US20090010964A1 - Lipid and Nitrous Oxide Combination as Adjuvant for the Enhancement of the Efficacy of Vaccines - Google Patents

Lipid and Nitrous Oxide Combination as Adjuvant for the Enhancement of the Efficacy of Vaccines Download PDF

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US20090010964A1
US20090010964A1 US11/794,503 US79450306A US2009010964A1 US 20090010964 A1 US20090010964 A1 US 20090010964A1 US 79450306 A US79450306 A US 79450306A US 2009010964 A1 US2009010964 A1 US 2009010964A1
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vaccine
acid
antigen
formulation
adjuvant
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Anne Grobler
Abraham Frederik Kotze
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Exhausto Inc
North West University
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Assigned to NORTH-WEST UNIVERSITY reassignment NORTH-WEST UNIVERSITY ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: GROBLER, ANNE, KOTZE, ABRAHAM FREDERIK
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/05—Actinobacteria, e.g. Actinomyces, Streptomyces, Nocardia, Bifidobacterium, Gardnerella, Corynebacterium; Propionibacterium
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/205—Rhabdoviridae, e.g. rabies virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/29—Hepatitis virus
    • A61K39/292—Serum hepatitis virus, hepatitis B virus, e.g. Australia antigen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55544—Bacterial toxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates to pharmaceutical preparations (which expression is herein intended to include veterinary preparations) for use in the prevention of disease by inoculation against infective organisms afflicting the animal body (which expression is herein intended to include the human body).
  • compositions comprising the combination of nitrous oxide [N 2 O] and at least one fatty acid, or lower alkyl ester thereof, in a dermatologically acceptable carrier medium, are useful in the treatment of a variety of skin, muscle and joint disorders. It further disclosed therein that such combinations might beneficially also include additional active ingredients.
  • active ingredients are specifically mentioned in this regard: coal tar solution, collagen, nicotinamide, nicotinic acid, lanolin, vitamin E, methyl salicylate, arnica and H1-antagonist antihistamines of which only diphenhydramine chloride is specifically mentioned.
  • analgesic, anti-inflammatory and anti-pyretic drugs may be enhanced by administering such drugs in conjunction with a medium which comprises 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, and any of the C 1 to C 6 alkyl esters of such long chain fatty acids, mixtures of such acids and mixtures of such esters.
  • the medium may comprise the mixture known as Vitamin F Ethyl Ester and may optionally further comprise eicosapentaenoic acid [C20:5 ⁇ 3] and decosahexaenoic acid [C22:6 ⁇ 3].
  • WO 02/05850 there is disclosed that the effect of anti-infective drugs may be enhanced by formulation thereof in the same carrier medium.
  • WO 02/05849 it is disclosed that the same carrier medium may also beneficially be used for the transportation of nucleic acid compounds across cell membranes.
  • Antigens for use in forming vaccines are not amongst the active ingredients mentioned in the aforementioned patents and patent applications as being capable of being formulated with beneficial effect with the aid of the carrier medium therein disclosed.
  • vaccine as used herein is intended to have its extended meaning as compound(s) contributing in the prevention of infectious disease by any method or mechanism of priming of the body, and to include viral-based, peptide-based, bacterially-based, VLP-based, and synthetic compound-based formulations, but to exclude anti-infective agents used for the treatment of disease.
  • therapeutic vaccine is further intended to cover vaccines which serve to prevent and/or treat an existing infection by eliciting and/or enhancing a specific immune response to the infective agent without the use of antimicrobial, antifungal or antiviral agents.
  • the expression is hence intended to be understood in the wider sense of the immune response, namely all compounds that contribute in eliciting or enhancing an immune response against specific microscopic and sub microscopic organisms.
