Patent Application
20190224311
The present invention provides adjuvants for use with antigens and vaccines. The adjuvants described herein comprise sialic acid binding molecules and are useful as mucosal adjuvants where they can be used to ensure not only a subject mucosally-administered an antigen raises a strong and suitable mucosal immune response, but that that subject also raises a systemic immune response to the same antigen.
The majority of vaccines in use today are delivered by injection and the resulting systemic immunity represents an important aspect of the host's armoury against pathogens; however, this route is less effective in inducing an immune response at mucosal surfaces. A mucosal immune response is desirable as vaccines often target pathogens that enter their host through the mucosal surfaces. For example, a number of pathogens that cause or contribute to respiratory diseases, including for example influenza, attach to and ultimately enter host cells via receptors present on the surface of cells of the mucosal membranes. Mucosal immune responses (which in humans comprise an IgA response) therefore represent a first line defence against these pathogens and thus vaccines, which induce protective mucosal immune responses, are desirable.
The advantages of a mucosal vaccine over vaccines administered by injection include, for example, ease of administration, a non-invasive nature and the ability to induce immunity across a large tissue surface area.
While systemic and mucosal immunity are important in their own right, a truly effective and protective immune response should comprise both mucosal and systemic immune responses. In humans, the aim might be to induce IgA, IgM and IgG immune responses via a single vaccine and a single route of administration.
However, while vaccines delivered by injection often don't raise sufficient (or indeed any) mucosal immune response, vaccines that are delivered mucosally may not induce a sufficient systemic immune response.
Adjuvants can be used to augment, improve, expand and/or modify the immune response raised by a vaccine. For example by combining a vaccine composition (which may comprise one or more antigens) with an adjuvant, it is possible to induce a better (more protective) immune response and perhaps even an immune response which extends through multiple tissues, systems and sites—for example an immune response which comprises both a systemic (blood borne) and a mucosal immune response. Any improvement in the immune response induced by a vaccine combined with an adjuvant would be in comparison to the level or extent of immunity induced by the vaccine alone (i.e. without adjuvant).
There is a need for adjuvants, which can be combined with vaccines to ensure that the vaccines deliver the required immune response. There is a further need for adjuvants, which can be administered with mucosal vaccines to ensure that the vaccine induces not only a local mucosal immune response, but also a systemic immune response.
Disclosed herein are novel adjuvants, which may be used in combination with one or more antigens to augment, modulate or enhance a host immune response to the one or more antigens. It should be noted that while the adjuvants disclosed herein can be combined with any type of antigen, the adjuvants, which are the subject of this disclosure are especially for use with antigens that are to be administered mucosally. Accordingly, the adjuvants of this invention may be referred to hereinafter as “mucosal adjuvants”.
It should be understood that throughout this specification, the terms “comprise”, “comprising” and/or “comprises” is/are used to denote aspects and embodiments of this invention that “comprise” a particular feature or features. It should be understood that this/these terms may also encompass aspects and/or embodiments which “consist essentially of” or “consist of” the relevant feature or features.
The adjuvants disclosed herein comprise compounds, which exhibit an ability to (or are capable of) binding sialic acid and/or moieties (for example cell receptors and other molecules) comprising the same. Compounds of this type shall be referred to hereinafter as sialic acid binding molecules.
Thus, disclosed herein are adjuvants, for example mucosal adjuvants, which comprise, consist essentially of, or consist of, sialic acid binding molecules.
