HK1118013B - Lipid and nitrous oxide combination as adjuvant for the enhancement of the efficacy of vaccines - Google Patents

Description

lipid and nitrous oxide combination as adjuvant for enhancing vaccine efficacy

Technical Field

The present invention relates to pharmaceutical formulations (which expression herein includes veterinary formulations) for use in the prevention of disease by vaccination against infectious organisms that afflict the body of an animal (which expression herein includes the human body).

Background

In EP93912877.3 and U.S. Pat. No. 5,633,284 and equivalents thereof, dermatological or topical compositions comprising nitrous oxide [ N ] in a dermatologically acceptable carrier for the treatment of various skin, muscle and joint disorders are disclosed₂O]And at least one fatty acid or lower alkyl ester thereof. It is also disclosed herein that these compositions may also beneficially contain other active ingredients. The following active ingredients are specifically mentioned in this regard: coal tar solution, collagen, niacinamide, nicotinic acid, lanolin, vitamin E, methyl salicylate, arnica and H1-antagonist antihistamines specifically mentioned only for diphenhydramine hydrochloride. In WO97/17978 and U.S. Pat. No. 6,221,377 and their corresponding applications and patents, it is also disclosed that the action of analgesics, anti-inflammatory agents and antipyretics may be enhanced by administering these agents together with a carrier comprising nitrous oxide and at least one long chain fatty acid selected from the group consisting of oleic acid, linoleic acid, alpha-linolenic acid, gamma-linolenic acid, arachidonic acid, the C of said long chain fatty acid₁To C₆Any of alkyl esters, mixtures of these acids, and mixtures of these esters. The carrier may comprise a mixture known as Vitamin F Ethyl Ester and optionally may further comprise eicosapentaenoic acid [ C20: 5 omega 3]And docosahexaenoic acid [ C22: 6 omega 3]。

In WO02/05850, it is disclosed that the efficacy of anti-infective drugs can be enhanced by formulating in the same carrier.

It is disclosed in WO02/05851 that the effect of known agents affecting the central and/or peripheral nervous system can be similarly enhanced by formulation in the same carrier.

It is disclosed in WO02/05849 that the same vector can also be used advantageously for transporting nucleic acid compounds across cell membranes.

The antigens used to form the vaccine are not among the active components mentioned in the aforementioned patents and patent applications, since they enable the formulation of beneficial effects by means of the vectors disclosed therein.

The foregoing disclosure is not to be understood as implying that the nitrous oxide and fatty acid composition has any adjuvant contribution in the prophylactic effect against diseases caused by infectious agents. In the context of the disclosures in the above patent families, the person reading this text (notificaladdress) is most likely, as the present inventors have understood the role of anti-infectious agents in treating patients who have suffered an infection.

It has now surprisingly been found that the above-mentioned mediators, or mediators associated therewith, can themselves act as adjuvants, thereby increasing the immunogenicity of known vaccines.

The expression "vaccine" as used herein refers to a compound having its extended meaning as contributing in the prevention of infectious diseases, e.g. by any method or mechanism of stimulating the body, and to anti-infective agents including virus-based, peptide-based, bacteria-based, VLP-based and synthetic-based compound preparations, but not including for the treatment of diseases.

The exclusion of anti-infective agents from the scope of the present invention is not an admission that the aforementioned patents and applications contain any disclosure of any prophylactic properties of these excluded compounds or that these properties are apparent from the disclosure in these patents or applications. These inferences are expressly rejected. The exclusion is introduced only to avoid potential obstacles to the grant of patents intended as part of the underlying subject, which is not considered to be dispute in itself during the inspection process, since it may unduly delay the performance of the practice of the important features of the invention. The remaining large number of subjects of the present invention are expected to play a great role in the ready availability of vaccines for the prevention of widespread infections, significantly reducing costs in the case of e.g. hepatitis b.

The expression "therapeutic vaccine" is also meant to include vaccines for preventing and/or treating an existing infection by eliciting and/or enhancing a specific immune response to an infectious agent without the use of antimicrobial, antifungal or antiviral agents. The term is therefore to be understood in the broader sense of the immune response, i.e. all compounds that play a role in eliciting or enhancing an immune response against specific microscopic (microscopical) and sub-microscopic organisms. The term also specifically includes all antigens or natural and synthetic biologicals that fall within 26 classes (biologicals) of the pharmaceutical classification (pharmaceutical classification) used in the medical specialties ("MIMS") published by Times Media in South Africa.

It therefore comprises:

an antibacterial vaccine;

an antifungal vaccine;

antiviral vaccines (including antiretroviral vaccines);

an antiprotozoal agent;

and anti-helicobacter vaccines.

The adjuvanticity of the above mentioned carriers was found in the context of the fact that there appears to be no suggestion in the previous literature that nitrous oxide itself or the addition of nitrous oxide to the long chain fatty acids used in the above formulations has an additional stimulatory effect on the immunogenicity of the vaccine.

In recent years, it is used inThe development of new vaccine systems for prophylactic and therapeutic purposes is constantly becoming increasingly attractive. Adjuvant formulation strategies and uses that can affect the immune response in both quantity and quality have attracted a great deal of attention from those familiar with the problems in drug delivery. Early efforts focused on parenteral vaccines and emphasized the role of controlled release techniques for biodegradable microspheres^1-3。

The primary purpose of vaccination is to prevent disease. Historically, vaccination has been the only strategy that has led to the elimination of viral disease, i.e., smallpox. Although most pathogens are more biologically hostile to vaccine development than smallpox, some vaccines do protect humans and animals to varying degrees from the relevant pathogens. An indirect correlation between vaccine immunogenicity and safety has been observed. The human immune response to synthetic and recombinant peptide vaccines administered with standard adjuvants tends to be weak; there is therefore an urgent need for effective vaccine adjuvants that enhance the immunogenic and immunostimulatory properties of vaccines, even imperfect vaccines can deliver public health and economic benefits and provide additional insight into prophylactic and therapeutic strategies. Although microbicides can effectively expand prophylactic options and serve as a valuable prototype for vaccine development, it is unclear whether these microbicides can be delivered to everyone at risk for sustained delivery.

Targeted vaccine action against diseases such as hepatitis b generally does not affect morbidity. To maximize public health and menstrual benefits, general immunization against children and young animals may be necessary. This means that there is a need for a very high level of safety compared to the vaccines currently in widespread use (vaccines provided to children worldwide). These considerations favor the use of vaccines based on relatively small fractions of pathogens.

Of course, there is a much greater potential in vaccines that have been shown to be able to potentially induce relevant immune responses than vaccines that are not able to potentially induce relevant immune responses. Animal studies and laboratory measurements of human immune responses can be used to provide 'protective correlates' that accelerate other studies and developments.

Vaccines mainly use harmless forms of the pathogen or some of its components to induce a protective immune response, including one or both aspects of the immune system (arm): humoral and/or cell-mediated immunity. Humoral immunity is based on antibodies and the B cells that produce them. The antibody recognizes a specific target (usually a sub-portion of a protein of an infectious microorganism). The 'neutralizing' antibodies play an important role in combating infection, while cytotoxic T cells or CD8+ cells play a major role in cell-mediated immunity. Cytotoxic T cells are able to destroy most pathogen infected cells, which are identified by the presence of very small fragments of the pathogen protein displayed on the cell surface that bind to cellular proteins. Helper T cells (CD4 cells) recognize fragments of the pathogen displayed on the surface of specialized 'Antigen Presenting Cells (APCs)'. These cells produce proteins that activate B cells and/or cytotoxic T cells. When the immune system is activated by vaccination, memory T cells and sometimes memory B cells are generated. When encountered by the pathogen itself, these cells are capable of carrying out a rapid and effective immune response, thereby preventing infection and/or disease.

