuvant molecules of heterogeneous molecular size having a coefficient sedimentation in Svedbergs (S) from approximately 6.43S to 24.03S.

[0040] In related embodiments, the polynucleotide adjuvant composition molecules in the composition have a heterogeneous molecular weight, where the molecular size is from about 12.85S to 24.03S, or from about 6.43S to 18.31S, or from about 12.8S 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 at 24.03S, or from about 16.14S 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 at 39.1S, or from about 39.1S to 41.54S, or from about 41.54S to 43.83S or from about 43.83S to 45.95S.

[0041] In still other related embodiments, polyriboinosinic-polyribocytidylic acid (PIC), kanamycin, and calcium have a mean sedimentation coefficient greater than 9, or greater than 12, or greater than 13.5, or greater than 15, or greater. 16, or over 17, or over 18, or over 19, or over 20, or over 21, or over 22, or over 25 or over 30.

[0042] In some embodiments the invention provides a polynucleotide adjuvant composition comprising polyriboinosinic-polyribocytidylic acid (PIC), kanamycin, and calcium, where it may be preferable for the composition to exclude molecules, especially at a level such that molecules that do not have a significant immunogenic effect, where the excluded molecules have a molecular weight of approximately or less than 30,000 Daltons, approximately or less than 40,000 Daltons, approximately or less than 50,000 Daltons, approximately or less than 60,000 Daltons, approximately or less than 70,000 Daltons, approximately or less than 80,000 Daltons, approximately or less than 90,000 Daltons, approximately or less than 100,000 Daltons, approximately or less than 150,000 Daltons, approximately or less than 200,000 Daltons, approximately or less than 250,000 Daltons, approximately or less than 300,000 Daltons, about or less than 350,000 Daltons, about or less than 400,000 Daltons, about or less than 450,000 Daltons, about or less than 500,000 Daltons, about or less than 600,000 Daltons,

ES 2 380 481 T3 about or less than 700,000 Daltons, about or less than 800,000 Daltons, about or less than 900,000 Daltons, about or less than 1,000,000 Daltons.

In some embodiments the invention provides a polynucleotide adjuvant composition comprising polyriboinosinic-polyribocytidylic acid (PIC), kanamycin, and calcium, where it may be preferable for the composition to exclude molecules, especially at a level such that molecules that do not have a significant immunogenic effect, where the excluded molecules have a molecular size of approximately or less than 4.49S, approximately or less than 5.12S, about or less than 5.67S, about or less than 6.16S, about or less than 6.6S, about or less than 7.02S, about or less than 7.4S, about or less than 7.77S, about or less than 9.34S, approximately or less than

10.64S, approximately or less than 11.78S, approximately or less than 12.8S, approximately or less

13.73S, approximately or less than 14.59S, approximately or less than 15.39S, approximately or less

16.14S, approximately or less than 17.54S, approximately or less than 18.81S, approximately or less

19.99S, about or less than 21.09S, about or less than 22.12S.

[0044] In an aspect of special interest, the invention provides an immunogenic composition for enhancing the antigenicity of an antigenic compound comprising the polynucleotide adjuvant composition.

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

[0046] In related embodiments, the source of the antigen is a human antigen, a non-human animal antigen, a plant antigen, a bacterial antigen, a fungal antigen, a viral antigen, a parasite antigen, or a cancer antigen.

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

[0048] In certain embodiments, the antigens may have been purified from a natural source, synthesized by solid phase synthesis, or may be obtained by recombinant genetic technology. The antigen can comprise a fragment of a protein that comprises one or more immunogenic regions of the molecule. Antigens can also be provided by whole cells or microorganisms (e.g. g., whole virus particles) that may be alive, attenuated or truncated, or dead.

[0049] In other embodiments, the antigens include one or more infectious agent agents, a plant antigen, carcinogens, allergens, and other human antigens such as for the development of autoimmune diseases. In other embodiments, the antigens include one or more infectious agents from any virus, bacteria, Mycobacterium, fungal, and parasite.

[0050] The polynucleotide adjuvant composition of the present 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 that encode the antigen can be naked or contain a delivery system, such as liposomes.

[0051] In still other related embodiments, The rabies virus antigen is selected from human diploid cell vaccine (HDCV) or purified inactivated rabies vaccine obtained from hamster kidney cells (HKC-IPRV) or inactivated raw rabies vaccine obtained from hamster kidney cells ( HKCICRV) or purified rabies vaccine from vero cells (PVRV) or purified from chicken embryo cells (PCEC) or purified duck embryo vaccine (PDEV) or rabies antigen purified inactivated obtained from hamster kidney cells (HKC-IPRA) or inactivated raw rabies antigen obtained from hamster kidney cells (HKC-ICRA).

[0052] In an aspect of special interest, the invention provides an immunogenic composition for enhancing the antigenicity of an antigenic compound comprising the polynucleotide adjuvant composition that is capable of inducing an antigen-specific cell-mediated immune response.

[0053] In an aspect of special interest, the invention provides an immunogenic composition for enhancing the antigenicity of an antigenic compound comprising the polynucleotide adjuvant composition that is capable of inducing an antigen-specific B-cell immune response.

IS 2 380 481 T3

In an aspect of special interest, the invention provides an immunogenic composition for enhancing the antigenicity of an antigenic compound comprising the polynucleotide adjuvant composition that is capable of inducing an antigen-specific combined T and B cell immune response.

[0055] In an aspect of special interest, the invention provides an immunogenic composition for enhancing the antigenicity of a compound comprising the polynucleotide adjuvant composition and an inactivated purified rabies antigen obtained in hamster kidney cells, where the presence of the rabies antigen should reach a minimum quantity, such as more than one international unit (IU).

[0056] In related embodiments, the immunogenic composition comprises the polynucleotide adjuvant composition and the inactivated purified rabies virus antigen obtained from hamster kidney cells where the presence of the rabies virus antigen should reach a minimal amount, at most than 0.25 international units, is more than 0.5 international units, is more than 1.2 international units, is more than 1.4 international units, is more than 1.6 international units, is more than 1.8 international units, is more than 2.0 international units, is more than 2.2 international units, is more than 2.4 international units, is more than 2, 6 international units, it is more than 2.8 international units, it is more than 3.0 international units, it is more than 3.2 international units, it is more than 3.4 international units, it is more than 3.6 international units, is more than 3.8 international units or is more than 4.0 international units.

[0057] In an aspect of special interest, the invention provides an immunogenic composition for enhancing the antigenicity of a compound comprising the polynucleotide adjuvant composition and an inactivated purified rabies virus antigen obtained in hamster kidney cells, where the presence of the adjuvant and the rabies virus antigen is in a ratio of approximately 1 to 1.

[0058] In related embodiments, the immunogenic composition comprises the polynucleotide adjuvant composition and an inactivated purified rabies virus antigen obtained in hamster kidney cells where the presence of the adjuvant and the rabies virus antigen is a ratio of less 1 to 10, about 1 to 9, about 1 to 8, about 1 to 7, about 1 to 5, about 1 to 4, about 1 to 3, about 1 to 2, about 2 to 1, about 3 to 1, about 4 to 1, about 5 to 1, about 6 to 1, about 7 to 1, about 8 to 1, about 9 to 1, approximately 10 to 1, or greater than 10 to 1.

[0059] In an aspect of special interest, the invention provides an adjuvant composition or an immunogenic composition where the immunogenic composition, or the adjuvant composition contained in the immunogenic composition, is in solid or liquid form or in solution or suspension.

[0060] In an aspect of special interest, the invention provides an adjuvant composition or an immunogenic composition comprising an adjuvant composition where the adjuvant composition or the immunogenic composition is lyophilized.

[0061] In related embodiments the invention provides a kit comprising the adjuvant composition and an antigenic compound.