Sialic acid binding molecules with utility as adjuvants may otherwise be used to neutralise or prevent infection by pathogens that are able to bind sialic acid and/or which exploit sialic acid containing receptors on the surface of host cells. For example, respiratory pathogens such as viruses belonging to the Orthomyxoviridae or Paramyxoviridae families and certain Streptococcus bacteria exploit the presence of sialic acid in cell surface receptors as a means to bind and gain entry to specific cell types in a variety of mammalian tissues. Without wishing to be bound by theory, a molecule which binds to sialic acid may interfere with and/or inhibit, prevent and/or block the interaction between a pathogen and a sialic acid moiety and/or a cell surface receptor containing sialic acid; the sialic acid binding molecule may therefore prevent the pathogen from binding and/or adhering. Thus compounds, for example proteins and peptides, which bind to sialic acid moieties and/or to molecules (for example cell surface receptors) comprising the same, may therefore be used in the treatment and/or prevention of diseases caused or contributed to by pathogens which exploit cell surface sialic acid containing receptors.
The inventors have now discovered that in addition to binding to sialic acid and interfering with or blocking, preventing and/or inhibiting the interaction between a pathogen and, for example, a sialic acid containing molecule or cell surface receptor, sialic acid binding molecules possess adjuvant properties and when administered together with one or more antigens, has an adjuvant effect.
Compounds with adjuvant properties (i.e. adjuvants) are often added to vaccines. Many different types of vaccine are currently in use and many of these are formulated/combined or administered (concurrently or separately) with adjuvants to improve, augment or modify the nature of the immune response raised against the antigen component of the vaccine. In some cases, an adjuvant is exploited as a means to improve an immune response in a human or animal host. For example an adjuvant may be used to improve the immune response raised by low (sub-optimal) doses of antigen. In this regard, an adjuvant/adjuvant composition of this invention may be combined with sub-optimal doses of a vaccine or antigen—the vaccine or antigen serving to increase the immune response raised by the sub-optimal antigen dose. When administered together or with an adjuvant or adjuvant composition of this invention, the immune response raised by a sub-optimal vaccine/antigen dose, may be comparable to an immune response raised by administration of an optimal dose of vaccine/antigen alone (i.e. without adjuvant). Adjuvants are particularly useful when the antigen component of a vaccine is poorly or insufficiently immunogenic in its own right. Antigens which are sufficiently immunogenic may not require to be administered in combination with an adjuvant as administration of the antigen alone raises or induces, for example, a protective and/or neutralising immune response (a “neutralising immune response” comprising, for example, antibodies which bind to certain parts of a pathogen and to prevent them from binding their usual receptors and a “protective immune response” comprising antibodies which are able to neutralise a pathogen and resolve an infection but possibly also lead to the opsonisation and/or clearance/destruction of a pathogen).
However, certain types of antigen may be poorly or insufficiently immunogenic. For example, certain proteinaceous antigens, in particular those that contain concealed epitopes or domains, which mimic certain host (or self) peptides and/or protein domains, may exhibit an insufficient level of immunogenicity when administered. Additionally, antigens that comprise significant amounts of carbohydrate material (or which consist (essentially) of carbohydrate material) might be less immunogenic than antigens, which are more proteinaceous in nature. Where the antigen (carbohydrate in nature or otherwise) is not sufficiently immunogenic in its own right, an adjuvant might be used to improve, augment or modify the immune response raised or induced upon administration of the antigen to a host.
In other instances, an adjuvant might be used to ensure that an immune response is raised in a specific tissue or at a specific site. One of skill will appreciate that whereas administration of an antigen might easily induce a local immune response—the locality of the immune response being restricted to the site of administration, that immune response may not be widespread in the host. For example, an antigen administered to a mucosal tissue may only raise a mucosal immune response. The induced mucosal immune response may offer adequate protection to the host throughout the mucosal surfaces/tissues, but the induced immune response may not also embrace a systemic immune response. In such cases an adjuvant may be used to ensure that any immune response raised is not merely a local immune response and confined to the site or tissue to which the antigen is administered. By way of example, an adjuvant may be used with an antigen that is to be administered mucosally for the purpose of inducing not only a local mucosal immune response, but also a systemic response.
In humans, certain antigens administered mucosally might only induce an IgA response (IgA being the predominant antibody isotype of the mucous membranes). When administered mucosally and in combination with an adjuvant, that same antigen might also induce a systemic IgM and/or IgG based response.