The main obstacle that has always hampered an effective mass immunization program (mass immunization program) is the inability to induce a suitable, protective immune response. For example, for vaccines against intracellular pathogens, cell-mediated immunity is required⁴Cell-mediated immunity as characterized by cytolytic T lymphocyte activity. Such reactions are particularly difficult to initiate, particularly via recombinant, soluble protein subunits. This drawback is due to the inability of these antigens to enter the machinery of the appropriate antigen processing pathway. Having improved understanding of the mechanisms behind this process and the recognition that delivery systems can affect the resulting immune response qualitatively and quantitatively, a great deal of research effort in this field has been witnessed in the last 10 years^4，8. The novel adjuvant formulations now comprise primarily a carrier for delivery of the antigen to antigen presenting cells.

Examples of carriers are generally particlesSuch as emulsions, microparticles, immunostimulating complexes and liposomes and microfluidized emulsion of squalene in water^4-8. The primary function of these delivery systems is to target the bound antigen to Antigen Presenting Cells (APCs), including macrophages and dendritic cells. Has been shown to be of a certain size (<5 microns) is effective in enhancing the immunogenicity of weak antigens in animal models. Two new adjuvants that have significant potential for the development of new vaccines include oil-in-water microemulsions and polymeric microparticles.

The parenteral route remains the most common route for antigen administration. However, the induction of an effective local immune response relies on the presence of air or food borne pathogens on mucosal surfaces, which can lead to the production of neutralizing antibodies. Furthermore, the products provided by syringes are inherently more expensive than products that can be ingested by mouth or, for example, nasal spray. The risk of needle reuse in underdeveloped countries is a complex factor.

Mucosal tissues encounter most of the antigens entering the host, and infections of the intestinal, respiratory and genito-urinary tracts are the most common cause of mortality and morbidity in humans². With regard to mucosal vaccination, it is possible to stimulate two aspects of the immune system and provide both humoral (antibodies) and cell-mediated responses (cytotoxic lymphocytes)¹. Despite the urgent need for an effective mucosal vaccine, its introduction is hampered by antigen degradation during transport to and low absorption by mucosa-associated lymphoid tissue (MALT). To overcome these problems, antigens for mucosal vaccine delivery can be combined or co-administered with adjuvants that simultaneously act as an effective delivery system^3，9。

Because each part of MALT has its own specific barrier, each route of administration requires its own vaccine delivery system. Oral immunization is the most complex as the antigen is degraded by the acidic environment in the stomach and by enzymes in the intestine. Furthermore, soluble antigens are not always efficiently taken up by M cells of Gut Associated Lymphoid Tissue (GALT). By entrapping the antigen in a particulate adjuvant, the antigen can be entrapped in the mucosal tissueProtect it from degradation and target it efficiently to M cells for uptake by M cells^10-13。

Nasal immunization is complicated primarily by the rapid clearance and low uptake of antigen by Nasal Associated Lymphoid Tissue (NALT). With respect to antigen transport across the nasal epithelial barrier, three pathways are available: co-administration of an antigen and an adjuvant that promotes the immune response while being absorbable by the nasal mucosa, co-administration of an antigen and an absorption enhancer or capture thereof into a particulate system to stimulate M cells also present in NALT, thereby internalizing the antigen^14，15。

Many strategies have been developed in the past to generate protective immune responses. These strategies include:

a) live attenuated vaccines are harmless to subjects as are defective pathogens such as nef-deficient viruses. In some cases it is not safe to use these types of vaccines.

b) Inactivated or "killed" vaccines. These vaccines still cannot be fully evaluated for their ability to protect against pathogens. For example, a challenge virus grown in a cell and matched to the host cell may or may not strip its envelope proteins during inactivation. This type of vaccine is exemplified in the development of more potent rabies viruses.

c) Recombinant subunit vaccines-or peptide vaccines-seek to stimulate antibodies against the pathogen by mimicking proteins on their surface (e.g., the proposed hepatitis b vaccine). Subunit vaccines studied to date are strain-specific and produce a lower antibody response. In recent years, intensive research into adjuvants has opened up new areas of research into envelope vaccines, some of which are capable of inducing neutralizing antibodies effective against a wide range of pathogen strains.

d) Recombinant vector vaccines use delivery systems to integrate genes or portions of genes of pathogens into established vaccines. The delivery system may comprise a live but harmless virus, such as canarypox virus. Vector vaccines have been shown to produce pathogen-specific cytotoxic T cell responses in subjects. These responses can be enhanced by priming with DNA vaccines.

e) DNA vaccines and replicons-include gene sequences that are injected into a subject to induce cells to express an antigen. In the case of replicons, these sequences are packaged in an outer shell of an unrelated virus.

f) Combination vaccine or 'prime and boost vaccine'. These vaccines provide strategies to combine two or more different vaccines to expand or enhance the immune response. Examples include vectors with an antigen that elicits a T cell response and a subunit that produces an antibody to boost the antigen, or vectors that deliver DNA followed by a gene or gene sequence that expresses the same gene or gene sequence. It is possible to provide two different vaccines at the same time, one of which acts more rapidly than the other. This will result in a 'prime-boost' effect from a single dose.

g) An important recent development in vaccine design is the use of synthetic genes to maximize their expression in human cells. This technology has been used in the design of HIV vaccines to enhance the immune response in animals and at least three vaccines using this technology are now entering early stage clinical trials. It is very important to understand that evidence of an immune response in a subject does not necessarily indicate that the vaccine prevents infection. Prevention of infection must be confirmed in animal and human trials. The above problems associated with vaccines have led to investigations associated with the present invention.

Fatty acid/nitrous oxide based technologies include unique submicron emulsion type formulations in which stable multivesicular structures or particles are formed. Among others, it is pointed out in the above-mentioned WO97/17978 that nitrous oxide is also a synthetically produced natural gas, also colloquially called "laughing gas", which has been used for many years as inhalation anesthetic and analgesic, in particular in dental medicine.

Nitrous oxide is known to be soluble in water and 1 liter of gas has been reported to dissolve in 1.5 liters of water at 20 ℃ and 2 atmospheres, see The Merck Index 10 th edition ed.p.6499.

Apart from the above-mentioned patents and patent applications, it seems that there is no mention in the literature that solutions of nitrous oxide may have any effect on humans or animals. To the best of the applicant's knowledge, no suggestion has been made that nitrous oxide could be used as an adjuvant with fatty acids to enhance the immune response against antigen-specific diseases.

It is known in the pharmaceutical field to formulate antigens in so-called lipid-based formulations. None of these lipid-based formulations is used with nitrous oxide, unlike the present invention where the combination of nitrous oxide and fatty acids and esters thereof form the basis of a microemulsion adjuvant system. As will be shown below, investigations confirm the essential role of nitrous oxide in the stimulation of immune responses. The combination of nitrous oxide and fatty acids as adjuvants for the vaccines of the invention described herein shows significant differences to fatty acid-based adjuvants alone.

Objects of the invention

It is an object of the present invention to provide adjuvants having the characteristic of enhancing the action of an antigen and to provide pharmaceutical preparations of these adjuvants and antigens which lead to an increased specific immune response, e.g. an increase in specific neutralizing antibodies, compared to preparations of known adjuvants comprising the same antigen.

Description of the invention

According to the present invention there is provided a method of enhancing a direct or subsequent immune response in a vaccine formulation, the method comprising the step of administering an antigen with an adjuvant comprising a solution of nitrous oxide gas formulated in a pharmaceutically acceptable carrier for the nitrous oxide gas and comprising at least one fatty acid or ester or other suitable derivative thereof selected from the group consisting of oleic acid, linolenic acid, alpha-linolenic acid, gamma-linolenic acid, arachidonic acid, eicosapentaenoic acid [ C20: 5 ω 3), docosahexaenoic acid [ C22: 6 omega 3), ricinoleic acid and derivatives thereof, selected from the group consisting of C1 to C6 alkyl esters thereof, glycerol-polyethylene glycol esters thereof and hydrogenated natural oils consisting essentially of ricinoleic acid-based oils, such as the reaction products of castor oil and ethylene oxide.