[0062] In an aspect of special interest, the invention provides the use of a polynucleotide adjuvant composition for the preparation of a medicament to enhance the immunogenic response of a host.

[0063] In an aspect of special interest, the invention provides a method for enhancing immune responses to an antigenic compound, which comprises administering to a host an immunogenic composition to enhance the antigenicity of an antigenic compound comprising the polynucleotide adjuvant composition.

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

IS 2 380 481 T3

[0065] The disclosure provides a method for enhancing immune responses to an antigenic compound, which comprises administering to a host an immunogenic composition to enhance the antigenicity of an antigenic compound, the composition comprising a polynucleotide adjuvant where the host is human.

[0066] In an aspect of special interest, the invention provides a method for enhancing immune responses to an antigenic compound, which comprises administering to a host an immunogenic composition to enhance the antigenicity of an antigenic compound, the composition comprising a polynucleotide adjuvant, wherein the host is an animal.

These and other features and advantages of the invention will be apparent from the following detailed description of the preferred embodiments thereof in connection with the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

[0068]

The relative molecular weight for the Av-PICKCa and PIKA samples is shown in Figure 1.

In Figure 2 it is shown that PIKA induces a dose dependent production of a specific interferon gamma cytokine.

DETAILED DESCRIPTION OF THE EXAMPLES OF EMBODIMENTS OF THE INVENTION

[0069] The present invention may be more readily understood by reference to the following detailed description of certain embodiments of the invention and the Examples included herein.

[0070] Unless defined otherwise, all technical and scientific terms used in this document have the same meaning as normally understood by a person skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, preferred methods and materials are described herein. All publications mentioned in this document are incorporated by reference to discover and describe procedures and / or materials in connection with the publications cited.

[0071] It should be appreciated that, as used herein and in the appended claims, the singular forms a / an and the / a include plural referents as long as the context does not dictate otherwise. Thus, for example, reference to a text includes a variety of such texts and reference to segment includes reference to one or more segments and equivalents thereof known to those skilled in the art, and so on. Furthermore, it will be appreciated that the claims may be defined to exclude any optional elements. As such, this statement is intended to serve as basic antecedent for the use of exclusive terminology such as "exclusively", "only" and the like in connection with the wording of the elements of the claims, or the use of a negative limitation.

DEFINITION OF TERMS

[0072] Before establishing further details of the present invention, it may be helpful for an understanding of the present invention to describe the definitions of the various terms that are used in this document.

[0073] The term "adjuvant" as used herein refers to any substance or mixture of substances that enhances or diversifies the immune response of a host against an antigenic compound. Specifically:

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

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

3. "PIKA" refers to a composition of the invention comprising poly I: C, an antibody (kanamycin), and a positive ion (calcium), where PIKA is characterized by physical characteristics (eg, molecular weight, size, and the like). as described herein) so that upon administration, PIKA exhibits characteristics of an adjuvant with reduced adverse side effects (eg, reduced toxicity) relative to, for example, PICKCa, and higher potency (eg. g., stimulates an enhanced immune response) relative to, for example, Av-PICKCa.

IS 2 380 481 T3

[0074] "PIC-containing molecule" or "PIC-containing compound refers to, without limitation, PIC that may optionally be complexing or otherwise combined with at least one or both of an antibody (eg, kanamycin. ) and a positive ion (eg, calcium) present in a composition containing the PIC-containing molecule.

[0075] "Heterogeneous as used herein in the context of the adjuvant compositions of the invention indicates that the components of the composition, e.g. For example, PIC-containing molecules are not uniform with respect to a physical characteristic of molecular weight, size, or both.

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

The term "antibody" includes polyclonal and monoclonal antibodies, as well as fragments of these antibodies that bind to the antigenic compound including Fab, F (ab ') 2, Fc, Fv fragments and single chain derivatives thereof. Furthermore, the term "antibody" includes naturally occurring antibodies as well as non-natural antibodies such as, for example, chimeric, bifunctional and humanized antibodies, and related synthetic isoforms.

[0078] As used herein, the term "antigenic compound" refers to any substance that can be recognized by the immune system (eg, bind to an antibody or be processed in a way that induces a cellular immune response) under the proper conditions.

[0079] An "antigen" refers to a substance, including compositions in vaccine form in which the vaccine itself comprises an antigenic compound and may or may not comprise an adjuvant other than PIKA, which when administered by an appropriate route (eg, parenterally), induces an immune response, eg, the formation of antibodies, including antibodies that specifically bind to the antigen. Two of the characteristics of antigens are their immunogenicity, that is, their ability to induce an immune response in vivo, and their antigenicity, that is, their ability to be selectively recognized by antibodies originating from antigens.

[0080] The terms "cell-mediated immunity" and "cell-mediated immune response" are intended to refer to the immune defense provided by lymphocytes, such as the defense provided by T lymphocytes when in proximity to their victim cells. A cell-mediated immune response typically includes lymphocyte proliferation. When lymphocyte proliferation is measured, the ability of lymphocytes to proliferate in response to a specific antigen is measured. Lymphocyte proliferation is intended to refer to the proliferation of B cells, T helper cells, or cytotoxic T lymphocytes (CTL).

[0081] An "effective amount of an antigenic compound" refers to an amount of an antigenic compound that, in optional combination with an adjuvant, will cause the subject to elicit a specific immune response to the antigenic compound.

The term "enhanced immune response" or the like, means that the immune response is elevated, enhanced or enhanced for the benefit of the host relative to the previous state of immune response, for example, prior to administration of an immunogenic composition. of the invention.

The terms "humoral immunity" and "humoral immune response" refer to the 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 compound by the immune system of a vertebrate subject. Examples of immune responses include, but are not limited to, cellular, as well as humoral, local, and systemic immunity, such as CTL responses, including antigen-specific induction of CD8 + CTL, T helper cell responses including proliferative cell responses. T and cytokine release and B cell responses including anti-body response.

[0085] The term "eliciting an immune response" is used herein generally encompasses the induction and / or enhancement of an immune response.

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

IS 2 380 481 T3

[0087] The term "enhancing an immune response" refers to a pre-existing immune response that is enhanced, promoted, supplemented, amplified, enhanced, augmented, or prolonged.

[0088] The term Poly I: C or PIC refers to a composition containing polyriboinosinic and polyribocytidyl nucleic acids that can also be referred to as polyinosinic acid-polycytidylic acid, respectively.

[0089] The term "immunogenic amount" refers to an amount of antigenic compound sufficient to stimulate an immune response, when administered with the composition of the invention, compared to the immune response observed in the absence of the polynucleotide adjuvant.

[0090] The term "immunopotentiating amount" refers to the amount of adjuvant necessary to effect an increase in antibody titer and / or cell-mediated immunity when administered with an antigenic compound in a composition of the invention, in compared to the increase in the level of antibody and / or cell mediated immunity in the absence of the polynucleotide adjuvant.

[0091] As used herein, the term "mixing" includes any method of combining the components of the composition; These procedures include, but are not limited to, mixing, dispersing, dissolving, emulsifying, coagulating, suspending, or otherwise physically combining the components of the composition.

[0092] A "pharmaceutically acceptable salt" of a compound means a salt that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. These salts include: (1) Acid addition salts formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic 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'-methylenebis (3-hydroxy-2-en-1) carboxylic acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary 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 an acidic proton is present in the parent compound whether it is substituted by a metal ion, e.g. eg, an alkali metal ion, an alkaline earth ion, or an aluminum ion, or coordinated with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like.

[0093] The term "treatment" encompasses any treatment of a disease in a vertebrate animal, especially a human, and includes: (i) preventing the onset of a disease in a subject who may be predisposed to said disease but has yet to diagnosed as having it; (ii) inhibit the disease, that is, stop its development or (iii) alleviate the disease, that is, cause it to regress.