By combining antigens with the sialic acid binding molecule-based adjuvants of this invention, and administering the combination mucosally (for example intranasally), it is possible to ensure the induction not only of a mucosal immune response to the antigen component of the vaccine but also immune responses to the antigen in or at other tissues, systems and sites. For example the combined mucosal administration of an antigen and a sialic acid binding molecule-based adjuvant may result not only in a mucosal immune response to the antigen but also a systemic immune response to the same antigen.
In view of the above, the present disclosure provides an adjuvant comprising a molecule capable of binding sialic acid (a “sialic acid binding molecule”).
The adjuvants of this disclosure may comprise one or more different types of sialic acid binding molecule.
In a second aspect, the disclosure provides the use of sialic acid binding molecules as adjuvants.
In a third aspect, the disclosure provides an adjuvant composition comprising a sialic acid binding molecule, any necessary additional adjuvant components including, for example, other adjuvants approved for mucosal administration. Suitable “other” adjuvants will be known to one of skill in this field and may include, for example; microbial toxin/toxoid based adjuvants, TLR ligands, non-TLR immunostimulants, novel small molecules, oil-based adjuvants and organic adjuvants. An adjuvant composition of this disclosure may further comprise a pharmaceutically or physiologically acceptable excipient, diluent, solvent or carrier.
As stated, the sialic acid binding molecule-based adjuvants described herein may be added to vaccines and/or vaccine compositions. As such, the present invention provides vaccines (for human and/or animal use), comprising or formulated with, a sialic acid binding molecule-based adjuvant of this invention. The vaccine may be further formulated for mucosal administration. For example, the vaccine may be formulated for administration to a mucosal surface. For example, the vaccine may be formulated for oral, intranasal, vaginal and/or rectal delivery. Thus the invention provides sialic acid binding molecule-based adjuvant supplemented mucosal vaccines. Without wishing to be bound by theory, the sialic acid binding protein-based adjuvants disclosed herein may be added to a vaccine or vaccine composition as a means to improve, augment or modify the effect of the vaccine/vaccine composition when it is administered. For example, a vaccine to which an adjuvant of this disclosure is added may induce a better (for example more protective and/or more widespread) immune response to the antigen component thereof as compared to the immune response raised by the same vaccine administered without the adjuvant.
Mucosal vaccines (that is, vaccines that are administered mucosally and which raise a mucosal immune response) represent an advantage over other, perhaps parenterally administered vaccines. In addition to the fact that mucosal vaccines are sometimes easier to administer than vaccines administered by other routes, a strong, effective and/or protective mucosal immune response is often desirable as it represents a first line defence against a number of pathogens, which enter the host via the host mucosal surfaces. Parenterally administered vaccines often do not induce a sufficient level of (or indeed any level of) mucosal immunity. Further, mucosal vaccines often only induce local, mucosal immune responses and while a mucosal response is often effective against those fungal, bacterial and/or viral pathogens, which colonise and invade through or via a mucosal surface (including, for example those collectively referred to as respiratory pathogens), it is often preferable that vaccines raise both local and systemic immune responses (for example mucosal and systemic immune responses).
The provision of an adjuvant, which can be combined with a mucosal vaccine, allows the user to benefit from the convenience of a mucosal route of administration so as to generate a strong mucosal immune response but with the confidence that the vaccine (although only administered mucosally) will also raise a systemic immune response.
For example, a mucosal vaccine comprising an antigen or antigens from one or more pathogens/respiratory pathogen(s) may be supplemented with a sialic acid-based adjuvant of this invention. The respiratory pathogen(s) from which the antigen(s) is/are derived may cause or contribute to a disease or condition which affects some part of the respiratory system including, for example the respiratory mucosal surfaces and/or the lungs. In this way, the vaccine will induce not only a mucosal (IgA (in humans) based) immune response, but also a systemic (IgM and/or IgG (in humans) based) immune response. One of skill will appreciate that while a mucosal response may be sufficiently protective, a systemic immune response offers an additional level of protection, which can help resolve infections that manage to get beyond the mucosal surfaces and into deeper tissues and/or systems.