According to a further aspect of the present invention there is provided a pharmaceutical formulation suitable for use as a vaccine comprising an antigen, the formulation being formulated with an adjuvant comprising a solution of nitrous oxide (formulated in a pharmaceutically acceptable carrier solvent for the gas) and which comprises at least one fatty acid or ester or other suitable derivative thereof selected from the group consisting of oleic acid, linolenic acid, alpha-linolenic acid, gamma-linolenic acid, arachidonic acid, eicosapentaenoic acid [ C20: 5 ω 3), docosahexaenoic acid [ C22: 6 omega 3), ricinoleic acid and derivatives thereof selected from the group consisting of C1 to C6 alkyl esters thereof, glycerol-polyethylene glycol esters thereof and hydrogenated natural oils consisting essentially of ricinoleic acid-based oils, such as the reaction products of castor oil and ethylene oxide.

The antigen may be selected from all possible antigens.

In a preferred form of the invention, the antigen used in the method or formulation may comprise different types of antigens as defined herein, namely: any one or more antigens of peptides, inactivated viruses, inactivated bacteria and Virus Like Particles (VLPs), or any combination thereof.

The antigen may be any antigen suitable for eliciting an immune response against the pathogen of the disease or an infection by an agent selected from the group consisting of: BCG vaccine, cholera, Haemophilus B, meningococcus, pertussis, pneumococcus, tetanus, typhoid, diphtheria, hepatitis A, hepatitis B, influenza, measles, mumps, poliomyelitis, rabies, rubella, Tick-borne Encephalitis (Tick-borne Encephalitis), varicella, and yellow fever.

The invention therefore relates to vaccines of the following type:

bacterial vaccines

BCG vaccine

Bacillus calmette-guerin vaccine for skin

Cholera vaccine

Haemophilus B conjugate vaccine

Meningococcal polysaccharide vaccine

Pertussis vaccine

Pneumococcal polysaccharide vaccine

Tetanus vaccine

Typhoid (stratin Ty 21a) live vaccine (oral)

Typhoid polysaccharide vaccine

Typhoid vaccine

Bacterial toxins

Diphtheria bacterin

Tetanus vaccine

Viral vaccines

Hepatitis vaccine family (inactivated peptides, VLP)

Human papilloma virus Vaccine (VLP)

Inactivated influenza vaccine (intact virosomes)

Inactivated influenza vaccine (split virosome)

Inactivated influenza vaccine (surface antigen)

Live measles vaccine

Live vaccine for parotitis

Inactivated polio vaccine

Poliomyelitis live vaccine (oral)

Rabies vaccine

Rubella live vaccine

Inactivated tick encephalitis vaccine

Varicella live vaccine

Vaccine for yellow fever

Mixed vaccine

Diphtheria-tetanus vaccine

Diphtheria, tetanus and pertussis vaccines

Diphtheria, tetanus and pertussis (acellular component) vaccines

Diphtheria, tetanus and pertussis (acellular component) and haemophilus type B conjugate vaccines

Diphtheria, tetanus and pertussis (acellular component) and

diphtheria, tetanus and pertussis (acellular component) and

inactivated polio vaccine

Hepatitis A (inactivated) and hepatitis B (peptide) vaccines

Live measles, mumps and rubella vaccine

It is envisaged that the list will expand as new antigens or different forms of antigens and new combinations are developed.

Depending on the particular antigen, the adjuvant may comprise eicosapentaenoic acid [ C20: 5 ω 3] and/or docosahexaenoic acid [ C22: 6 ω 3] or a modification of these, to which at least one further component of the carrier as defined above is added.

The reaction product of hydrogenated natural oils, which consist predominantly of ricinoleic acid-based oils, with ethylene oxide is preferably produced from castor oil, the fatty acid fraction of which is known to consist predominantly of ricinoleic acid. The product can be modified to allow hydrogenation, ethylation, and the addition of groups such as polyethylene glycol. Many of these products are marketed by BASFA under the trade Specification of Cremophor grades.

The carrier solvent for the nitrous oxide gas may be any one of water or a pharmaceutically acceptable alcohol, ether, oil or polymer, such as polyethylene glycol or the like. The oil may be an organic or mineral oil. The organic oil may be a requisite oil based on long chain fatty acids (fatty acids having 14 to 22 carbon atoms). The oil may also be of natural or synthetic origin and, if of natural origin, it may be a vegetable or animal oil. As the vegetable oil, vegetable oil rich in gamma linolenic acid [ GLA ] is preferable, and as the animal oil, whipped cream (dairy cream) can be used.

In a preferred form of the invention, the solution is a nitrous oxide saturated aqueous solution. The oil component and the water component may be packaged separately and mixed directly just prior to application. The water is preferably deionized and purified to be free of microorganisms and endotoxins.

When the antigen-containing formulations are provided in the form of liquids for oral administration (including encapsulated liquids), or in the form of nasal or bronchial or pulmonary sprays, or in injectable formulations, these formulations may incorporate as part of the administration medium water or other liquid in which the nitrous oxide is dissolved, or in which the fatty acid or ester thereof is dissolved or suspended or emulsified with the antigen by formulating it with the antigen. Likewise, when the antigen is administered to a patient as a topical agent, buccal or vaginal cream, ointment, spray, lotion or as a suppository, the formulation used to make these creams, ointments, sprays, lotions or suppositories may be incorporated and the antigen formulated therewith integrated with some water or other liquid containing or saturated with carbon monoxide, long chain fatty acids or esters thereof and the antigen formulated therewith and in addition such additional excipients and carriers such as are conventionally used in the pharmaceutical trade in making these dosage forms.

The carrier solvent for nitrous oxide gas that may be present in the alternative formulation of the present invention is therefore substantially non-aqueous and comprises at least one fatty acid or ester thereof selected from the group consisting of oleic acid, linolenic acid, alpha-linolenic acid, gamma-linolenic acid, arachidonic acid, eicosapentaenoic acid [ C20: 5 ω 3), docosahexaenoic acid [ C22: 6 omega 3), ricinoleic acid and derivatives thereof, selected from the reaction products of hydrogenated or non-hydrogenated natural oils and ethylene oxide, consisting essentially of C1 to C6 alkyl esters thereof, glycerol-polyethylene glycol esters thereof and oils based on ricinoleic acid.

Formulations suitable for transdermal administration, whether as an injection, ointment, cream or lotion, or in the form of a skin patch providing a reservoir for the formulation, are preferred dosage forms of the invention.

The essential fatty acid component of the composition preferably comprises a mixture of esters of the fatty acids listed above. Thus, in the most preferred dosage form of the invention, the fatty acid component of the composition consists of a complex known as Vitamin F, and in this regard, the ester form of Vitamin F known as Vitamin F ethyl ester is preferably used. This product is commercially available from CLR Chemicals laboratory Rister Dr.Kurt Richter GmbH of Berlin, Germany under the product description of Vitamin F Ethyl Ester CLR 110000 Sh.L.U./g. The typical fatty acid partitioning of this product is as follows:

<C₁₆：0

<C_16.0：8.3％

C_18.0：3.5％

C_18.1：21.7％

C_18.2：34.8％

C_18.3：28.0％

C₁₈：1.6％

unknown substance: 2.1 percent of

More preferably, a salt known as eicosapentaenoic acid [ C20: 5 ω 3] and docosahexaenoic acid [ C22: 6 omega 3] long chain fatty acids. These product compositions are commercially available from Croda under the trade designation "incomega".

Microscopic analysis shows that the formulations of the antigens and adjuvants described herein form microstructures within or attached to which the antigens are contained in a stable form and from which the antigens are delivered to the site of action.

It is a further aspect of the invention that the formulation may be prepared so as to be suitable for mucosal administration, in particular for nasal administration. It therefore includes mucosal immunogenicity.

The present invention has not been proven by empirical work to be applicable to all antigens. However, no negative results have been observed so far for these antigens, regardless of the biological and chemical diversity of the antigens which have been investigated, which have been formulated with the aforementioned adjuvants of the invention and evaluated by different methods with regard to the expected increase in immunogenicity and different routes of administration. Based on these preliminary observations of products representing many kinds of these antigens, applicants therefore confidently expect that the present invention will find general use within the pathogen spectrum of some of the examples encompassed and shown below by these terms as defined herein throughout.