The term "dosage unit form, as used herein, refers to physically discrete units suitable as unit doses for humans and / or animals, each unit containing a predetermined quantity of compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically / physiologically acceptable diluent, carrier or vehicle.

OVERVIEW OF THE INVENTION

[0095] The present invention is directed to compounds and methods useful for enhancing an immune response that may be humoral and / or cell-mediated, in a human, animal, or cell culture. In general, the composition comprises an immunogenic composition containing an adjuvant. The presence of the adjuvant enhances or modifies the immune response. Therefore, humoral and / or cell-mediated immune responses are more effective in the presence of the adjuvant. Additionally, the adjuvant can alter the quality of the immune response by affecting the subclasses (isotypes) of the immunoglobulins and cytokines produced.

[0096] The key characteristics of the adjuvant are its ability to stimulate a desired level and type of immune response without inducing adverse side effects. There are currently only a limited number of adjuvants approved for use in humans that have this combination of characteristics. Safety standards for immunogenic substances and especially vaccines are strict and rigorously enforced.

Thus, a significant limitation to the development of an effective adjuvant is the development of a product that is potent enough to elicit an appropriate immune response without inducing adverse side effects.

IS 2 380 481 T3

[0097] The preferred embodiment of the invention is a polynucleotide adjuvant in which the polynucleotide is polyriboinosinic-polyribocytidylic acid (PIC). PIC alone has been shown to be an effective adjuvant but shows an unacceptable safety profile and is unstable in humans and primates. The current invention provides a PIC composition combined with an antibiotic and a positive ion that enhances the desired immunogenic attributes of an adjuvant while improving the safety and stability profile.

[0098] The present invention is further based on the discovery that the physical and biological characteristics of the PIKA molecules of the adjuvant composition influence the characteristics of the immune response and adverse side effects. In the course of the research studies it was unexpectedly discovered that adjusting certain characteristics of the polynucleotide adjuvant it becomes more or less potent and / or more or less toxic in the ways described in detail below. By defining the composition of the adjuvant in terms of its physical characteristics, it is possible to more precisely describe the attributes of the adjuvant composition that provide a preferable immunogenic response and a preferable safety / stability profile.

[0099] The adjuvant of the present invention, referred to herein for convenience as the PIKA adjuvant, is therefore defined entirely by a combination of its chemical composition plus the fundamental physical attributes of the molecules that make up the adjuvant. Thus, the particular form of PIKA that exhibits significantly superior immunogenic properties while being safe for use in animals and humans is best defined by one or more, usually a combination, specific attributes such as composition, molecular weight, molecular size, concentration, and pH.

[0100] PIKA generally comprises a polynucleotide, an antibiotic and a positive ion, where the polynucleotide is polyriboinosinic-polyribocytidylic acid (PIC) and the antibiotic is the aminoglycoside (kanamycin) and the ion is calcium.

[0101] "Aminoglycoside" antibiotics refer to antibiotics whose structure contains amino sugars attached to an aminocyclitol ring (hexose nucleus) via glycosidic linkages. Aminoglycoside antibiotics are derived from various species of Streptomyces and Micromonospora or are produced synthetically. For example, kanamycin is an aminoglycoside antibiotic obtained from the soil bacterium Streptomyces kanamycetis, used in the treatment of various infections, especially those produced by gram negative bacteria.

[0102] The PIKA composition is produced by mixing polyinosinic acid, polycytidylic acid, an antibiotic and the source of a positive ion in a sodium chloride / phosphate buffer solution having a pH between pH 6 and pH. Polyinosinic acid and polycytidylic acid are generally provided at a concentration of 0.1 to 10 mg / ml, preferably 0.5 to 5 mg / ml, and more preferably 0.5 to 2.5 mg / ml. The hyperchromicity value should be greater than 10%, preferably greater than 15%, and more preferably greater than 20%. The preparation of the PIC and the combination with kanamycin and calcium is preferably carried out under quality standards in accordance with the International Good Manufacturing Process.

[0103] In certain embodiments of the present invention, kanamycin in the polynucleotide adjuvant composition can be used in conjunction with other antibiotics, or substituted for one or more antibiotics, selected from the group including tobramycin, anthracyclines, butyrosine sulfate, gentamicins, hygromycin , amikacin, dibekacin, nebramycin, metrzamide, neomycin, puromycin, streptomycin, and streptozocin. The antibiotic (p. g., kanamycin and 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 more preferably from about 500 to 5,000 units / ml.

[0104] The positive ion (calcium) can be provided in the composition of the invention at a concentration in the range of about 10 pmol to 10 mmol / ml, preferably about 50 pmol to 5 mmol / ml, and more preferably about 100 pmol to 1 mmol / ml.

[0105] As noted above, the positive ion can be provided in the form of any suitable organic salt or complex, including, but not necessarily limited to, chloride, fluoride, hydroxide, phosphate or sulfate salts. For example, when the positive ion is calcium, said ion can be in the form of calcium carbonate, calcium chloride, calcium fluoride, calcium hydroxide, calcium phosphates, or calcium sulfate.

IS 2 380 481 T3

[0106] When the positive ion in the adjuvant composition of the invention is calcium, it can be in combination with, or substituted for, other positive ions, including cadmium, lithium, magnesium, cerium, cesium, chromium, cobalt, deuterium, gallium, iodine, iron and zinc, where the ions can be in the form of inorganic salts or organic complexes.

[0107] The resulting composition is further transformed into PIKA by an additional manufacturing process involving the isolation of molecules of a defined size and / or molecular weight. Separation of polynucleotide molecules of special characteristics using filtration, chromatography, heat treatment, centrifugal separation, electrophoresis, and similar procedures that are standard processes, is known to those skilled in the art.

[0108] In certain embodiments of the present invention, the polynucleotide adjuvant composition is further defined by the physical attribute of molecular weight. In the course of the investigation it was surprisingly found that there is a positive correlation between the molecular weight and the efficacy of the polynucleotide adjuvant composition. The observed potency level of an immunogenic composition containing the polynucleotide adjuvant composition, including the ability to trigger the production of immunoglobulins and cytokines, increases as the molecular weight of the polynucleotide adjuvant composition increases. 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 section below, the inventor has found that vaccine compositions containing a PIKA adjuvant of various molecular weights showed a direct correlation between molecular weight and antigen-specific protective potency (see Example 2). Furthermore, the inventor has discovered that there is a direct correlation between the molecular weight of PIKA adjuvant compositions and the ability to trigger interferon gamma production when administered to a host in combination with a rabies virus antigen (see Example 3).

[0110] The inventor further identified, during 1996 human clinical trials in China using a rabies vaccine with an adjuvant containing PICKCa of a particularly high molecular weight specification, so that the resulting composition surprisingly exhibited an unacceptable level of effects adverse side effects. Results from previously unpublished 1996 clinical trials are presented in Example 4. The investigation into molecular weight is presented in Examples 5 and 6. The study was conducted under the jurisdiction of the Chinese Food and Drug Administration. Therefore, the adjuvant should not have been administered to humans in a controlled clinical trial setting if these side effects had been anticipated based on our knowledge at the time.

[0111] The inventor has found that the PIKA adjuvant compositions of the invention in preclinical studies with molecular weights up to 1.0 x 10⁶ and vaccine compositions including PIKA adjuvant compositions with molecular weights up to 5.5 x10⁵ have shown a broader margin of safety in specific toxicity tests (see Example 7). PIKA with a maximum molecular weight of 1.2 x 10 has been used successfully in preclinical research⁶ (see Example 3). Further research by the inventor demonstrates the safety of PIKA when used in conjunction with an antigenic compound in the form of a vaccine (see Example 8).