Examples of pathogens (for example respiratory pathogens) from which antigen(s) might be derived (for use in vaccines) and against which mucosal and/or systemic immune responses might be effective, include (but are not limited to) viruses belonging to the group known as the ‘retroviruses’ (including, for example HIV), the Picornavirales (including Rhinovirus species), Flaviviridae, Coronaviridae, Adenoviridae (including Adenovirus species), Orthomyxoviridae (including influenza viruses A, B and C) or Paramyxoviridae families, certain bacteria including, for example those species belonging to the Streptococcus, Staphylococcus, Corynebacterium, Klebsiella, Pseudomonas, Haemophilus, Legionella, Mycobacterium, and Neisseria genera and certain fungal pathogens including, for example, those of the Aspergillus genus. As stated, the adjuvants of this disclosure may be used in combination with any antigens (whether or not derived from the above list of pathogens) and thus this list of respiratory pathogens should not be construed as in anyway limiting. In effect, the adjuvants described herein may be used in combination with any antigen that can be administered mucosally.
The term “antigens” may also embrace cancer or tumour antigens. Thus the adjuvants of this invention may be used in combination with one or more cancer or tumour antigens.
As described in more detail later, the adjuvants described herein may be administered together or concurrently with, an antigen(s) or a vaccine. Concurrent administration may involve administering the antigen(s)/vaccine and adjuvant together and at the same time (co-administration) or together in the form of a conjugate or fusion. For example, the adjuvant may be fused or conjugated to the antigen (or alternatively, the antigen(s) may be fused or conjugated to the adjuvant). Where the sialic acid binding molecule based adjuvant is fused to the antigen, the fusion may be at the N- and/or C-terminal end of the antigen. Alternatively (or additionally) the adjuvant may be placed somewhere within the antigen. Where there are multiple antigens, one or more of the antigens may be provided as a fusion and the fusions (in terms of where the fusion between the antigen and adjuvant occurs (at the N- and/or C-terminal end of the antigen or internally within the antigen)) may be the same or different. The adjuvants disclosed herein comprise sialic acid binding molecules and the term “sialic acid” embraces all forms of N- or O-substituted neuraminic acid and includes all synthetic, naturally occurring and/or modified forms thereof. Sialic acids may be found as components of cell surface molecules, glycoproteins and glycolipids. Most often, sialic acids are present at the end (terminal regions) of sugar chains connected to cell membranes and/or proteins. For example, some cells of the human upper respiratory tract comprise α-2,6-linked sialic acid receptors and other cells of the upper and lower respiratory tracts comprise α-2,3-linked sialic acid receptors. The sialic acid family encompasses a number (approximately 50) of derivatives that may result from acetylation, glycolylation, lactonisation and methylation at C4, C5, C7, C8 and C9. All such derivatives are to be embraced by the term “sialic acid”.
Furthermore, sialic acids are found linked α(2,3) or α(2,6) to Gal and GalNAc or α(2,8) or α(2,9) to another sialic acid. Accordingly, it is important to understand that while the term “sialic acid” is used throughout this specification, it encompasses all derivatives, analogues or variants (either naturally occurring or synthetically generated) thereof as well as monomers, dimers, trimers, oligomers, polymers or concatamers comprising the same.
Thus an adjuvant of this disclosure comprises a sialic acid binding molecule, which exhibits an affinity for sialic acid—including all forms of sialic acid described above and sialic acid present on the surface of mammalian cells.
It should be understood that molecules, which exhibit an affinity for sialic acid, bind to or otherwise couple to or associate with sialic acid moieties. Thus the term “sialic acid binding molecule” may further encompass any fragment of a whole sialic acid binding molecule, which retains an ability to bind to or otherwise couple or associate with sialic acid.