  • This term is further specifically intended to include all antigens or native and synthetic biologicals falling within class 26 (Biologicals) of the pharmacological classification employed in the Monthly Index of Medical Specialities (“MIMS”) published by Times Media in South Africa. It is thus intended to include:
  • anti-bacterial vaccines anti-fungal vaccines
  • anti-viral vaccines including anti-retroviral vaccines
  • anti-protozoa agents anti-protozoa agents
  • anti spirochaete vaccines anti-bacterial vaccines
  • anti-fungal vaccines anti-fungal vaccines
  • anti-viral vaccines including anti-retroviral vaccines
  • anti-protozoa agents anti-protozoa agents
  • anti spirochaete vaccines anti-bacterial vaccines
  • anti-fungal vaccines including anti-retroviral vaccines
  • anti-protozoa agents anti-protozoa agents
  • anti spirochaete vaccines anti-protozoa agents
  • the primary aim of vaccination is to prevent disease. Historically, vaccination is the only strategy that has ever led to the elimination of a viral disease, namely smallpox. While the biology of most pathogens is less favourable than smallpox to vaccine development, some vaccines do, to varying degrees, protect humans and animals against related pathogens. An indirect relationship has been observed for vaccine immunogenicity and safety. Human immune responses to synthetic and recombinant peptide vaccines administered with standard adjuvants tend to be poor; hence there is an urgent need for effective vaccine adjuvants to enhance the immunogenicity and immunostimulatory properties of vaccines, although even imperfect vaccines could deliver public health and economic benefits and provide further insights for prevention and treatment strategies. While microbicides may usefully extend prevention options and serve as valuable prototypes for vaccine development, it is not clear that these can be delivered sustainably to everyone at risk.
  • Vaccines primarily use a harmless form of a pathogen, or some component of it, to induce a protective immune response involving one or both arms of the immune system: humoral and/or cell-mediated immunity.
  • Humoral immunity is based on antibodies and the B cells that produce them. Antibodies recognise a specific target, usually a sub-part of a protein of the infective organism. ‘Neutralising’ antibodies play an important role in fighting off infections whereas 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, identified by the presence of very small fragments of pathogen proteins that are displayed on the cell surface, bound to cell proteins.
  • Helper T cells (CD4 cells) recognise fragments of pathogens, displayed on the surface of specialised ‘antigen presenting cells (APC)’. These produce proteins, which 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 produced. These cells enable a rapid and effective immune response when the pathogen itself is encountered, preventing infection and/or disease.
  • APC antigen presenting cells
  • Examples of vehicles are generally particulate e.g. emulsions, microparticles, iscoms and liposomes, and microfluidized squalene-in-water emulsions 4-8 .
  • the main function of such delivery systems is to target associated antigens to antigen presenting cells (APC), including macrophages and dendritic cells.
  • a number of adjuvants that are particulates of defined dimensions have been shown to be effective in enhancing the Immunogenicity of weak antigens in animal models.
  • Two novel adjuvants that possess significant potential for the development of new vaccines include an oil-in-water micro-emulsion and polymeric microparticles.
  • the parenteral route is still the most common route used for the administration of antigens.
  • the induction of an efficient local immune reaction is dependent on the presence of air or food born pathogens at the mucosal surfaces, which presence can result in the production of neutralising antibodies.
  • products given by syringe are inherently more expensive than those which can be taken by mouth or—for example—as a nasal spray.
  • the danger of re-use of needles in underdeveloped countries is a compounding factor.
  • each administration route needs its own vaccine delivery system.
  • Oral vaccination is firstly complicated due to degradation of antigens by both the acidic environment in the stomach and the enzymes in the gut.
  • the soluble antigens are not always taken up efficiently by the M-cells of the gut associated lymphoid tissue (GALT).
  • GALT gut associated lymphoid tissue
  • Nasal vaccination is mainly complicated by the fast clearance of the antigen and the low uptake by the nasal associated lymphoid tissue (NALT).
  • NALT nasal associated lymphoid tissue
  • three different approaches can be followed: co-administration of the antigen with an adjuvant that contributes to the immune response but is at the same time absorbable by the nasal mucosae, co-administration of the antigen with an absorption enhancer, or entrapment into a microparticulate system to stimulate M-cells, which are also present in NALT, to internalise the antigen 14,15 .