[0112] The results of a subsequent experiment carried out by the inventor in 2002 in China also show that the use of PIKA provides a safe and effective adjuvant in humans. The results of this experiment, not previously published, are presented in Example 9.

[0113] Based on the above observations, the preferred embodiment of PIKA thus comprises molecules that have physical characteristics of molecular weight and / or size that provide potency and efficacy benefits while providing an adequate degree of safety margin of so that no adverse side effects are induced. The molecules present in Av-PICKCa, at the lower end of the molecular weight range, can be effective as an antiviral composition but are significantly less effective than the molecular composition of PIKA when used as an adjuvant in an immunogenic composition. Additionally, PIKA has been shown to have a safety profile that is preferable to PICKCa.

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

[0115] The PIKA composition of the invention generally comprises a collection or population of molecules, where the molecules have physical characteristics of, for example, molecular weight and / or size, which provide

ES 2 380 481 T3 a desired effect of eliciting an immune response while preferably mitigating or avoiding adverse side effects (such as those associated with the administration of PICKCa). PIKA molecules are generally heterogeneous in weight and / or molecular size.

[0116] As generally used herein and unless otherwise specifically stated, PIKA, the adjuvant composition of the invention, includes PIC that may be complexing with the antibiotic (kanamycin) and a positive ion (calcium). The molecules in PIKA are heterogeneous in molecular weight (eg, as assessed by the determination of Daltons) or size (eg, as assessed by the sedimentation coefficient).

[0117] When a range is used in reference to a heterogeneous characteristic of PIKA molecules (p. g., molecular weight or size), reference to such range in this document indicates the approximate lower and upper limits of the molecular weights or sizes of the PIKA molecules in the composition, but does not imply or intend that the composition contains a molecule. of PIKA with a molecular weight or size that is representative of any molecular weight or size in the range. Thus, for example, a molecular weight range of about 66,000 to 1,200,000 Daltons indicates that PIKA molecules of about 66,000 Daltons and about 1,200,000 Daltons are contained in the composition, but need not be in the composition. 88,000 Dalton PIKA molecules (although, in fact, these may be present).

[0118] When a physical characteristic of the PIKA molecules in the composition of the invention is defined by a range of molecular weights, the PIKA molecules are heterogeneous in terms of their molecular weight, where the molecular weight range is approximately 300,000 to 660,000 Daltons, from approximately 300,000 to 1,200,000 Daltons, from approximately 66,000 to 660,000 Daltons, or from approximately 66,000 to 1,200,000 Daltons.

[0119] The invention also contemplates heterogeneous, where in a weight range about about about about about PIKA molecules having molecular weights in the upper limits and within these ranges, are present in the composition.

300,000 aa

aaa

1.000.000 2.000.000 3.000.000 4.000.000

4.000.000

1.500.000

2.500.000

3.500.000

4,500,000 compositions having PIKA molecules of molecular molecular weight is Daltons, Daltons, Daltons, Daltons,

Daltons of approximately 300,000 to 2,000,000 approximately approximately approximately approximately

1.500.000

2.500.000

3.500.000

4,500,000 lower than

1.000.000

2.000.000

3.000.000

4.000.000

5.000.000

Daltons, Daltons, Daltons, Daltons, Daltons, Daltons.

of of of of of

500,000 aa

aa

at these intervals, as well as

[0120] When a physical characteristic of the PIKA molecules in the composition of the invention is defined by the average molecular weight, the PIKA molecules can have an average molecular weight equal to or greater than 150,000 Daltons, equal to or greater than 250,000 Daltons, equal to or greater than 350,000 Daltons, equal to or greater than 500,000 Daltons, equal to or greater than 650,000 Daltons, equal to or greater than 750,000 Daltons, equal to or greater than 1,000,000 Daltons, equal to or greater than 1,200,000 Daltons, equal to or greater than 1,500,000 Daltons or equal to or greater than 2,000,000 Daltons.

[0121] When a physical characteristic of the PIKA molecules in the composition of the invention is defined by the sedimentation coefficient, which is a measure of molecular weight and size, the PIKA molecules can have a sedimentation coefficient greater than 9, or greater than approximately 12, or greater than approximately 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 to 22, or greater than 25 or greater than 30.

[0122] In some embodiments the invention provides a polynucleotide adjuvant composition comprising polyriboinosinic-polyribocytidylic acid (PIC), kanamycin, and calcium, where the composition excludes a detectable amount of molecules having a molecular weight of about or less than about 30,000 Daltons, about or less than 40,000 Daltons, approximately or less than 50,000 Daltons, approximately or less than 60,000 Daltons, approximately or less than 70,000 Daltons, approximately or less than 80,000 Daltons, approximately or less than 90,000 Daltons, approximately or less than 100,000 Daltons, approximately or less than 150,000 Daltons, approximately or less than 200,000 Daltons, approximately or less than 250,000 Daltons, approximately or less than 300,000 Daltons, approximately or less than 350,000 Daltons, approximately or less than 400,000 Daltons, approximately or less than 450,000 Daltons, approximately or less than 500,000 Daltons, approximately or less than 600,000 Daltons, approximately or less than 700,000 Daltons, approximately or less than 800,000 Daltons, approximately or less than 900,000 Daltons, approximately or less than 1,000,000 Daltons . In this embodiment, the exclusion of these molecules from

Low molecular weight ES 2 380 481 T3 is especially of interest up to a level such that the exclusion of such molecules has no significant immunogenic effect.

[0123] The inventor has shown that PIKA comprising molecules with a molecular weight of up to 1.0 x 10⁶Daltons are safe in animals in specific toxicity tests (see Example 7). PIKA comprising molecules up to a molecular weight of 1.2 x 10⁶ Daltons have been used safely in preclinical studies (see Example 3). PIKA has also been shown to be safe when used in an immunogenic composition (see Example 8). This composition of PIKA provides benefits in terms of efficacy. PIKA comprising molecules up to a molecular weight of 6.6 x 10⁵ Daltons also elicit an effective immune response with a wider margin of safety when used in humans and animals. Raising the molecular weight of the smallest molecules present up to 6.6 x 10⁵ Daltons and preferably up to 3.0 x 10⁵ Daltons improves the efficacy of the adjuvant without compromising safety standards.

[0124] It has further been discovered 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 found that increasing the concentration of the polynucleotide adjuvant can cause coalescence (or aggregation) of the PICKCa molecules, resulting in molecules with a higher molecular weight. This process has been shown to be irreversible. Therefore, subsequent dilution of the polynucleotide adjuvant composition in a suitable medium does not result in a reduction in the molecular weight of the adjuvant molecules. As seen in Example 6, when the largest concentrated molecular form of the polynucleotide adjuvant composition is combined with the rabies virus antigen, the result is a composition that retains its high molecular weight range. A rabies vaccine obtained in this way showed adverse side effects in human clinical trials (see Example 4).

[0125] The PIKA composition of the invention provided in any physiologically acceptable buffer can be used herein although phosphate buffers are preferred. Other acceptable buffers, such as acetate, tris, bicarbonate, carbonate, and 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 preferable to adjust the pH of the system between 6 and 8.5, where this pH does not significantly reduce the stability of other components of the composition and does not is physiologically inappropriate in any other way. In certain embodiments, the aqueous portion of the immunogenic composition is in a buffered saline solution. When these compositions are intended to be for parenteral administration, it is preferable to make such solutions so that the tonicity, i.e. osmolality, is essentially the same as normal physiological fluids to prevent post-administration inflammation or rapid absorption of the composition due to at the differential ionic concentrations between the composition and the physiological fluids.

[0127] The amount of buffered saline used in these compositions will be the amount necessary to bring the value of the composition to unity. That is, an amount of buffered saline sufficient to make 100% will be mixed with other components to make up the volume of the composition.