The adjuvants disclosed herein may comprise a single molecule capable of binding sialic acid (a monomeric or monovalent molecule, for example) or, alternatively, two or more sialic acid binding molecules (which may all be the same or different—a polymeric or multivalent molecule, for example).
The adjuvants may comprise one or more sialic acid binding molecules known as “carbohydrate binding modules” (CBMs). CBMs suitable for use as adjuvants may exhibit an affinity for sialic acid. Exemplary carbohydrate binding modules for use in this invention may comprise the sialic acid binding domain of Vibrio cholerae NanH sialidase (VcCBM) and/or the equivalent (or homologous) domain from Streptococcus pneumoniae NanA sialidase (SpCBM). Of course, similar or homologous sialic acid binding modules present in other organisms are to be encompassed within the scope of the term “CBM”. This may include, for example CBMs with a function and/or sequence which is homologous to any of the specific CBMs described herein.
Thus, this disclosure provides adjuvants, which comprise, consist essentially of or consist of one or more CBM(s).
The disclosure further provides an adjuvant or vaccine/vaccine composition comprising:
The disclosure further provides vaccines and vaccine compositions comprising one or more antigens and one or more adjuvants selected from the group of adjuvants which comprise, consist essentially of or consist of:
By way of example vaccines and vaccine compositions against, for example, forms of ‘flu’, diphtheria, Whooping cough, measles, mumps, rubella, polio, tuberculosis, meningitis, tetanus, yellow fever, rabies (to name a few) may be combined, used or administered with any of the adjuvants described herein. The adjuvants of this disclosure may also be combined or used with any mucosally administered vaccine, including, for example, those mucosally administered influenza vaccines.
An exemplary Vibrio cholerae NanH sialidase amino acid sequence is deposited under accession number A5F7A4 and is reproduced below as SEQ ID NO: 1 (781 amino acids).
The CBM region of SEQ ID NO: 1 is from amino acid residue 25 to 216—this sequence may be SEQ ID NO: 2.
An exemplary Streptococcus pneumoniae NanA sialidase amino acid sequence has been deposited under accession number P62575 and is reproduced below as SEQ ID NO: 3 (1035 amino acids).
The CBM region of SEQ ID NO: 3 is from amino acid residue 121 to 305—this sequence may be SEQ ID NO: 4.
As such, the adjuvants disclosed herein may comprise a protein or peptide having the sequence of SEQ ID NO: 1 or 2 or a sialic acid binding fragment thereof. For example, a useful adjuvant may comprise a proteinaceous moiety encoded by the sialic acid binding domain of the nanH gene (encoding sialidase) of V. cholerae or an equivalent or homologous gene present in another organism (for example the equivalent/homologous nanA sialidase gene of S. pneumoniae). For example, a sialic acid binding molecule of this invention may comprise from about residue 1, 5, 10, 15, 25 or 30 (i.e. from 1-30 or from any amino acid residue there between) to about residue 150, 175, 200, 210, 216, 220-781 (to any residue from 150 to 781 including any residue therebetween) of the V. cholerae sialidase molecule of SEQ ID NOS: 1 and 2. For example an adjuvant for use may comprise a peptide having a sequence corresponding to residue 25 to about residue 216 of SEQ ID NO: 1 above.
A further suitable adjuvant may comprise a protein or peptide having the sequence of SEQ ID NO: 3 or 4 or a sialic acid binding fragment thereof. For example, a useful adjuvant may comprise a proteinaceous moiety encoded by the sialic acid binding domain of the Streptococcus pneumoniae nanA gene (encoding sialidase). For example a sialic acid binding molecule of this invention may comprise from about residue 80, 90, 100, 110, 120, 121 to 130 (i.e. from any of about residues 80 to 130 including any residue therebetween) to about residue 250, 275, 300, 305, 310, 320-1035 (i.e. to any residue from about 250-1035 including to about any residue therebetween) of the S. pneumoniae sialidase molecule of SEQ ID NOS: 3 and 4. For example and adjuvant for use may comprise a peptide having a sequence corresponding to residue 121 to about residue 305 of SEQ ID NO: 3 above.