  • fatty acid/nitrous oxide-based technology comprise of a unique submicron emulsion type formulation within which stable vesicular structures or particles are formed. It was pointed out, inter alia, in WO97/17978 referred to above that nitrous oxide is a natural gas which is also produced synthetically, that it is also known by the trivial name “laughing gas”, and that it has been in use for many years as an inhalation anaesthetic and analgesic, particularly in dentistry.
  • Nitrous oxide is known to be soluble in water and it has been reported that at 20° C. and 2 atm pressure one litre of the gas dissolves in 1,5 litres of water, see The Merck Index 10th Ed. p. 6499.
  • nitrous oxide may be used in conjunction with fatty acids as an adjuvant to enhance the immune response against antigen-specific diseases.
  • lipid-based formulations None of these lipid-based formulations are used in combination with nitrous oxide, unlike the present invention in which the combination of nitrous oxide and fatty acids and esters thereof forms the basis of the micro-emulsion adjuvant system. As will be shown below, investigation confirmed the essential role of nitrous oxide in the stimulation of the immune response.
  • the combination of nitrous oxide and fatty acids as an adjuvant for vaccines according to the present invention as described herein shows significant differences to that based on the fatty acids only.
  • a method of enhancing direct or subsequent immunological responses to an antigen in a vaccine formulation comprising the step of administering the antigen with an adjuvant which adjuvant comprises a solution of nitrous oxide gas in a pharmaceutically acceptable carrier solvent for the gas and which adjuvant includes at least one fatty acid or ester or other suitable derivative thereof selected from the group consisting of oleic acid, linoleic acid, alpha-linolenic acid, gamma-linolenic acid, arachidonic acid, eicosapentaenoic acid [C20: 5 ⁇ 3], decosahexaenoic acid [C22: 6 ⁇ 3], ricinoleic acid and derivatives thereof selected from the group consisting of the C 1 to C 6 alkyl esters thereof, the glycerol-polyethylene glycol esters thereof and the reaction product of hydrogenated natural oils composed largely of ricinoleic acid based oils, such as castor oil with
  • a pharmaceutical preparation suitable for use as a vaccine comprising an antigen, which is formulated with an adjuvant which adjuvant comprises a solution of nitrous oxide in a pharmaceutically acceptable carrier solvent for the gas and which includes at least one fatty acid or ester or other suitable derivative thereof selected from the group consisting of oleic acid, linoleic acid, alpha-linolenic acid, gamma-linolenic acid, arachidonic acid, eicosapentaenoic acid [C20: 5 ⁇ 3], decosahexaenoic acid [C22: 6 ⁇ 3], ricinoleic acid and the derivatives thereof selected from the group consisting of the C1 to C6 alkyl esters thereof, the glycerol-polyethylene glycol esters thereof and the reaction product of hydrogenated natural oils composed largely of ricinoleic acid based oils, such as castor oil, with ethylene oxide.
  • an adjuvant which adjuvant comprises a solution of nitro
  • the antigen may be selected from the group comprising all possible antigens.
  • the antigens utilised in the method or formulation may comprise any one or more of the different types of antigens as herein defined, namely: peptides, inactivated viruses, inactivated bacteria and virus-like particles (VLPs) or any combination thereof.
  • the antigen may be any antigen suitable to elicit an immunogenic response against the causative agent of an ailment, or infection by an agent, selected from the group consisting of: Bacillus Calmette-Guérin Cholera, Haemophilus Type B, Meningococcal, Pertussis, Pneumococcal, Tetanus, Typhoid, Diphtheria, Hepatitis A, Hepatitis B, Influenza, Measles, Mumps, Poliomyelitis, Rabies, Rubella, Tick-borne Encephalitis, Varicella and Yellow Fever.
  • the invention is thus concerned with the following types of vaccines:
  • the adjuvant may include the eicosapentaenoic acid [C20: 5 ⁇ 3] and/or decosahexaenoic acid [C22: 6 ⁇ 3] or modifications of these as additional long chain fatty acids to at least one of the other components of the carrier medium defined above.