This is part of the applicant's current hypothesis by which an understanding of the present invention is sought and which is not intended to be limited to the stage in which the administration medium of the present invention is used to most effectively transport an adjuvanted antigen formulated therewith through the human or animal body, the adjuvant also playing an important role in transferring the antigen to cells of the immune system by a mechanism that has not been explained so far, thereby eliciting an effective immune response. In this regard, the applicants believe that the present invention will find general utility regardless of the variety of types, mechanisms and applications of the antigen.

Precondition assumptions for operating mechanisms

Mechanisms by which the present invention can achieve an effect of enhancing immunogenicity are currently being studied. Some observations about this aspect have been recorded above. In addition, some other possible interpretable prerequisite observations are recorded. The applicant again does not wish to be bound by any hypothetical explanation that may be presented at this point. However, it is noted that it is clear that long chain fatty acids and the nitrous oxide of the formulation of the invention or at least some of these components may form small stable vesicles or microsponges (hereinafter referred to as "fatty acid based particles") in the formulation during production of the formulation.

1. Structural features of the formulation of the article

The nitrous oxide and the unsaturated long chain fatty acid forming part of the administration vehicle are formulated by mixing with the specified antigen to form particles comprising said antigen. The particles comprise a lipid phase that is adjuvant in nature, (a) a synthetic particulate polymer and gas, nitrous oxide (which appears to activate or render effective the combination of these particles). Several of the characteristics of the particles contribute to their use as effective vaccine adjuvants:

the capture capacity and delivery efficiency of the particles;

complex (polyphenolic) nature of particles-the particles are well suited to capture a combination of hydrophilic and hydrophobic molecules, for example in the capture of antigens and immune modulatory molecules;

passive targeting of particles and affinity between particles and lipid structures such as lipid rafts in cell membranes; and

these particles appear to carry unsafe or toxic hazards.

These features and the results obtained with some antigens used with the particles are discussed below.

2. Composition, number and size of particles

The results obtained with the present invention and the literature concerning the field indicate that different routes of administration require different sized particles to deliver an effective vaccine. Furthermore, the type and amount of antigen loaded into the particles and the absorptive capacity of the particles are largely determined by the composition, number and size of the particles. The size of the various types of antigens (e.g. compared to peptides and viruses) is very different and needs to be adjusted (accommoded). The ability to repeatedly manipulate the size and number of particles is therefore very important. The relationship between size and number of particles in the present invention does not appear to be directly proportional but can be manipulated by:

the degree of saturation of nitrous oxide which has been shown to have an effect on the size and number of particles formed;

addition of various polyunsaturated fatty acids;

change in the proportion of fatty acids used;

modification of the fatty acid or alteration of a derivative used;

addition of biomolecules such as peptides; and

use of synthetic polymers of various sizes.

Two important observations have been made in this regard:

a) it was found that when the unsaturated long chain fatty acid used was 20 carbons or more, the microstructure formed was spherical with similar sub-compartments as seen in the sponge.

These structures are stable and we believe and observe that antigens (particularly peptides) are well-suited to these sub-compartments so that they are able to bind to specific epitopes or receptors on the surface of target cells. When unsaturated long chain fatty acids of 16 to 20 carbons are used, the microstructures are in the form of vesicles having a kinetic field that moves the autofluorescent particles around the vesicles.

3. Stability of

The particles appear to maintain structural integrity after 24 months at room temperature. Any loaded compound remains trapped during this time. This stability characteristic is believed to be very important in the use of vaccines.

4. Lack of cytotoxicity

The particles do not have significant cytotoxicity or toxicity at applicable concentrations, which has been shown in cell culture, animal and human studies.

5. Mechanism of action

5.1 Loading efficiency:

the high loading effect of the particles can be demonstrated by the high capture of Diphtheria Toxoid (DT) and inactivated rabies virus in the particles of the invention, as illustrated by laser confocal scanning microscopy (CLSM). The inactivated virus was a generous gift from the SA State vaccine entity, now the BIOVAC entity.

5.2 absorption and transport:

the particles of the invention appear to act as an absorption machine in the case of intranasal and oral administration and as a transport machine in the case of parenteral administration to deliver antigens to immunocompetent cells. Delivery efficiency is related to tissue penetration, cell adsorption, interactions between cell membranes and components of the particle, internalization of the particle by the cell, and intracellular stability.

5.3, releasing:

the result of the high delivery efficiency is the release of antigen not only at membrane sites but also at intracellular sites, resulting in an enhanced efficiency of the vaccine. The particles act synergistically with the antigen to achieve enhanced immunogenicity. The release rate of the particles is influenced by their composition. To combine priming and boosting factors for vaccination, extended and/or controlled release particles may be used.

5.4 flexibility and elasticity:

laser confocal scanning microscopy (CLSM) shows that the configuration of the particles can be altered by their environment. For example, as vesicles move across biological barriers such as circulating capillaries, changes in configuration that occur to accommodate extravascular conditions have been observed microscopically.

The contribution of the unsaturated long chain fatty acid component to the cellular integrity is its contribution to membrane maintenance. The nitrous oxide component of the particles of the present invention enhances membrane fluidity, which presumably has a positive effect on adsorption, absorption and other membrane binding processes. The compositions of the invention have been found to have a beneficial effect on the immunogenicity of antigens.

These beneficial effects are believed to be due to the dynamic nature of the fatty acid based particles.

5.5. Relationship between dynamic lipid (inter-lipid) vesicles:

it has been shown that both particle lipid-to-particle and particle/cell relationships do exist. The particles may be combined to continuously re-adjust their own size without compromising their stability. The membrane characteristics of these interactions optimize the movement of vesicles through the cell.

Despite the interrelationship between particles, particles have been shown to be stable in blood and body fluids for up to 5 hours.

Examples of the invention

Without limiting the scope of the invention, some examples will now be described to illustrate the invention.

Preparation 1

Preparation of FAA-1 for parenteral rabies vaccine and intranasal Diphtheria Toxoid (DT) vaccine

Step 1: buffer solutions available for specific antigens were saturated with nitrous oxide at ambient temperature using a high pressure vessel and a nebulizer. The buffer used in the case of rabies virus is Phosphate Buffered Saline (PBS), and in the case of DT for intranasal administration, distilled water is used.

Step 2: the following fatty acids were heated to 70 ℃: 21% oleic acid, 34% linolenic acid and 28% linoleic acid. These fatty acids are modified by esterification with a carboxyl-terminal vinyl group. The pegylated, hydrogenated fatty acid, ricinoleic acid (INCI name also known as PEG-n-hydrogenated castor oil) was heated to 80 ℃ and mixed with the first set of fatty acids at 70 ℃. The ratio of the first set of fatty acids to the latter fatty acids was 3: 1.

and step 3: the buffer was heated to 70 ℃ and mixed with the fatty acid mixture to a final concentration of 1.85%. This fatty acid mixture constitutes an adjuvant and is referred to herein as FAA-1(μ). The μ symbol represents a fine size range of particles, the particle size being between 2-5 μm, as determined by particle size analysis on a size sorter. FAA-1(n) was prepared from FAA-1(μ) by sonication (short term) or by increasing the ricinoleic acid component (long term).

And 4, step 4: capture of antigen in adjuvant: each antigen was captured in different adjuvant formulations by thorough mixing in Vibramix for 3 hours (rabies virus) or 4 hours (DT) at room temperature.

Preparation 2

Preparation of FAA-2 for parenteral hepatitis B vaccine

To the fatty acid contained in FAA-1 above

1. D 1-alpha-tocopherol as antioxidant

2. Additional ethylated fatty acids DHA (docosahexaenoic acid) and EPA (eicosapentaenoic acid). The preferred amount of the two fatty acids used in the present invention is 0.2%.