An adjuvant for use may comprise one or more CBMs. For example, suitable adjuvants may comprise single CBMs—for example, a single VcCBM or a single SpCBM. Alternatively, the adjuvants may comprise a plurality or multiple (i.e. two or more) CBMs. Sialic acid binding molecules, which comprise a plurality of CBMs may be termed “multivalent sialic acid binding molecules” or “multivalent CBMs”. A multivalent CBM may, for example, comprise two or more VcCBMs or two or more SpCBMs. A multivalient CBM may comprise a mixture of different CBMs, for example one or more VcCBMs with one or more SpCBMs. Thus an adjuvant disclosed herein may comprise a multivalent CBM.
For example, multivalent CBM molecules, (for example a Vc4CBM molecule) may be prepared as constructs comprising multiple CBMs linked by amino acid/peptide linkers. Each CBM (for example VcCBM) may be linked to another by, for example, peptides comprising 5, 10 or 15 amino acids. By way of example any one or more of the following peptides may be used to link two or more CBMs to produce a multivalent CBM:
An adjuvant for use may further comprise an oligomerisation domain. Suitable oligomerisation domains may exhibit an ability to self-associate to form multimer structures, for example trimers. An oligomerisation domain for use as an adjuvant of this disclosure may comprise any molecule with the above mentioned oligomerisation properties or any functional fragment thereof. For example one or more (for example two) sialic acid binding molecules (for example CBMs) may be bound, coupled or fused to an oligomerisation domain—the resulting sialic acid binding molecule/oligomerisation domain “fusion” may then be used (with one or more other such “fusions” as an adjuvant).
Suitable oligomerisation domains may be derived from, for example, Pseudomonas aeruginosa pseudaminidase. An exemplary Pseudomonas aeruginosa pseudaminidase sequence amino acid sequence has been deposited under accession number PAO579 and is reproduced below as SEQ ID NO: 5 (438 amino acids).
The oligomerisation domain of SEQ ID NO: 5 is from amino acid residue 333 to 438—this sequence may be SEQ ID NO: 6.
Thus an oligomerisation domain for use in an adjuvant of this disclosure may comprise from about residue 250, 275, 300, 310, 320, 333, 340 to 350 (i.e. from about residue 250 to about residue 350 including from about any residue therebetween) to about residue 400, 410, 420, 430 or 438 (i.e. to about any residue from about residue 400 residue 438 including to about any residue therebetween) of the P. aeruginosa pseudaminidase trimerisation domain (PaTD) provided by SEQ ID NO: 5. For example, a useful adjuvant may exploit oligomerisation domains comprising residues 333 to 438 of SEQ ID NO: 6.
Thus this invention provides adjuvants comprising, for example, one or more VcCBM or SpCBM for use in or with vaccines and vaccine compositions.
An adjuvant may comprise one or more of the CBM based molecules presented in
For example, a suitable adjuvant may comprise (consist essentially of, or consist of) two or more VcCBMs optionally fused, bound or conjugated to an oligomerisation domain (such as a PaTD or oligomerisation fragment thereof). The adjuvant may comprise, consist or consist essentially of two fused (or bound) VcCBMs, which are in turn, fused to an oligomerisation domain (see, for example, molecule Vc2CBMTD shown in
Other adjuvants for use may comprise two or more SpCBMs optionally fused, bound or conjugated to an oligomerisation domain (such as a PaTD or an oligomerisation fragment thereof). The adjuvant may comprise, consist or consist essentially of two fused (or bound) SpCBMs, which are in turn, fused to an oligomerisation domain (see, for example, molecule Sp2CBMTD shown in
It should be understood that the various multivalent CBMs (including Vc2CBMTD and Sp2CBMTD as shown in
Also provided, are sialic acid-binding molecule (for example CBM)-based adjuvants for use in enhancing immune responses in human or animal subjects. As stated, the adjuvants described herein may be administered together or concurrently with, an antigen(s) or a vaccine. The adjuvants may be formulated together with a vaccine for administration to a human or animal subject. However, the adjuvants for use may be formulated as separate compositions (together with suitable pharmaceutical carriers, diluents and/or excipients) to be administered to human or animal subjects before or after administration of a vaccine.