  • the reaction product of hydrogenated natural oils composed largely of ricinoleic acid based oils with ethylene oxide is preferably produced from castor oil of which the fatty acid content is known to be predominantly composed of ricinoleic acid.
  • the product may be modified as to the extent of hydrogenation, ethylation and the addition of groups such as polyethylene glycol. A range of such products is marketed by BASF under the trade description of Cremophor grades.
  • the carrier solvent for the nitrous oxide gas may be water or any of the pharmaceutically acceptable alcohols, ethers, oils or polymers such as a polyethylene glycol or the like.
  • the oil may be organic or mineral oil.
  • the organic oil may be an essential oil based on long chain fatty acids having between 14 and 22 carbon atoms in the fatty acid.
  • the oil may also be of either natural or synthetic origin and, if of natural origin, it may be either plant oil or animal oil. As plant oils those rich in gamma linolenic acid [GLA] are preferred and as animal oil dairy cream may be used.
  • the solution is an aqueous solution saturated with nitrous oxide.
  • the oil component and aqueous component may be separately packaged and only mixed directly before administration.
  • the water is deionised and purified to be free of microbes and endotoxins.
  • such formulation may incorporate, as part of the administration medium, water or acceptable other liquid into which the nitrous oxide is dissolved and in which the fatty acid(s) or ester(s) thereof is either dissolved or suspended or emulsified along with the antigen by being formulated therewith.
  • the formulation used in making up such cream, ointment, spray, lotion or suppository may incorporate, along with the antigen formulated therewith, a quantity of water or other liquid containing, and preferably saturated with, nitrous oxide, the long chain fatty acid(s) or ester(s) thereof and the antigen formulated therewith, and, further, such additional excipients and carriers as are conventionally used in the pharmaceutical trade in making up such dosage forms.
  • the carrier solvent for the nitrous oxide gas may thus in an alternative formulation according to the invention be essentially non-aqueous and composed of at least one fatty acid or ester thereof selected from the group consisting of oleic acid, linoleic acid, alpha-linolenic acid, gamma-linolenic acid, arachidonic acid, eicosapentaenoic acid [C20: 5 ⁇ 3], decosahexaenoic acid [C22: 6 ⁇ 3], and ricinoleic acid and derivatives thereof selected from the group consisting of the C1 to C6 alkyl esters thereof, the glycerol-polyethylene glycol esters thereof and the reaction product of hydrogenated or unhydrogenated natural oils composed largely of ricinoleic acid based oils with ethylene oxide.
  • a formulation suited to transdermal application whether as an injectable, ointment, cream or lotion or in the form of a skin patch providing a reservoir for the formulation is also a preferred form of the formulation according to the invention.
  • the essential fatty acid component of the composition preferably comprises a mixture of esters of the fatty acids listed above.
  • the fatty acid component of the composition is constituted by the complex known as Vitamin F and in this regard it is preferred to make use of the ester form of Vitamin F known as Vitamin F Ethyl Ester.
  • Vitamin F Ethyl Ester This product is commercially available under the trade description of Vitamin F Ethyl Ester CLR 110 000 Sh.L. U./g from CLR Chemicals Laboratorium Dr. Kurt Richter GmbH of Berlin, Germany.
  • the typical fatty acid distribution of this product is as follows:
  • Microscopical analyses showed that the formulation of the antigen with an adjuvant as herein described gives rise to the formation of microstructures, within which, or attached to which the antigen is contained in a stable form and from which it is delivered at the site of action.
  • the formulation may be prepared to be adapted for mucosal administration, and in particular for nasal administration. It will thereby include mucosal immunogenecity.
  • Nitrous oxide and the unsaturated long chain fatty acids forming part of the administration medium are formulated by being mixed with designated antigens to form the particles containing the antigen.