3. Capture of the hepatitis b peptide occurred by mixing in Vibramix for 30 minutes at ambient temperature. Stable particles of fairly uniform size, varying in size from 20nm to 50 μm, can be easily produced on a large scale. The size and shape of the particles can be reproducibly controlled. The use of FAA-1 and FAA-2 in animal studies, which is within the present invention, is described below. The following antigens (which, although not exhaustive of the scope of the vaccines to which the invention relates, are considered representative and thus illustrative) were used in cell and animal studies to confirm the invention:

toxoid as antigen (diphtheria)

Inactivated virus (rabies) as antigen

Protein/peptide as antigen (hepatitis B Hep B)

Some examples of studies and their results are described in the following examples:

example 1

FAA-1/DT vaccine induces systemic immune response after oral and intranasal administration, respectively Determination of the ability to answer

This example relates to the increase in immune response to diphtheria toxoid in animals, in particular in vaccines administered intranasally and orally, compared to the gold standard-aluminium hydroxide (alum) -based parenteral vaccine currently used.

1. The research aims are as follows:

the primary objective of this study was to evaluate when administered by parenteral route

a) PBS salt solution

b) Alum

Compared to the antigen in (b), the inventive FAA-derived formulation enhances the efficacy in systemic immune responses after oral and intranasal administration of the model antigen DT.

Desai et al¹⁶Chitosan particles that are shown to be taken up by M cells depend on the size of the particles and the hydrophobic/hydrophilic character of the particles. It has been determined that particles having a size in the nanometer range are more readily taken up by M cells located in peyer's patches. Second purpose is to determine

a) Whether the size of the FAA particles has any effect on the increase in immune response to DT and

b) whether the immune response by oral or intranasal administration is comparable to that obtained using alum as adjuvant using the parenteral route of administration.

2. Study background:

the parenteral route remains the most common route for administering antigens. Although the introduction of effective oral or intranasal vaccines increases patient compliance and reduces costs and the need for personnel eligible to administer the antigen, most vaccines still must be administered parenterally. Depending on the prevalence of AIDS, alternative routes of administration are beneficial, particularly in developing countries where nurses inject up to 170 children in school with the same needle during an immunization campaign, as evidenced, for example, by recent reports from these developing countries. Furthermore, mucosal vaccination induces local immune responses as well as systemic immune responses, in contrast to parenteral vaccination, which results in the induction of only systemic immune responses. After induction of an effective local immune response, air or food borne pathogens can be neutralized when they reach the mucosal surface.

The literature shows that various studies on the design and application of different adjuvants have been examined in mouse models. The model is thus described in detail. Few tested adjuvants were found to be comparable in efficacy to the currently used aluminum hydroxide adjuvanted parenteral vaccines. For example, Van der Lubben et al¹⁷The results indicated that chitosan microparticles were less effective in stimulating immune responses after intranasal administration, and only half of intranasally vaccinated mice showed immune responses.

3. General methodology of inoculation

3.1 intranasal inoculation of mice

The following groups of mice received intranasal administration of the formulation.

I. Positive control 1: intranasal administration of 40Lf DT in PBS

Positive control 2: 40Lf DT adsorbed to alum (aluminium hydroxide) administered by subcutaneous injection (registered dosage form).

FAA-1 (. mu.) and 40Lf DT

FAA-1(n) and 40Lf DT

V. negative control 1: FAA-1 (u) without DT

Negative control 2: FAA-1(n) without DT

In each group, 10 SPF balb/c female mice 6 weeks old were inoculated. Balb/c mice have been used previously in oral and intranasal vaccination studies using diphtheria toxoid as the antigen, and the results show that this animal model is suitable for these studies. To obtain a blood sample for the igg assay at week 4, half of the mice were sacrificed by decapitation (5). The other half of the mice were treated similarly at week 6. In each case, DT adsorbed to alum was injected subcutaneously as a positive control.

Vaccine administration:intranasal formulations were provided in a volume of 10 μ l/day (5 μ l per nostril). The total dose of DT was distributed over 3 consecutive days on weeks 1 and 3.

Collecting a sample:blood and nasal washes were collected in applicable containers after decapitation. No anticoagulant is present. Serum was prepared by centrifugation. The samples were stored at-20 ℃.

3.2 oral immunization in mice.

Mice in the following groups were inoculated during the study, 6 per group:

I. positive control 1: oral administration of 40Lf DT in PBS

Positive control 2: 40Lf DT adsorbed to bright vanadium (aluminium hydroxide) administered by subcutaneous injection. (registered forms)

FAA-1 (. mu.) with 40Lf DT

FAA-1(n) with 40Lf DT

V. negative control 1: FAA-1 (u) without DT

Negative control 2: FAA-1(n) without DT

Administration of the vaccine: the oral dosage formulation was fed intragastrically with a blunt needle. Mice were inoculated on three consecutive days in weeks 1 and 3. The doses were divided and the total volume fed was less than 300. mu.l.

Blood sampling:according to the literature, an immune response should be observable at week 6. Blood for the intranasal study was therefore collected at the end of week 6 to determine IgG titration. Samples were analyzed by antigen-specific enzyme-linked immunosorbent assay (ELISA).

4. Design, calculation and statistical evaluation

i) Experiment design: the experimental design for the intranasal and oral dosing study was a parallel design in which animals were arranged according to treatment groups, one treatment per experimental animal.

ii) number of groups of experimental animals the number of animals was consistent with that in the previously published study and was discussed and confirmed by Department of Statistics of the North-West University, SouthAfrica.

iii) random assignment of experimental animals in group: all mice were approximately the same age and body size. Animals were randomly grouped, 10 for intranasal study and 6 for oral study. Mice were placed in numbered containers. Treatments were randomly assigned to each animal group. The study was a non-blind study, as were the investigators preparing and administering the toxoid, collecting the samples and performing the analysis. All dosing and collection were instructed and checked by a qualified researcher. To reduce the chance of any possible bias, oral and nasal administration studies, including analyses, were performed by two different investigators. Background variables were reduced as much as possible by taking measures such as using a single batch of mice from a supplier, performing all analyses using the same laboratory equipment and obtaining blood samples and nasal washes of all mice on the same day. The oral and intranasal administration studies each included their own control (see negative control). Animals in the breeding facility are monitored and care taken to remain pathogen free.

iv) statistical methods: analysis of collected samples is performed by enzyme-linked immunosorbent assay (ELISA), a sensitive and specific assay widely used for the analysis of biological samples. In both oral and intranasal studies, IgG titers (systemic immune response) obtained with ELISA assay were statistically compared to controls with p < 0.05. The two treatments were compared to each other using the Pearson test.

5. Results

5.1 intranasal inoculation

Studies conducted with adjuvant formulation FAA-1, including binding of DT antigen prepared as described in formulation 1 of the invention, showed significant improvement in vaccine efficacy compared to the positive PBS-DT control formulation. The immune response obtained was comparable to that produced by alum-adjuvanted parenteral administration.

Table 1 reflects the results obtained for one dilution of the serial dilutions of the ELISA assay:

as expected, the negative control showed no local or systemic antibody response at any time. Positive controls (PBS saline solution and antigen) showed a small but observable systemic immune response as reflected by the determined IgG titers. Unfortunately, two of the FAA-1(n) group of mice died at week 4 due to the vaccination method. This death is independent of the particular adjuvant used.

Figure 1 illustrates the systemic immune response against DT as reflected by the titers of anti-DT neutralizing antibodies (found in blood after 4 and 6 weeks). Titers are shown on the Y-axis on a logarithmic scale.

Figure 2 illustrates the increase in production of a particular antibody caused by the formulation of DT antigen and adjuvant. The positive control PBS-DT was used as reference and divisor. The enhancement relative to the positive control was more than 2000-fold for alum and FAA- (μ) and more than 1000-fold for FAA-1(n) 4 weeks after the initial vaccination. By week 6, the enhancement of the reaction had decreased to just over 500 times in the case of alum and FAA-1(μ) and 200 times in the case of FAA-1 (n). These results demonstrate that the particle size plays a role in the degree of systemic immune response elicited following intranasal administration. The covariance between the alum-based and FAA-1(μ) -based immune responses was 676800.7901, whereas the covariance between the alum-based and FAA-1(n) -based immune responses was 377679. The statistical difference between the PBS-DT control and the three adjuvanted groups was below 0.05 in each case.