[0128] In certain embodiments, the antigens may have been purified from a natural source, synthesized by solid phase synthesis, or may be obtained by recombinant genetic technology. The antigen can comprise a fragment of a protein that comprises one or more immunogenic regions of the molecule. Antigens can also be provided by whole cells or microorganisms (eg. g., whole virus particles) that may be alive, attenuated or truncated, or dead.

[0129] In other embodiments, antigens include one or more infectious agent agents, a plant antigen, carcinogens, allergens, and other human antigens, such as for the development of autoimmune diseases. In other embodiments, the antigens include one or more infectious agents from any of the viruses, bacteria, Mycobacterium, fungi, and parasites.

[0130] The polynucleotide adjuvant composition of the present 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 that encode the antigen can be naked or contain a delivery system, such as liposomes.

IS 2 380 481 T3

[0131] In certain embodiments, the polynucleotide adjuvant composition can be used in combination with vaccines. It does not matter if the vaccine contains adjuvants or not. The classes of vaccines included are anti-infective, anti-cancer, anti-allergic, anti-autoimmune diseases, and immunocontraception.

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

[0133] In certain embodiments, the rabies virus antigen can be inactivated raw rabies virus antigen such as an inactivated raw rabies virus antigen obtained from hamster kidney cells (HKC-ICRA) or a Inactivated purified rabies virus antigen as an inactivated purified rabies virus antigen obtained from hamster kidney cells (HKC-IPRA).

[0134] In certain embodiments, the polynucleotide adjuvant composition can be used with a rabies vaccine. There are suitable rabies vaccines available on the market or under investigation including inactivated vaccines, recombinant and peptide subunits such as human diploid cell vaccine (HDCV), o purified hamster kidney cell-derived rabies vaccine (HKC-IPRV) o raw inactivated rabies vaccine obtained from hamster kidney cells (HKC-ICRV) o purified vero cell (PVRV) or purified rabies vaccine obtained chicken embryo cells (PCEC) or purified duck embryo vaccine (PDEV). However, not all rabies vaccines induce a cell-mediated immune response, which is important in pre- and post-exposure immunizations. When the polynucleotide adjuvant composition (eg, PIKA) is administered with the rabies vaccine, the induced immune response includes: non-specific responses (eg, increased macrophage functions), humoral responses (eg. g., increased production of specific antibodies) and cell-mediated responses (eg, production of cytokines including interferon and interleukin-2).

[0135] In certain embodiments the invention provides a kit comprising the polynucleotide adjuvant and an antigenic compound.

[0136] An immunogenic composition that includes PIKA is capable of inducing a specific immune response in two ways: i) humoral immunity, which includes the stimulation of B cells and the production of antibodies or immunoglobulins (other cells are also involved in the generation of an anticorpal response, p. g., antigen presenting cells (APC, including macrophages) and helper T cells (Th1 and Th2)) and ii) cell-mediated immunity, which generally involves T cells including cytotoxic T lymphocytes (CTL), although they are also other cells involved in generating a CTL response (eg, Th1 and / or Th2 cells and APC). Procedures for evaluating humoral and / or cellular immune responses in an individual are well known in the art (see Examples 10, 11, 12 and 13).

[0137] Additionally, the polynucleotide adjuvant composition can alter the quality of the immune response by affecting the subclasses (isotypes) of the immunoglobulins produced (IgG1, IgG2, IgG3 and IgG4 for human IgG; IgG1, IgG2a, IgG2b and IgG3 for human Mouse IgG) as well as their affinities.

[0138] A Th1 cell-regulated response in mice will induce IgG1, IgG2a, IgG2b and, to a lesser extent, IgG3, and will also promote a cell-mediated immune response against an antigen. If the IgG response to an antigen is regulated by Th2-type cells, it will predominantly enhance the production of IgG1 and IgA.

[0139] NIH potency tests using a PIKA adjuvant composition and an inactivated purified rabies virus antigen obtained in hamster kidney cells surprisingly showed that the immunogenic potency of the composition requires a minimal presence of the virus antigen of rabies (see Example 14). The potency of the composition increases rapidly in relation to the presence of additional rabies virus antigen that exceeds 1 IU of antigen. Therefore, the rate of increase in the potency of the composition was found to be greater with about 1.5 IU to 2.5 IU of the rabies virus antigen present in the composition. The NIH potency test is described in: Laboratory Techniques in Rabies, edited by FX Meslin, MM Kaplan, H Koprowski 4^to ISBN 92 4 1544 Edition 1.

[0140] Tests using a PIKA adjuvant composition and an inactivated purified rabies virus antigen obtained in hamster kidney cells demonstrated that the immunogenic potency of the composition increased as the amount of adjuvant present exceeded the amount of antigen Present. The potency increased when the ratio of PIKA to the inactivated purified rabies virus antigen obtained in hamster kidney cells increased with the preferred ratio being greater than 3: 1 (see Example 15).

IS 2 380 481 T3

[0141] The invention contemplates methods of using the polynucleotide adjuvant of the invention with an antigen to, for example, induce an antigen-specific humoral response and / or a specific cellular response (eg, T cells) in a subject. The immune response elicited may be a response to an antigen in a subject not previously exposed to the antigen or it may serve to enhance an existing immune response (eg, as during a reimmunization).

[0142] In certain embodiments, the PIKA adjuvant composition and an immunogenic composition comprising the PIKA adjuvant and an antigenic compound may be freeze-dried (lyophilized) for long-term stability and storage in solid form. The lyophilization procedure is known to those of skill in the art. Reconstitution of the immunogenic composition containing PIKA and an antigenic compound demonstrated a maintained level of efficacy (see Example 16).

[0143] The immunogenic composition can be prepared as an injectable liquid solution, suspension or emulsion. The preparation of formulations of a desired immunogenic composition is generally described in New Trends and Developments in Vaccines, edited by Voller et al., University Park Press, Baltimore, Md., USA, 1978. The immunogenic composition of the present invention can be used in forms such as capsules, liquid solutions, emulsions, suspensions or elixirs for oral administration, or sterile liquid forms such as solutions, emulsions or suspensions. Any inert vehicle is preferably used, such as saline, or phosphate buffered saline, or any vehicle in which the compounds used in the process of the present invention have solubility properties suitable for use in the processes of the present invention. .

[0144] The immunogenic composition of the present invention can be administered to a subject using various procedures known in the art. In certain embodiments, the immunogenic composition can be administered parenterally, by injection, as intramuscular, intraperitoneal, intravenous, or subcutaneous injection, or by inhalation. In other embodiments, the immunogenic composition can be administered rectally, vaginally, nasal, orally, ophthalmic, topical, transdermal, or intradermal. When the mode of administration is by injection, the encapsulated antigenic compound can remain at the injection site for up to two weeks, thus providing a reservoir of antigen that provides sustained release or pulsatile release in vivo. This delivery system can allow single shot immunogenic formulations to produce antigenic compounds that might otherwise require multiple injections to induce an immune response.

[0145] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and first making the diluent liquid isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous and intraperitoneal administration. In connection with the foregoing, the sterile aqueous media that can be employed will be known to those skilled in the art in light of the present disclosure. Examples of injection media that can be used in the present invention include a buffer with or without dispersing agents and / or preservatives and edible oil, mineral oil, cod liver oil, mono-, di-, or triglycerides, and a mixture of the themselves.

[0146] The exact amount required of these compositions will vary from subject to subject, depending on the species, age, weight and general conditions of the subject, the severity of the disease, infection or condition being treated or prevented, the compound in concrete used, its mode of administration and the like. One skilled in the art can determine the appropriate amount using only routine experimentation as described in this document. Following an initial administration, subjects may receive one or more suitably spaced booster immunizations.