  • the particles contain a lipid phase which is adjuvantic in nature, (a) synthetic polymer(s) which is particulate nature, and a gas, nitrous oxide, which seems to activate or potentiate the combination of these particles.
  • the particles at applicable concentrations, have no apparent cytotoxicity or toxicity, as has been shown in cell culture, animal and human studies.
  • Stable particles of fairly homogeneous sizes ranging from 20 nm to 50 ⁇ m can be manufactured with ease 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 as it pertains to this invention is described below.
  • a toxoid as antigen diphtheria
  • An inactivated virus as antigen Ros
  • a protein/peptide as antigen Hep B
  • the primary objective of this study was to assess the efficacy of FAA-derived formulations of the present invention in enhancing the systemic immune response after oral and nasal administration of the model antigen DT when compared with antigen administered in
  • the parenteral route is still the most common route to administer antigens. Although the introduction of effective oral or nasal vaccines would improve patient compliance and diminish costs and the need for qualified personnel to administer antigens, most vaccines still have to be administered parenterally. In light of the AIDS epidemic an alternative administration route would be beneficial, especially in developing countries, as is evidenced for instance by recent reports from such developing countries that nurses have injected up to 170 school children with the same needle during immunisation campaigns. Furthermore, mucosal vaccination induces both a local as well as a systemic immune response in contrast to parenteral vaccination that results in the induction of a systemic immune response only. After the induction of an efficient local immune reaction, air or food born pathogens can be neutralized upon arrival at the mucosal surfaces.
  • mice received nasal administrations of the formulations.
  • mice 10 SPF balb/c female mice, aged 6 weeks, were vaccinated.
  • Balb/c mice have previously been used in oral and nasal vaccination studies with diphtheria toxoid as antigen and results showed that this animal model is suitable for these studies.
  • Half of the mice (5) were sacrificed by decapitation in order obtain blood samples for IgG determination in week 4. The other half was similarly treated in week 6.
  • the DT adsorbed to alum was injected subcutaneously as positive control.
  • Vaccine Administration Nasal formulations were given in a volume of 10 ⁇ l/day (5 ⁇ l in each nostril). The total dose of DT was distributed over three consecutive days in week 1 and 3. Sample collection: Blood and nasal washes were collected in applicable containers after decapitation. No anticoagulant was present. Serum was prepared by centrifugation. Samples were stored at ⁇ 20° C.
  • mice The following groups of 6 mice each were vaccinated during the study:
  • mice were of approximately the same age and were comparable in physical size. The animals were divided randomly into groups of 10 for the nasal study and 6 for the oral study. The mice were placed into numbered containers. Treatment was randomly assigned to each animal group. The study was not blind, as the same researcher prepared and administered the toxoid, collected the samples and performed the analyses.
  • Table 1 reflects the results obtained for one of the serial dilutions of the ELISA assay:
  • the negative controls showed no antibody response at any time, either locally or systemically.
  • the positive control PBS saline with antigen
  • the positive control showed a small but observable systemic immune response, as reflected by the determined IgG titre.
  • two of the mice in the week 4 FAA-1 (n) group died due to the inoculation procedure. The deaths were not related to the specific adjuvant used.
  • FIG. 1 illustrates the systemic immune response against DT as reflected by the titres of neutralizing antibodies against DT found in the blood after 4 and 6 weeks.
  • the titres are set out on the Y-axis on logarithmic scale.
  • FIG. 2 illustrates the enhancement of specific antibody production due to the formulation of the DT antigen with the adjuvants.
  • the positive control PBS-DT was used as reference and divider.
  • the enhancement relative to this positive control was >2000 fold for Alum and FAA-( ⁇ ) and >1000 fold for FAA-1 (n) 4 weeks after the initial inoculation.
  • the enhancement in response has decreased to just more than 500 fold in the case of Alum and FAA-1 ( ⁇ ) and 200 fold in the case of FAA-1 (n).
  • the covariance between the alum-based and FAA-1 ( ⁇ )-based immune response was 676800.7901, whereas that between the alum-based and FAA-1 (n) was 377679.