5.2 oral vaccination

The results of oral vaccination showed that the immune response observed was much lower than in the case of intranasal vaccination, but the enhancement was statistically significant in the case of alum and Faa-1(n), as shown in figure 3 below:

the results demonstrate the importance of the adjuvant particle size: in contrast to intranasal administration, the nano-sized particles of the present invention enhance the systemic immune response by 40-fold, but the micro-sized particles increase only by 2-fold. The increase in specific antibodies after oral administration is lower than after intranasal vaccination, presumably for some of the following reasons: enhancement was determined relative to the positive PBS-DT control, which was 2.9-fold higher in the oral study, and diphtheria toxoid was sensitive to low pH, which is the pH to which it was exposed in the stomach. However, the response of alum-based vaccines is also significantly lower than after intranasal administration and alum-based vaccines are administered parenterally and thus are not exposed to factors such as low pH. Thus, both studies indicate that intranasal vaccination results in a better immune response than oral vaccination for this particular toxoid. However, vaccination with alum-based and FAA-1(n) -based toxoids resulted in a statistically significant response (>10AU/ml) that complied with international health organization's international requirements for vaccine efficacy against diphtheria (levels >0.01AU/ml are protective in humans according to WHO).

6. And (4) conclusion:

FAA is administered by intranasal and oral routes of administration in the form of a solution comprising micro-or nanoparticles in which the toxoid is captured. No response was observed for unloaded FAA-1 (. mu.) or FAA-1(n) in the oral or intranasal studies. After vaccination with the positive control, PBS-DT, the immune response was low and did not comply with the requirements of the set standards. Furthermore, only 2 out of 5 mice showed some immune responses in the intranasal administration and 1 out of 6 mice showed some immune responses in the oral administration study after inoculation with PBS-DT.

These studies clearly demonstrate the role that adjuvants play in increasing the efficacy of vaccines. Parenteral alum-based vaccination results in a significant systemic immune response, similar to that described in the literature. Similarly, FAA-1(μ) and FAA1- (n) showed comparable and statistically significant systemic immune responses after intranasal administration, whereas FAA-1(n), but not FAA-1(μ), showed comparable and statistically significant immune responses after oral administration.

The results of these studies indicate that the present invention should be able to be vaccinated using the intranasal route instead of the parenteral route, thereby eliminating the need for needles and injections. The present invention will therefore contribute to a safer, cheaper and more environmentally benign vaccine.

Example 2

Efficacy of FAA-1 based rabies vaccine compared to that of commercially available vaccine Enhanced determination:

animal studies in example 1 show that the fatty acid adjuvant based FAA-1 described in the present invention is effective in enhancing specific immune responses (IgG antibodies) systemically against diphtheria after intranasal and oral administration. All mice vaccinated with DT bound to FAA-1 micron or nanoparticles produced sufficient neutralizing antibodies to protect them from diphtheria toxin.

This example relates to the enhancement of immune response to inactivated rabies virus used to formulate rabies vaccine with higher efficacy than that of the commercially available parenterally administered vaccines currently used. In animal studies, the effective delivery of rabies antigen by parenteral administration was examined using the rabies vaccine formulations shown above. Mice were challenged with either inactivated rabies virus (control) or FAA-conjugated inactivated virus by intraperitoneal or subcutaneous injection and their survival measured.

1. The research aims are as follows:

the first purpose of these studies was to determine the efficacy of the parenteral adjuvants of the invention. The above example 1 does not illustrate the efficacy of fatty acid based adjuvants for parenteral administration. These studies involve a direct comparison of the adjuvanticity of the adjuvant of the present invention with the commercially used adjuvant alum.

The second objective of the study included;

● example 1 describes a model system using model antigens. In this example, the antigens studied are antigens of the industry used to prepare commercial vaccines.

● one of the purposes was to increase the number of animals per study and to demonstrate the enhanced reproducibility of efficacy observed in animals.

● it is determined whether the number of administrations can be reduced in the invention.

● it was determined whether the fatty acid-based adjuvant itself promoted an immune response.

2. Background of the study

Rabies is acute, progressiveViral encephalitis affecting humans and animals^1-3. The pathogens are the neurotropic RNA viruses of the rhabdoviridae family, the lyssavirus genus, which have as hosts carnivore and bat species. Viral transmission is primarily transmitted through animal bites and once the virus is deposited in a peripheral wound, centripetal migration toward the central nervous system occurs. After viral replication, centrifugal diffusion to the major exit, salivary glands, creates a pathway for infection of the next host^1-3。

Despite continuing efforts at medical intervention, rabies remains an uncertain feature as an infectious disease with the highest fatality rate (dubiousdiction)³. Every year at least 50000 people die from rabies, more than ten million receive post-exposure vaccination against the disease, while more than 25 hundred million live in areas where rabies is endemic. These data are underestimated because areas are not easily accessible in some places, resulting in incomplete reporting. Preliminary monitoring indicated that one person died from the disease every 15 minutes, and another 300 persons were exposed to the disease. Once the symptoms of the disease occur, the infection of humans by animals suffering from rabies is almost certainly fatal. Although latency is on average 1-3 months, disease development has also been documented days or years after exposure. Children 5 to 15 years of age are at a particularly dangerous stage.

Rabies was found in all continents except antarctica. More than 99% of all people in asia, africa, and south america die after rabies; 30000 deaths were reported separately in india each year. From a global perspective, rabies is the most important viral infectious disease of animals given its widespread distribution, public health considerations, veterinary recommendations and economic burden⁵. The WHO encouraged carefully designed studies involving the feasibility and impact of integrating modern rabies vaccines in the early immunization program of infants and children (living in communities where rabies is a major health problem).

The most effective and cost-effective method of control is vaccination. Historically, many rabies vaccines are derived from infected brain tissue. Although relatively inexpensive, their efficacy levels vary. With the development of cell culture propagation, the efficacy and safety of rabies vaccines have been greatly improved in the last 20 years. However, in some countries, the only vaccine available is from the nervous tissue of sheep, goats or rodent babies.

WHO has signed a complete suspension of unpurified neural tissue vaccines for replacement with cell culture vaccines in developing countries by 2006. For these countries, the only approach may be to have the right to use inexpensive high quality cell culture vaccines. Standard cell culture vaccination protocols (e.g., Essen protocol) consist of administering the vaccine in the deltoid muscle of children or in the pre-thigh area on days 0, 3, 7, 14, and 28. A typical intradermal administration regimen (8-0-4-0-1-1) consists of administering the vaccine at 8 sites on day 0, followed by 4 intradermal inoculations on day 7 and one site immunization on days 28 and 90.

Vaccines for post-intradermal exposure prophylaxis have included human diploid cell vaccines, Vero cell rabies vaccine, purified chick embryo cell vaccine and purified duck embryo cell vaccine. After aventis pasteur, the south africa Biovac Institute (BI) is the second laboratory in the world to adapt rabies virus for growth in Human Diploid Cells (HDC). The HDC vaccine is considered to be "gold standard"⁶. It produces high serological titers in patients and does not contain foreign animal tissue, producing fewer adverse effects, but is very expensive to produce. A HDC vaccine with higher efficacy would reduce costs. HDC rabies vaccine is a weak antigen whose potency can be enhanced by the use of an adjuvant⁷. Although RVA (rabies vaccine adsorbed onto aluminum phosphate) is commercially available in the united states, most human rabies vaccines are formulated without adjuvant. However, recent studies comparing the effects of aluminum-and non-aluminum-containing rabies vaccines in animals have not shown to have the advantageous aspect of adjuvant presence⁸. However, the use of a suitable adjuvant may be the best way to increase the potency of HDC rabies vaccine, which is generally applicable to other inactivated vaccines as wellViral vaccine of⁷. In preliminary studies, the fatty acid-based adjuvant described together with this resulted in a significant increase in the level of protection against rabies (using HDC antigen) in mice (9-fold increase in antibody titer compared to non-adjuvanted rabies vaccine).