[0147] The above description generally describes the present invention. The following examples will aid in the understanding of the present invention. These examples are described for illustration purposes only and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are viewed as circumstances that may suggest or lead to a record. Although specific terms have been used, it is intended that these terms have a descriptive sense and are not limiting.

IS 2 380 481 T3

EXAMPLES

EXAMPLE 1: DETERMINING THE MOLECULAR WEIGHT OF PIKA AND Av-PICKCA

[0148] This example shows how the molecular weight of PIKA adjuvant can be determined in comparison to Av-PICKCa.

[0149] Agarose gel electrophoresis is known to those skilled in the art, so this invention only describes the details in this document. The agarose gel used in the present invention had a 1.5% agarose concentration. Molecular weight markers were 100 bp DNA staggered from 100 bp to 1,000 bp corresponding to a molecular weight range of 6.6 x 10⁴ to 6.6 x 10⁵ Daltons. Loading samples were 4 µΙ at 1 mg / ml. Figure 1 shows a representative image of the results of the samples in the agarose gel following the indications in this paragraph. The five (5) different batches tested showed a wide distribution range of their molecular weights. The upper limits of their molecular weights ranged from 2.3 x 10⁵ Daltons for Av-PICKCa at 5.28 x 10⁵ Daltons for PIKA.

EXAMPLE 2: IMMUNE EFFECTIVENESS OF PIKA COMPARED TO Av-PICKCA

[0150] This example shows the difference between the potency of Av-PICKCa with a maximum molecular weight of 230,000 Daltons and samples of PIKA with a maximum molecular weight of up to 528,000 Daltons.

[0151] Three lots of PIKA adjuvant of different molecular weight and one lot with molecules of the molecular weight corresponding to that of Av-PIKA were mixed with the inactivated purified rabies virus antigen obtained from hamster kidney cells (HKC-IPRA) . The resulting compositions were subjected to the NIH efficacy test.

[0152] The NIH test is a rigorous and extensive comparative study between the investigational rabies vaccine and a standardized rabies vaccine. The vaccinated mice are infected with a live rabies virus strain and their survival rate is determined. Different dilutions of the rabies vaccine were administered to the different groups of mice. A comparison of survival rates between groups of mice exposed to the experimental and standardized vaccines determines the potency of the experimental vaccine (Laboratory Techniques in Rabies, Edited by FX Meslin, MM Kaplan H Koprowski, 4^to Edition, ISBN 92 4 1544 1).

[0153] The efficacy of each combination vaccine has been normalized relative to the standard non-combined vaccine where the efficacy of the non-combined vaccine was designated as 1, and the relative efficacy was designated as the times the efficacy of the combined was increased relative to to the uncombined. Table 1 summarizes the results. As can be seen in Table 1, the higher the molecular weight of the PICKCa adjuvant, the greater the value increasing efficacy of the rabies vaccine.

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

<td>Type of adjuvant</td><td>Antigen</td><td>Sample number</td><td>Upper molecular weight limit of adjuvant</td><td>ED50</td><td>Potency (IU / ml)</td>

<td>PIKA</td><td>HKC-IPRA</td><td> 20000304</td><td>5.28 x 10⁵</td><td> 2,10</td><td> 5,00</td>

<td>PIKA</td><td>HKC-IPRA</td><td> 20000907</td><td>4.62 x 10⁵</td><td> 2,00</td><td> 3,98</td>

<td>PIKA</td><td>HKC-IPRA</td><td> 990202</td><td>3.96 x 10⁵</td><td> 1,98</td><td> 3,80</td>

<td>Av-PICKCa</td><td>HKC-IPRA</td><td> 000703</td><td>2.30 x 10⁵</td><td> 1,88</td><td> 3,00</td>

<td></td><td>HKC-IPRA</td><td>Control vaccine</td><td></td><td> 1,40</td><td> 1,00</td>

EXAMPLE 3: COMPARISON OF INTERFERON PRODUCTION BETWEEN PIKA AND Av-PICKCA

[0154] This example shows the difference in the ability to cause interferon production between Av-PICKCa samples with a maximum molecular weight of 230,000 Daltons and PIKA samples with a maximum molecular weight of up to 1,200,000 Daltons.

[0155] Two lots of PIKA with upper molecular weight limits of 1.2 x 10 were compared⁶ Daltons and 4.6 x 10⁵Daltons with a batch of Av-PICKCa with an upper molecular weight limit of 2.3 x 10⁵ Daltons.

[0156] The PIKA and Av-PICKCa compositions were combined with an inactivated purified rabies virus antigen obtained from hamster kidney cells (HKC-IRPA). The compositions were injected subcutaneously into mice. After two hours, the presence of interferon was determined in each mouse. The general procedure for determining interferon is known to those of skill in the art. Briefly, in a 96-well plate, each well was inoculated with L929 cells at 0.15 ml / well with approximately 30,000 cells. After three (3) days when the cells had grown to confluence, samples of

ES 2 380 481 T3 the sera (0.1 ml / well) to the wells in which the sera had been diluted from 1:20 to 1: 640. Three wells were used for each diluted sample. The wells were incubated overnight at 37 ° C. Serum samples were removed by washing. Vesicular stomatitis virus (VSV) particles were used to detect interferon production. Table 2 shows the interferon production induced by the mixtures. As can be seen in Table 2, the higher the molecular weight of the PIKA samples, the better the induction of interferon production.

Table 2. Relationship between molecular weight and interferon production

<td>Type of adjuvant</td><td>Lot number</td><td>Upper range of molecular weight in Daltons</td><td>HKC-IPRA Lot No.</td><td colspan="2">PIKA: HKC-IPRA ratio</td><td>Interferon production value</td>

<td>PIKA</td><td> 20010601</td><td>1.20 x 10⁶</td><td> 20001205</td><td> 4</td><td> 1</td><td> 868,6</td>

<td>PIKA</td><td> 200009-7</td><td>4.62 x 10⁵</td><td> 20001205</td><td> 4</td><td> 1</td><td> 530,6</td>

<td>Av-PICKCa</td><td> 000703</td><td>2.30 x 10⁵</td><td> 20001205</td><td> 4</td><td> 1</td><td> 46,4</td>

EXAMPLE 4: 1996 HUMAN VACCINE CLINICAL TRIAL (WITH TOXIC SIDE EFFECTS)

[0157] This example demonstrates that the PICKCa adjuvant when combined with a vaccine generates an unacceptable level of side effects when administered for use in humans.

[0158] The objective of the study was to evaluate the safety and immune response of a rabies vaccine comprising the adjuvant PICKCa at a concentration of 11.95 mg / ml and a molar mass of 69,700 (note that in this case , the molar mass is not equivalent to Daltons; see Example 5) and an inactivated raw rabies virus antigen obtained from hamster kidney cells (HKC-ICRA). The results and conclusions of the previous clinical trial have not been previously presented in public.

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

[0160] From a safety perspective, observations of body temperature and local and systemic symptoms were made at 24 hours, 48 ​​hours, and 72 hours after each injection. The following observations were made:

Table 3. Side effects after injection of HKC-ICRA with alum or PICKCa

<td>Secondary effect</td><td>Group</td><td>Number of volunteers</td><td>Number with side effect</td>

<td>Local</td><td>PICKCa plus HKC-ICRA</td><td> 20</td><td> 6</td>

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

<td>Systemic</td><td>PICKCa plus HKC-ICRA</td><td> 20</td><td> 4</td>

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

[0161] Systemic adverse effects include: fever (1), rash (2), joint pain (2), lymph nodes (1), throat swelling (1). Local adverse effects include: redness of the skin at the injection site (6).

[0162] The inventor's subsequent research attributed the observed side effects to the molecular size of the adjuvant molecules (see Examples 5 and 6).