  • the statistical differences between the PBS-DT control and the three adjuvanted groups were less than 0.05 in each case.
  • the enhancement of the specific antibody was lower after oral administration than after nasal vaccination, presumably for some of the following reasons: the enhancement is determined relative to the positive PBS-DT control, which was 2.9 higher in the oral study, and the diphtheria toxoid is sensitive to low pH, to which it was exposed in the stomach.
  • the response of the Alum-based vaccine is also significantly lower than after nasal administration and was parenterally administered and thus not exposed to factors such as low pH.
  • the FAA was administered by the nasal and the oral route as solution, which contained either micro- or nano-particles within which the toxoid were entrapped. No response was observed for unloaded FAA-1 (p) or FAA-1 (n) in either the oral or nasal study. After vaccination with the positive control, PBS-DT, the immune response was low and did not comply with the set requirements. In addition, only two of the 5 mice in the nasal and 1 of the 6 mice in the oral study showed some immune response after vaccination with PBS-DT.
  • This example pertains to the enhancement of the immune response to inactivated rabies viruses for the formulation of a rabies vaccine with a higher efficacy than that of the currently used commercially available parenteral vaccine.
  • the efficient delivery of the rabies antigen by parenteral administration was investigated in animal studies, using the rabies vaccine formulation shown above. Mice was injected either intraperitoneally or subcutaneously with inactivated rabies virus (control), or FAA-associated inactivated virus, challenged and their survival measured.
  • Example 1 did not address the efficacy of the fatty acid-based adjuvant for parenteral administration.
  • These studies pertain to a direct comparison of the adjuvanticity of the adjuvant according to this invention and the commercially used adjuvant alum.
  • Rabies is an acute, progressive, incurable viral encephalitis that affects both humans and animals 1-3 .
  • the causative agents are neurotropic RNA viruses in the family Rhabdoviridae, genus Lyssavirus that use carnivores as well as bat species as hosts. Viral transmission occurs mainly via animal bite, and once the virus is deposited in peripheral wounds, centripetal passage occurs towards the central nervous system. After viral replication, there is centrifugal spread to major exit portals, the salivary glands, creating a channel for the infection of the next host 1-3 .
  • rabies retains the dubious distinction of being the infectious disease with the highest case-fatality ratio 3 . At least 50 000 people die from rabies annually, more than 10 million receive post-exposure vaccination against this disease, whilst more than 2.5 billion people live in regions where rabies is endemic 4 . These figures are an underestimation, as some of the endemic regions are not easily accessible, causing underreporting.
  • Rudimentary surveillance indicates that one person dies from the disease every 15 minutes, and more than 300 others are exposed. Infection of humans from rabid animals is almost invariably fatal once symptoms of disease occur. Although incubation periods average 1-3 months, disease occurrence days or years after exposure has been documented. Children aged 5-15 years are at particular risk.
  • Rabies is found on all continents except Antarctica. More than 99% of all human deaths from rabies occur in Asia, Africa and South America; India alone reports 30 000 deaths annually. From a global perspective, given the widespread distribution, public-health concerns, veterinary implications, and economic burdens, rabies is the most important viral zoonosis 5 .
  • the WHO encourages carefully designed studies on the feasibility and impact of incorporating modern rabies vaccines in the early immunization programmes of infants and children in communities where rabies is a major health problem.
  • rabies vaccines were derived from infected brain tissue. Although relatively cheap, they are of varying levels of efficacy. The potency and safety of rabies vaccines have greatly improved in the past 20 years with the development of cell-culture propagation. Nonetheless, in some countries the only available vaccine is of nerve-tissue origin from sheep, goats, or suckling rodents.
  • a standard cell-culture vaccination regimen (e.g. the Essen schedule) consists of a vaccine on days 0, 3, 7, 14, and 28 administered in the deltoid or in the anterior thigh for children.