3. General methodology

Different preparations of inactivated rabies virus were subjected to different in vitro comparisons and animal studies. By using challenge experiments in mice rather than in vitro assays based on antibody content¹⁰Determining the potency of rabies vaccine (NIH assay)⁹) Because the in vitro assay cannot detect a reduction in the potency of rabies vaccine (partially degraded by heat). The evaluation of the potency of inactivated rabies vaccines has been the subject of many investigations; the most widely used is the NIH titer test, which produces variable results but is the only test currently accepted by the WHO¹². The animal experiment took 30 days to perform, including immunization of mice with test and reference antigens, followed by intracerebral challenge with a standard rabies vaccine strain. Rabies virus was cultured in lung fibroblasts and then inactivated by the SA State Vaccine Institute according to a novel method developed by Dr Woolf Katz. The method for culturing the virus does not form part of the present invention.

Generally, mice are injected intraperitoneally or subcutaneously with each of the two vaccines. A third group of mice injected with phosphate buffer served as controls. Mice were administered 6 dilutions (up to 1: 2500 dilutions) of each vaccine on the first day, 10 mice per dilution (60 mice in total for each vaccine), and vaccination was repeated on day 15. After another 14 days, mice were challenged with intracerebral injections of live rabies virus. Mice that are non-resistant or have a weak immune response against the virus die within a few days. Commonly used animal studies are described below:

3.1 preparation of sample:

the titer of the vaccine was determined in most studies using serial dilutions of 1/20, 1/100, 1/500 and 1/2500 of the vaccine. The titer of the vaccine is directly proportional to the number of mice protected from death at each serial dilution. The results of the three studies resulted in the design of the study described below. The animal study contained the following groups of mice:

I.positive control 1:a standard vaccine; two (2) vials of the standard vaccine were reconstituted in water to the original concentration (to provide the vaccine) and diluted in PBS according to the dilution series above and administered twice according to the standard mouse vaccination protocol.

II.Test vaccine 1:FAA-1 diluted in PBS: 2 vials of the standard vaccine were re-dissolved in FAA-1 and diluted in PBS in the dilution series above, and two injections were performed according to the standard mouse vaccination protocol.

III.Test vaccine 2:dilution of FAA-1 in PBS: two bottles of the standard vaccine were re-dissolved in FAA-1 and diluted in PBS according to the dilution series above, with one injection according to standard mouse vaccination methods.

IV.Test vaccine 3:two bottles of the standard vaccine were re-dissolved in FAA-1 and diluted in FAA-1 according to the dilution series above, injected once according to the standard mouse vaccination protocol.

V.Positive control 2:the aluminum-adjuvanted vaccine with high titer provided by the BIOVAC Institute was diluted serially according to the above dilution series and injected twice according to the standardized mouse vaccination method.

VI to VIII: not to receive vaccinationNegative control group。

3.2 administration and challenge of the vaccine:

mice were grouped, 10 mice per cage. Each group received a dilution of one of the vaccine formulations according to the NIH standard rabies vaccine test method and the vaccine administration protocol shown in table 2. Three groups of mice received no vaccine preparation and served as negative controls, and were titrated for Challenge Virus (CVS) on day 14. A total of 180 balb/c mice were used in this study: all groups contained 4 subgroups, each for 4 serial dilutions, inoculated and challenged with each of 10 balb/c mice, thus 32 mice per group.

Table 2: administration and challenge regimens

Group injection challenge

Day 0, day 7, day 14

I 0.5ml i.p. 0.5ml i.p CVS

II 0.5ml i.p. 0.5ml i.p CVS

III.5 ml i.p. non-injected CVS

IV 0.5ml i.p. non-injected CVS

V 0.5ml i.p. 0.5ml i.p CVS

VI-VIII non-injection CVS titration

After administration of the different vaccines, an intracerebral challenge with live infectious virus CVS was performed in all mice at the applicable dilution two weeks according to the NIH assay.

4. Results

Measurement of relative efficacy of adjuvant formulations using NIH assay (BI) was determined by survival of mice. Figure 4 below illustrates the survival of 4 different series of dilutions of mice in each group.

The relative efficacy of the groups determined according to WHO recommendations was expressed as IU/ml and is reflected in figure 5. Non-vaccinated mice and mice with a weak immune response died within 6 days (bars indicate groups, not survival of animals) (ii) a Most mice vaccinated with the existing aluminum-based vaccine died, however only two of the mice receiving the lowest dilution (1: 2500) of the FAA-based vaccine died; all experiments were performed according to WHO's instructions. (who. grades: Human Vaccines, (2004) [ Web:]http://www.who.int/ rabies/vaccines/human vaccines/en[ Date of use: 2004, 1 month, 27 days])。

For rabies vaccine WHO a relative titer of 2.5IU/ml is required.

5. Conclusion

Rabies vaccination has three major problems: repeated administration (5X), parenteral administration and most importantly the development of highly effective vaccines in cell culture. In the case of antigens prepared using cell culture, the fatty acid-based adjuvants described herein provide adjuvants that significantly increase the immunogenicity index.

The results of the previous studies and the results shown here illustrate that:

a) among the vaccines tested containing HDC antigens, only the vaccine containing the fatty acid adjuvant described in the present invention meets the criteria set by WHO;

b) a higher immunogenicity index of the vaccine may facilitate fewer vaccinations as shown by the group IV mice that received the FAA-1 based antigen diluted with FAA-1 only once, but still resulted in the highest survival and protection of the mice. Reducing the number of inoculations should limit costs and increase user friendliness.

c) Thus, FAA-based vaccines are far more effective than commercially available vaccines and show intrinsic immunostimulatory activity, and after the initial immunization of animals by the first vaccination with FAA-1 and antigen, they appear to be useful as boosters (boost) despite the absence of antigen.

d) FAA-based vaccines meet the international requirements of this particular vaccine in terms of efficacy and safety. The efficacy of FAA-based vaccines is on average 7 to 9 times higher than aluminium hydroxide-based vaccines.

e) The study was reproducible and proved to be effective in the study itself, and the results were statistically significant. Effective delivery of parenterally administered antigens was confirmed by similar animal studies using rabies vaccine formulations.

f) The role of FAA itself in enhancing the efficacy of a FAA-based rabies vaccine is determined by comparing the efficacy of the FAA-based rabies vaccine diluted with physiological buffer and the efficacy of the FAA-based vaccine diluted with FAA. The results show that the efficacy of the vaccine is significantly enhanced using dilutions of FAA, again suggesting that the FAA adjuvant has intrinsic immunostimulatory properties.

g) The effect of nitrous oxide was exemplified by freeze-drying and reconstitution studies-all nitrous oxide was removed under vacuum for freeze-drying. Reconstitution of the FAA vaccine resulted in a vaccine in which no dinitrogen monoxide was present. The results show that the efficacy of this vaccine is similar to that of alum adjuvanted vaccines, but not as good as the efficacy of FAA-based vaccines comprising nitrous oxide.

h) The possibility that the enhancement of vaccine efficacy is due to the inclusion of a heat source in the FAA-based formulation is similarly excluded by the fact that the freeze-dried FAA-based reconstituted rabies vaccine does not show such an enhancement. The reconstituted vaccine still contains a heat source, but the alteration of the FAA structure due to the lyophilization process results in a loss of vaccine efficacy.

Similar animal studies with various aspects were repeated 4 times. Formulated adjuvants contain components that are well recognized as being pharmaceutically safe. There is therefore an opportunity to use this Human Diploid Cell (HDC) culture antigen or other antigens in conjunction with an adjuvant to develop a high quality, low cost, immunologically effective rabies vaccine. With this adjuvant, the administration of the vaccine can also be extended to other routes of administration, thereby eliminating the use of parenteral routes.