EXAMPLE 5. RELATIONSHIP BETWEEN THE PICKCA CONCENTRATION AND ITS MOLECULAR WEIGHT

[0163] In this example it is shown that increasing the concentration of the PICKCa adjuvant results in a composition with an increase in molecular weight.

[0164] PICKCa can be made in different concentrations. It has been hypothesized that PICKCa could exist as a complex of polymers in different forms when prepared in different concentrations. Laser light scattering was used for this purpose. Laser light scattering has been widely used to determine weight mean molar mass (Mp) and radius of gyration (Rg). The kits are commercially available and the process is known to those of skill in the art. Table 4 shows that the observed molecular weight of PICKCa by laser light scattering correlates with its concentration.

Table 4. Molecular weight observed by laser light scattering | PICKCa concentration (mg / ml) | Average weight molar mass |

IS 2 380 481 T3

<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 PREVIOUS PICKCa CONCENTRATION AND VACCINE MOLECULAR WEIGHT

[0165] This example demonstrates a correlation between the increase in molecular weight of the PICKCa adjuvant and the molecular weight resulting from the composition including the PICKCa adjuvant and an inactivated raw rabies virus antigen obtained from hamster kidney cells. .

[0166] It was also suspected that the pre-pool concentration of PICKCa samples could affect antigens in vaccines. The PICKCa samples were combined with an inactivated raw rabies virus antigen obtained from hamster kidney cells. Laser light scattering was used for this purpose. Laser light scattering has been widely used to determine weight mean molar mass (Mp) and radius of gyration (Rg). The kits are commercially available and the process is known to those of skill in the art. Table 5 shows that an increase in pre-combination concentrations of PICKCa resulted in an increase in the Mw of rabies vaccines.

Table 5. Relationship between pre-combination concentration of PICKCa and Pm of rabies vaccines

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

<td> 11,95</td><td>29.6 x 10⁴</td><td>17.2 x 10²</td>

<td> 4,00</td><td>22.2 x 10⁴</td><td>15.0 x 10²</td>

<td> 2,00</td><td>13.8 x 10⁴</td><td>11.8 x 10²</td>

<td> 1,00</td><td>5.60 x 10⁴</td><td>7.55 x 10²</td>

<td> 1,00</td><td>5.29 x 10⁴</td><td>6.50 x 10²</td>

EXAMPLE 7: PIKA TOXICITY TEST

[0167] In this example the safety characteristic of the adjuvant PIKA is demonstrated when there is a limitation in the maximum molecular weight.

[0168] A toxicity test was performed according to the provisions of the China National Drug Standard (WS1-XG050-2000). Briefly, five (5) mice with a body weight of approximately 18-22 grams were injected intravenously with 0.5 ml / mouse of a sodium chloride solution containing 0.3 mg of a PIKA adjuvant having a molecular weight higher from about 525,000 to about 1,000,000 Daltons. Injected mice are observed for 7 days and weighed at the end of observation. Table 6 summarizes the results showing that the molecular weight of the PIKA adjuvant should be a maximum of 1.0 x 10⁶Daltons without obvious toxicity.

Table 6. PIKA adjuvant toxicity test,

<td>Lot number</td><td>Upper Molecular Weight Range (Daltons)</td><td>Weight of the mice before the test (g)</td><td>Amount injected into the tail vein</td><td>Status of the mice at the end of the test</td><td>Weight of the mice after the test</td><td>Observations</td>

<td> 20000304</td><td>5.25 x 10⁵</td><td> 18-19</td><td>0.5 ml / mouse</td><td>Healthy</td><td> 23-26</td><td>Satisfactory</td>

<td> 20010103</td><td>5.20 x 10⁵</td><td> 18-19</td><td>0.5 ml / mouse</td><td>Healthy</td><td> 22-25</td><td>Satisfactory</td>

<td> 20010816</td><td>5.20 x 10⁵</td><td> 18-19</td><td>0.5 ml / mouse</td><td>Healthy</td><td> 23-25</td><td>Satisfactory</td>

<td> 20010511</td><td>1.00 x 10⁶</td><td> 18-20</td><td>0.5 ml / mouse</td><td>Healthy</td><td> 24-26</td><td>Satisfactory</td>

EXAMPLE 8: PIKA IN THE VACCINE COMPOSITION TOXICITY STUDY

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

[0170] PIKA adjuvant (molecular weight 66,000 Daltons to 660,000 Daltons) was combined with inactivated purified rabies virus antigen obtained from hamster kidney cells (HKC-IPRA) in a PIKA: HKC-IPRA ratio of 4: 1.

[0171] The vaccine composition of PIKA and HKC-IPRA were compared to a commercially available purified inactivated rabies vaccine (IPRV) that includes an alum adjuvant.

IS 2 380 481 T3

[0172] Five (5) doses of the vaccine composition were administered to the mice on day 0, day 3, day 7, day 14, and day 28. The dose administered was equivalent to approximately 300 times the dose of an adult human under a normal human rabies immunization regimen.

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

Table 7: Safety Observations After Administration of Rabies Vaccine

<td>Formulations</td><td>Effect</td><td>Day 0</td><td>Day 3</td><td>Day 7</td><td>Day 14 1</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 appearance) / (total number)

[0174] The conclusion drawn is that the PIKA / HKC-IPRA combination is safer than the IPRV available on the market.

EXAMPLE 9. SAFE USE OF PIKA ADJUVANT IN HUMANS

[0175] In 2002, five (5) volunteers were immunized with a composition of PIKA (molecular weight 66,000 to 660,000 Daltons) and the inactivated purified rabies virus antigen obtained from hamster kidney cells (HKC-IPRA) . The vaccine composition was administered to volunteers on day 0, day 3, day 7, day 14 and day 30.

[0176] No local or systemic side effects were observed in any of the patients after each of the vaccinations.

[0177] The potency of the vaccine was determined using the standard NIH test with the results presented below in Table 8:

Table 8: Observations on the potency of the rabies vaccine

<td>Day</td><td>ED50</td><td>Neutralizing antibody IU / ml</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 triggers the production of protective neutralizing antibodies.

EXAMPLE 10: POST-EXPOSURE TEST (CELL MEDIATED IMMUNITY)

[0179] Post-exposure testing is the most definitive proof that the vaccine has the ability to eradicate pathogens from the host's body after infection. Thus, it is an indication of the vaccine-induced cell-mediated immune response.

[0180] In post-challenge testing, mice were infected with a wild-type rabies virus strain and subsequently inoculated with: an inactivated purified rabies virus antigen obtained from hamster kidney cells (HKC -IPRA) in combination with PIKA adjuvant (molecular weight range 1.65 x 10⁵ a 40 1.2 x 10⁶ Daltons) or HKC-IPRA in combination with an aluminum hydroxide adjuvant (alum), a purified rabies vaccine obtained from commercial vero cells (PVRV), or phosphate buffer solution (PBS). The results show conclusively that the adjuvant PIKA improved survival rates, see Table 9.

Table 9. Post-exposure stimulation test

<td colspan="3">9.1 Death rate after treatment</td>

<td>Groups</td><td>Default dose of 80% death</td><td>Default dose of 50% death</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 rate after treatment</td>

IS 2 380 481 T3

<td></td><td>Default at 80%</td><td>Default 50%</td>

<td>Groups</td><td>Death dose</td><td>Death 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] Mice infected with a subcutaneous injection of live rabies virus were treated with the vaccine at 6 hours, 1 day, 2 days and 3 days after infection.

EXAMPLE 11. PRODUCTION OF GAMMA INTERFERON IN ANTIGEN-SPECIFIC CELL-MEDIATED IMMUNE RESPONSE

[0182] The production of interferon gamma is an indicator of the activity of cell-mediated immunity.