  • a typical intradermal regimen (8-0-4-0-1-1) consists of vaccine administered at eight sites on day 0, followed by four intradermal inoculations on day 7, and vaccine at one site on days 28 and 90.
  • Vaccines used for intradermal postexposure prophylaxis have included human-diploid-cell vaccine, Vero-cell rabies vaccine, purified chicken-embryo-cell vaccine, and purified duck-embryo-cell vaccine.
  • Vero-cell rabies vaccine After Aventis Pasteur, the South African Biovac Institute (BI) is the second laboratory in the world to have adapted a rabies virus to grow in human diploid cell (HDC).
  • the HDC vaccine is considered to be the “gold standard 6 . It produces high serological titres in patients and contains no foreign animal tissue thus giving rise to fewer adverse effects, but is expensive to produce. HDC vaccines with a higher efficacy will reduce the cost.
  • HDC rabies vaccines are weak antigens and their potency may be enhanced by use of adjuvants 7 .
  • Most human rabies vaccines are not formulated with adjuvant although RVA (Rabies Vaccine Adsorbed onto aluminium phosphate) is available in the USA.
  • RVA Rabies Vaccine Adsorbed onto aluminium phosphate
  • a recent study in animals comparing the effect of aluminium adjuvant-containing and non-aluminium adjuvant-containing rabies vaccines showed no advantage to having adjuvant presents. Nevertheless, use of appropriate adjuvants may be the best way of increasing the potency of HDC rabies vaccines as is commonly used for other inactivated viral vaccines 7 .
  • the fatty acid based adjuvant herewith described resulted in dramatically enhanced levels of protection (9-fold increase in antibody titre compared to unadjuvanted rabies vaccine) against rabies in mice, using the HDC antigen.
  • rabies vaccine potency was determined by use of challenge experiments in mice (NIH test 9 ), rather than in vitro tests based on antigen content 10 as in vitro assays are not able to detect the decrease in potency of rabies vaccines partially degraded by heat 11 .
  • Potency evaluation of inactivated rabies vaccines has been the subject of much investigation; the most widely used is the NIH potency test which gives variable results but is the only test currently accepted by the WHO 12 .
  • This animal test takes 30 days to perform and involves immunisation of mice with test and reference antigens followed by intracerebral challenge with a standard strain of rabies vaccine. Rabies viruses were cultured in lung fibroblasts and then inactivated by the SA State Vaccine Institute according to novel procedures developed by Dr Woolf Katz. The procedure for the culturing of the virus does not form part of the present invention.
  • mice were injected either intraperitoneally or subcutaneously with each of the two vaccines.
  • Six dilutions (up to 1:2500 dilution) of each of the vaccines were administered to 10 mice each (60 mice in total for each vaccine) on day 1, and the inoculation was repeated on day 15.
  • mice were challenged by intracerebral Injection of the live rabies virus. Mice with no resistance or weak immune response against the virus died within a couple of days.
  • a typical animal study is described below:
  • mice Serial dilutions of 1/20, 1/100, 1/500 and 1/2500 of the vaccine were used in most studies to determine the potency of the vaccine.
  • the potency of the vaccine is directly proportionate to the number of mice protected against death at each of the serial dilutions.
  • mice were divided into groups of 10 mice per cage. Each group received one of the dilutions of one of the vaccine preparations as per the standard described rabies vaccine testing procedure of the NIH and the vaccine administration schedule set out in table 2. Three groups of mice received no vaccine preparation and were used as negative control and for the titration of the 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 the 4 serial dilutions, for each of which 10 balb/c mice were vaccinated and challenged, thus 32 mice per group.
  • CVS challenge virus
  • the administrations of the different vaccines were followed by an intracerebral challenge with live virus after two weeks with applicable dilutions of live infective virus CVS in all of the mice, according to the NIH test.
  • FIG. 4 illustrates the survival of mice for the four different serial dilutions of each group.
  • the relative potencies determined for the groups are expressed as IU/ml according to the recommendations of the WHO and are reflected in FIG. 5 .