Example 3

Proposed hepatitis B vaccine

It is estimated that 4 hundred million people are infected with chronic Hepatitis B Virus (HBV)¹³. Hepatitis B Virus (HBV) infection is caused by Small enveloped DNA viruses (Small enveloped DNA viruses) that infect the liver, causing immune-mediated necrosis and inflammation of hepatocytes. The infection may be acute or chronic. Clinical severity can range from (a) asymptomatic and complete remission to (b) symptoms with progressive and even fatal disease or (c) occasional fulminant hepatic failure. The course of infection appears to be determined by the host immune response. In most immunocompetent adults, acute infection results in acute hepatitis, followed by rapid clearance of the virus and generation of life-long immunity. However, if the infection occurs during neonatal or first year of birth, infection with HBV often becomes permanent. Chronic viral hepatitis infection leads to serious health hazards such as cirrhosis and hepatocellular carcinoma¹⁴. A prophylactic vaccine should allow the production of neutralizing antibodies effective in preventing infection in immunocompetent individuals. The present invention is used in animal studies to determine the applicability of FAA-based adjuvants in enhancing the efficacy of hepatitis b vaccines.

The surface antigen of hepatitis b (peptide) was captured in FAA and efficacy was measured by determining the specific antibody response obtained after vaccination with PBS and peptide (control) (currently used aluminium-based vaccine and FAA-based vaccine). Two weeks after inoculation, mice (10 animals/group) received a second inoculation. Blood was obtained from the tail of the animal after two weeks and the amount of antibody was determined. Mixing the raw materials in a ratio of 1: 1 dilution of antibodies obtained from FAA-based hepatitis b vaccinated mice to enable measurement. Figure 6 shows comparative efficacy of the proposed FAA-based vaccine against hepatitis b in mice. Figure 7 shows the relative titers of different vaccines obtained by using the results obtained with the peptide antigens alone as divisor. The Rec FAA was freeze dried and a FAA-based hepatitis vaccine was reconstituted.

The results thus show that trapping peptide antigens in FAA results in a 10-fold increase in b antibody production over the observed aluminum-based vaccine and 250-fold over the antigen without any adjuvant. As in the case of the proposed rabies vaccine, reconstituted or reconstituted FAA showed a similar response to the aluminum-based vaccine, without a significant increase.

Many modifications may be made to the present invention without departing from the spirit thereof.

Reference documents:

1.WHO.Rabies：Epidemiology，(2004)[Web：]http://www.who.int/rabies/epidemioloay/en/[Date of use：27Jan 2004]

2.O′Hagan，D.T.Drug Targets Infect.Di sord，1(2001)273-86.

3.Rupprecht，CE，Hanlon，A，and Hemachudha，T.The Lancet；Infectious Diseases，2(2002)101-9.

4.Singh J，Jain DC，Bhatia R，et al.Indian Pediatr 38(2001)1354-60.

5.Meltzer MI，Rupprecht CE.Pharmacoeconomics 14(1998)365-83.

6.Dreesen DW.Vaccine 15(Suppl)(1997)S2-S6.

7.Moingeon P，Haensler J，Lindberg A.Vaccine19(2001)4363-4372.

8.Lin Hand Perrin P.Zhonghua Shi Yan He Lin Chuang BingDu Xue Za Zhi 13(1999)133-5.

9.WHO Technical Report Series658，Annex2，Requirementsfor rabies vaccine for human use.WHO，Geneva，1981.

10.Hulskotte EGJ，Dings MEM，Norley SG and Osterhause ADME.Vaccine15(1997)1839-1845.11.Madhusudana SN，ShamsundarbR and Seetharamanc S.Int.J.Infect.Dis.8(2004)21-25.

12.Brarth R，Diderrich G and Weinmann E.NIHtest，aproblematic method for testing potency of inactivated rabiesvacci ne.Vaccine，1988，6：369-377.

13.Vaccines and Biologicals.WHO vaccine-preventablediseases：monitoring system；2002 global summary

14.Tiollais.P.，Pourcel，C.and Dejean，A.，The hepatitisB virus.Nature 1985.317，pp.489-495.

Claims (6)

1. Pharmaceutical preparation suitable for use as a vaccine comprising an antigen suitable for eliciting an immunogenic response against a pathogen of disease or against an infection caused by a pathogen, the pathogen being selected from diphtheria, hepatitis B and rabies, the vaccine being formulated with an adjuvant, wherein,

when the antigen is suitable for eliciting an immunogenic response against a pathogen of disease or against an infection caused by a pathogen, and the pathogen is selected from diphtheria and rabies, the adjuvant comprises: (1) a solution of nitrous oxide gas in a pharmaceutically acceptable carrier solvent for the gas, (2) a mixture of oleic, linolenic and linoleic acids modified by vinyl esterification of the carboxy terminus thereof, and (3) pegylated hydrogenated ricinoleic acid, and

when the antigen is suitable for eliciting an immunogenic response against hepatitis B, the adjuvant comprises: (1) a solution of nitrous oxide gas in a pharmaceutically acceptable carrier solvent for the gas, (2) a mixture of oleic, linolenic and linoleic acids modified by vinyl esterification of the carboxy terminus thereof, (3) pegylated hydrogenated ricinoleic acid, and (4) d1- α -tocopherol, ethylated docosahexaenoic acid and ethylated eicosapentaenoic acid.

2. A vaccine comprising the formulation of claim 1, wherein the formulation comprises one or more antigens that render it suitable for use as a vaccine selected from diphtheria, rabies and hepatitis b vaccines.

3. A pharmaceutical formulation suitable for use as a vaccine comprising an antigen selected from diphtheria toxoid, inactivated rabies virus and hepatitis b surface antigen, formulated with an adjuvant, wherein:

when the antigen is selected from diphtheria toxoid and inactivated rabies virus, the adjuvant comprises:

(1) a solution of nitrous oxide gas in a pharmaceutically acceptable carrier solvent for said gas, and

(2) a mixture of oleic, linoleic and linolenic acids modified by esterification of the vinyl group at its carboxyl terminus,

(3) pegylated hydrogenated ricinoleic acid; and

when the antigen is a hepatitis b surface antigen, the adjuvant comprises:

(1) a solution of nitrous oxide gas in a pharmaceutically acceptable carrier solvent for said gas,

(2) a mixture of oleic, linoleic and linolenic acids modified by esterification of the vinyl group at its carboxyl terminus,

(3) PEGylated hydrogenated ricinoleic acid, and

(4) d 1-alpha-tocopherol, ethylated docosahexaenoic acid and ethylated eicosapentaenoic acid.

4. The pharmaceutical formulation of claim 3, which is suitable for use as a diphtheria vaccine, the vaccine comprising an antigen in the form of diphtheria toxoid and being formulated with an adjuvant comprising:

a solution of nitrous oxide gas in a pharmaceutically acceptable carrier solvent for said gas,

a mixture of oleic, linoleic and linolenic acids modified by esterification of the vinyl group at the carboxyl terminus thereof, and

pegylated hydrogenated ricinoleic acid.

5. A pharmaceutical formulation according to claim 3, suitable for use as a rabies vaccine, the vaccine comprising an antigen in the form of an inactivated rabies virus and being formulated with an adjuvant comprising:

a solution of nitrous oxide gas in a pharmaceutically acceptable carrier solvent for said gas,

a mixture of oleic, linoleic and linolenic acids modified by esterification of the vinyl group at the carboxyl terminus thereof, and

pegylated hydrogenated ricinoleic acid.

6. A pharmaceutical formulation according to claim 3, suitable for use as a hepatitis b vaccine, said vaccine comprising an antigen in the form of a hepatitis b surface antigen, and being formulated with an adjuvant comprising:

a solution of nitrous oxide gas in a pharmaceutically acceptable carrier solvent for said gas,

a mixture of oleic, linoleic and linolenic acids modified by esterification of the vinyl group at its carboxyl terminus,

PEGylated hydrogenated ricinoleic acid, and

d 1-alpha-tocopherol, ethylated docosahexaenoic acid and ethylated eicosapentaenoic acid.