[0183] In this experiment, blood samples were taken from two clinical trial patients and two individual controls. Volunteer patients had been vaccinated with the PIKA rabies vaccine, which contained PIKA (molecular weight range 66,000 to 660,000 Daltons) and the hamster kidney cell inactivated purified rabies virus antigen (HKC-IPRA) 2, 5 years prior to collection of blood samples.

[0184] The results in Figure 2 show the significant difference in interferon gamma produced by the two clinical trial patients when compared to individual controls.

[0185] Monocytes isolated from blood samples were incubated with the same HKC-IPRA that was used in the original clinical trial. After 3 days of incubation, cell-free supernatants were collected and interferon gamma in the supernatants was determined by cytokine-specific ELISPOT. A dose-dependent effect was observed.

[0186] The conclusions of the previous observation are:

• the rabies vaccine that included the PIKA adjuvant of the invention had the ability to induce the production of interferon gamma and, consequently, to elicit a cell-mediated immune response • the interferon gamma response is specific (that is, the response is directed against the rabies virus antigen as opposed to a non-specific reaction). If the interferon gamma response were not specific, no variation would be observed in the level of interferon gamma production in the blood of vaccinated patients while the concentration of the stimulating antigen increases.

EXAMPLE 12: PIKA EFFECTIVENESS TEST

[0187] The object of this experiment is to demonstrate the ability of PIKA to trigger the production of interferon gamma and interleukin 12 (IL-12).

[0188] Splenocyte samples from normal healthy mice were incubated for a period of three days in the presence of PIKA (molecular weight range 66,000 to 660,000) in a clean environment. At the end of the period, the level of cytokines in the supernatants was checked with specific ELISA tests for IL-12 (p40) and interferon gamma. The results of the experiment are presented below in Table 10.

Table 10: In vitro cytokine production test

<td>PIKA pg / 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 / A</td>

<td> 250</td><td> 1.340</td><td>N / A</td>

[0189] The conclusion from the above experiment is that PIKA triggers a dose-dependent production of the cytokines interferon gamma and IL-12 and thus induces a cell-mediated immune response.

IS 2 380 481 T3

[0190] In a further experiment, four (4) mice were administered 500 pg / mouse PIKA (molecular weight range 66,000 to 660,000 Daltons) by peritoneal injection. A phosphate buffer solution was used as a negative control test. Five hours after injection a blood sample was drawn and serum was prepared. The level of cytokines in the serum was checked with specific ELISA tests for IL-12 (p40) and interferon gamma. The results of the experiment are presented below in Table 11.

Table 11: In vivo test for cytokine production

<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>PIKA</td><td> 410</td><td> 40</td>

[0191] The conclusion from the above experiment is that PIKA is effective in stimulating a cell-mediated immune response.

EXAMPLE 13: USE OF PIKA WITH PURIFIED INACTIVATED RABIES VIRUS ANTIGEN OBTAINED IN VERO CELLS

[0192] The objective of this experiment is to evaluate the efficacy of PIKA in combination with a purified, inactivated rabies vaccine obtained from vero cells (PVRV).

[0193] PIKA with a molecular weight range of 66,000 to 660,000 Daltons was combined with PVRV to form a rabies vaccine. The NIH test was used to evaluate the potency of the resulting vaccine composition. The results are presented below in Table 12.

Table 12: NIH Test Results for PIKA and Purified Inactivated Rabies Vaccine Obtained in Vero Cells

<td>Composition of vaccine</td><td>Antigen</td><td>Adjuvant</td><td>Composition potency (IU / ml)</td>

<td>PVRV</td><td>0.02 IU / ml</td><td>n / a</td><td> 0,46</td>

<td>PVRV plus PIKA</td><td>0.02 IU / ml</td><td>PIKA</td><td> 3,68</td>

[0194] The conclusion drawn is that PIKA increases the potency of the inactivated purified rabies vaccine in vero cells.

EXAMPLE 14. DOSE OF ANTIGEN

[0195] This experiment demonstrates the requirement to have a minimal amount of inactivated purified rabies virus antigen obtained from hamster kidney cells (HKC-IPRA) present in the composition together with the PIKA adjuvant (molecular weight range 66,000 to 660,000 Daltons) to elicit a substantially enhanced levels of specific immune response. Increasing amounts of rabies virus antigen were added to a constant amount of 0.1 mg of PIKA adjuvant. Potency was measured using the NIH standard rabies vaccine potency test. Following an initial predictable increase in potency, a clear and dramatic increase was observed before potency stabilized as expected with the addition of antigen (see Table 13).

Table 13: Potency of the vaccine with increasing amounts of inactivated purified rabies virus antigen obtained from hamster kidney cells

<td>HKC-IPRA (UI)</td><td>HKC-IPRA plus 0.1 mg 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 increase in potency is the increase in potency of the HKC-IPRA / PIKA vaccine observed for the addition of one IU of the present HKC-IPRA.

[0197] The conclusion drawn is that a minimal presence of antigen is necessary before inducing a substantial immune response. Furthermore, an excess amount of antigen greater than the trigger point produces only a marginal increase in potency.

IS 2 380 481 T3

EXAMPLE 15. ADJUVANT ANTIGEN RATIO

[0198] This experiment demonstrates the optimal mixture of the inactivated purified rabies virus antigen obtained from hamster kidney cells (HKC-IPRA) and the PIKA adjuvant (molecular weight range 66,000 to 660,000).

[0199] Various amounts of antigen were mixed with various amounts of adjuvant with added PBS to ensure a reproducible total 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 combined test results indicates that the optimal vaccine combination is a ratio of PIKA to antigen in the range of at least 3 to 1.

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

<td>Samples</td><td>HKC-IPRA (ml)</td><td>PIKA (ml)</td><td>PBS (ml)</td><td>Ratio (PIKA: HKC -IPRA</td><td>ED50</td><td>Potency (IU / ml)</td>

<td>TO</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>na</td><td>na</td><td> 2,52</td><td> 6,70</td>

EXAMPLE 16. LYOPHILIZED STORAGE OF PIKA AND PIKA COMBINED WITH ANTI-RABIC VACCINE

[0201] In this example PIKA is shown to be stable in lyophilized form.

[0202] Freeze-drying technology has been used for the long-term preservation of rabies vaccines for up to three years. The inventor sought to see if freeze-dried preservation of PIKA (66,000 to 660,000 molecular weight range) and rabies vaccines containing PIKA could be beneficial. The following compositions were used for the lyophilized preservation test: i) thawed PIKA 25 was added to an inactivated purified rabies virus antigen obtained from thawed and lyophilized hamster kidney cells (HKC-IPRA), ii) lyophilized composition of Reconstituted PIKA plus HKC-IPRA, iii) a reconstituted freeze-dried commercial rabies vaccine (without adding PIKA) and iv) a thawed commercial rabies vaccine. Table 15 shows that lyophilized PIKA and rabies vaccines containing PIKA were ideal for long-term preservation of rabies vaccines.

30. Table 15. Effects of lyophilized preservation on the potency of rabies vaccines

<td>Sample</td><td>ED50</td><td>Relative power</td><td>IU / ml</td>

<td>i) Freeze-dried rabies vaccines diluted with PIKA</td><td> 2,89</td><td> 2,34</td><td> 15,71</td>

<td>ii) Rabies vaccines containing lyophilized PIKA diluted in PBS</td><td> 3,00</td><td> 3,00</td><td> 20,23</td>

<td>iii) Lyophilized commercial rabies vaccines PIKA diluted in PBS</td><td> 1,85</td><td> 0,21</td><td> 1,43</td>

<td>iv) Thawed standard rabies vaccines</td><td> 2,52</td><td> 1,00</td><td> 6,70</td>