Also provided are methods of raising immune responses and/or methods of enhancing, modulating or augmenting immune responses in human or animal subjects, said methods comprising the step of administering a vaccine and a sialic acid binding molecule (for example CBM)-based adjuvant disclosed herein, to a human or animal subject in need thereof. The adjuvant may be administered mucosally and may be formulated with the vaccine such that it is administered to a human or animal host therewith or as a separate composition such that it can be administered separately from the vaccine or concurrently therewith.
It is stated above that the adjuvants may be provided with suitable excipients, diluents and/or carriers. Suitable excipients may include, for example oil and one of skill will appreciate that the term “oil” may include alkanes, alkenes, alkynes, and their corresponding acids and alcohols, the ethers and esters thereof, and mixtures thereof. The individual compounds of the oil are light hydrocarbon compounds, i.e., such components have 6 to 30 carbon atoms. The oil can be synthetically prepared or purified from petroleum products. The “oil” may have a straight or branched chain structure. It may be fully saturated or have one or more double or triple bonds. Some non-metabolizable oils for use in the present invention include mineral oil, paraffin oil, and cycloparaffins, for example.
The term oil is also intended to include “light mineral oil,” i.e., oil which is similarly obtained by distillation of petrolatum, but which has a slightly lower specific gravity than white mineral oil. Other “oil” for use as excipients may include metabolizable (non-toxic) oils. Oils of this type may include, for example vegetable oils, fish oils, animal oils or synthetically prepared oils which can be metabolized by the body of the subject (human or animal) to which the adjuvant will be administered and which are non-toxic. Sources for vegetable oils include nuts, seeds and grains.
One of skill will appreciate that any of the oil excipients described herein may be provided as oil-in-water, water-in-oil or water-in-oil-in-water emulsion forms.
An oil-in-water emulsion may comprise an AMPHIGEN® formulation. This formulation comprises an aqueous component, lecithin, mineral oil, and surfactants. Patents describing the components of the formulation include U.S. Pat. Nos. 5,084,269 and 6,572,861, the contents of which are incorporated herein by reference. Typically, the oil component of the present invention is present in an amount from 1% to 50% by volume; or in an amount of 10% to 45%; or in an amount from 20% to 40%.
Other components of the adjuvant compositions described herein can include pharmaceutically acceptable excipients, such as carriers, solvents, and diluents, isotonic agents, buffering agents, stabilizers, preservatives, vaso-constrictive agents, antibacterial agents, antifungal agents, and the like. Typical carriers, solvents, and diluents include water, saline, dextrose, ethanol, glycerol, oil, and the like. Representative isotonic agents include sodium chloride, dextrose, mannitol, sorbitol, lactose, and the like. Useful stabilizers include gelatin, albumin, and the like.
Surfactants are often used to assist in the stabilization of an emulsion selected to act as the carrier for the adjuvant and any antigen. Surfactants suitable for use with the adjuvants described herein may include natural biologically compatible surfactants and non-natural synthetic surfactants. Biologically compatible surfactants include phospholipid compounds or a mixture of phospholipids. Preferred phospholipids are phosphatidylcholines (lecithin), such as soy or egg lecithin. Lecithin can be obtained as a mixture of phosphatides and triglycerides by water-washing crude vegetable oils, and separating and drying the resulting hydrated gums. A refined product can be obtained by fractionating the mixture for acetone insoluble phospholipids and glycolipids remaining after removal of the triglycerides and vegetable oil by acetone washing. Alternatively, lecithin can be obtained from various commercial sources. Other suitable phospholipids include phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, phosphatidic acid, cardiolipin, and phosphatidylethanolamine. The phospholipids may be isolated from natural sources or conventionally synthesized.