  • Unvaccinated mice and those with a poor immune response died within 6 days (the small bar indicates the group, not the survival of an animal); most of the mice that were inoculated with the current aluminium-based vaccine died, whereas only two of the mice that received the lowest dilution (1:2500) of FAA-based vaccine died. All experiments were performed according to the specifications of the WHO. (WHO. Rabies: Human Vaccines, (2004) [Web:] http://www.who.int/rabies/vaccines/human_vaccines/en [Date of use: 27 Jan. 2004]).
  • the WHO requires a relative potency of 2.5 IU/ml for a rabies vaccine.
  • Rabies vaccination presents with three major problems: the repeated dosage (5 ⁇ ), the parenteral administration and most of all the development of a cell-cultured high efficacy vaccine.
  • the fatty acid-based adjuvant described herewith provides an adjuvant with a significantly increased immunogenic index, using cell-culture prepared antigen.
  • the formulated adjuvant contains components that have been recognized as pharmaceutically safe. There is thus an opportunity to use this human diploid cell (HDC) culture antigen or other antigens in concert with the adjuvant to develop a high quality, low-cost, immunologically effective rabies vaccine. Using this adjuvant, administration of the vaccine may also be expanded to other administration routes, eliminating the use of the parenteral route altogether.
  • HDC human diploid cell
  • Hepatitis B virus (HBV) 13 An estimated 400 million people are chronically infected with the hepatitis B virus (HBV) 13 .
  • Hepatitis B virus (HBV) infection is caused by a small enveloped DNA virus that infects the liver, causing immune mediated hepatocellular necrosis and inflammation.
  • the infection can be either acute or chronic.
  • Clinical severity may range from (a) asymptomatic and completely resolving to (b) symptomatic with progressive and even fatal illness or (c) occasional fulminant hepatic failure.
  • the course of the infection appears to be determined by the host's immune response. In most immunocompetent adults, acute infection leads to an acute hepatitis followed by rapid clearance of the virus and the development of lifelong immunity.
  • a surface antigen (peptide) of hepatitis B was entrapped in FAA and the efficacy was measured by assaying the specific antibody response obtained after inoculation with PBS with peptide (control), the currently used alum-based vaccine and the FAA-based vaccine.
  • the mice (10 animals/group) received a second inoculation. Blood was obtained from the tails of the animals two weeks later, and the number of antibodies determined.
  • the antibodies obtained from FAA-based Hepatitis B inoculated mice were diluted 1:1 to enable measurement.
  • FIG. 6 below illustrates the comparative efficacy of the proposed FAA-based vaccine against hepatitis B in mice.
  • FIG. 7 shows the relative potency of the different vaccines, using the results obtained with the peptide antigen alone as divider. Rec FAA is the freeze-dried and reconstituted FAA-based hepatitis vaccine.

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WO2019136038A1 (en) 2018-01-02 2019-07-11 Khloris Biosciences, Inc. Ipsc-based vaccine as a prophylactic and therapeutic treatment for cancer
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US9585955B2 (en) 2005-01-28 2017-03-07 North-West University Lipid and nitrous oxide combination as adjuvant for the enhancement of the efficacy of vaccines
EP3026059A1 (en) 2014-10-28 2016-06-01 ADMA Biologics, Inc. Compositions and methods for the treatment of immunodeficiency
EP4233903A2 (en) 2014-10-28 2023-08-30 ADMA BioManufacturing, LLC Compositions and methods for the treatment of immunodeficiency
EP3375789A1 (en) 2017-03-15 2018-09-19 ADMA Biologics, Inc. Anti-pneumococcal hyperimmune globulin for the treatment and prevention of pneumococcal infection
US10259865B2 (en) 2017-03-15 2019-04-16 Adma Biologics, Inc. Anti-pneumococcal hyperimmune globulin for the treatment and prevention of pneumococcal infection
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WO2022256277A1 (en) 2021-06-01 2022-12-08 Khloris Biosciences, Inc. Induced pluripotent stem cell-based cancer vaccines

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