Non-natural, synthetic surfactants suitable for use with the adjuvants described herein include sorbitan-based non-ionic surfactants, e.g. fatty-acid-substituted sorbitan surfactants (commercially available under the name SPAN® or ARLACEL®), fatty acid esters of polyethoxylated sorbitol (TWEEN®), polyethylene glycol esters of fatty acids from sources such as castor oil (EMULFOR®); polyethoxylated fatty acid (e.g., stearic acid available under the name SIMULSOL M-53®), polyethoxylated isooctylphenol/formaldehyde polymer (TYLOXAPOL®), polyoxyethylene fatty alcohol ethers (BRIJ®); polyoxyethylene nonphenyl ethers (TRITON® N), polyoxyethylene isooctyl phenyl ethers (TRITON® X). Generally speaking, the surfactant, or the combination of surfactants, if two or more surfactants are used, is present in the emulsion in an amount of 0.01% to 10% by volume, preferably, 0.1% to 6.0%, more preferably 0.2% to 5.0%.
As used herein, the term “a pharmaceutically-acceptable carrier” includes any and all solvents, dispersion media, coatings, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like that might be used with an adjuvant. The carrier(s) should be “acceptable” in the sense of being compatible with the other components of the compositions and not deleterious to the subject. Typically, the carriers will be sterile and pyrogen-free, and selected based on the mode of administration to be used. It is well known by those skilled in the art that certain formulations of pharmaceutically acceptable carrier comprise those carriers approved in the applicable regulations promulgated by the United States (US) Department of Agriculture or US Food and Drug Administration, or equivalent government agency in a non-US country. Therefore, the pharmaceutically accepted carrier for commercial production of an adjuvant composition according to this invention may be a carrier that is already approved or will be approved by the appropriate government agency in the US or foreign country.
Useful adjuvant compositions may optionally include compatible pharmaceutically acceptable (i.e., sterile or non-toxic) liquid, semisolid, or solid diluents that serve as pharmaceutical vehicles, excipients, or media. Diluents can include water, saline, dextrose, ethanol, glycerol, and the like. Isotonic agents can include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Stabilizers include albumin, among others.
Suitable adjuvant compositions can also contain antibiotics or other preservatives, including, for example, gentamicin, merthiolate, or chlorocresol. The various classes of antibiotics or preservatives from which to select are well known to the skilled artisan.
Adjuvant compositions provided herein may also comprise one or more additional adjuvant components. For example, an adjuvant composition may, in addition to the sialic acid binding molecules (for example CBMs) described herein, comprise one or more adjuvants selected from the group consisting of alum (comprising aluminium hydroxide/phosphate); oil-based adjuvants and organic adjuvants (for example squalene).
The adjuvant and/or adjuvant compositions described herein may be provided in lyophilised form or as lyophilised compositions. One of skill will appreciate that lyophilised adjuvant/adjuvant compositions of this invention may be reconstituted (for example prior to use) using any of the pharmaceutically acceptable carriers/diluents and/or excipients described herein.
Advantageously, the adjuvants and adjuvant compositions described herein may further comprise one or more preservative agents—including, for example, a cryopreservative agent.
The disclosure further provides a kit comprising an adjuvant of this invention and, optionally a vaccine and instructions for the preparation of the adjuvant, vaccine and/or a formulation for administration comprising the adjuvant and vaccine. The kit may comprise diluent, buffer, carrier, solvent and/or excipients for preparation of the adjuvant and/or vaccine. The kit may further comprise tools and equipment required for the preparation of the vaccine, adjuvant and/or vaccine/adjuvant composition and for the delivery or administration of the same to a subject. For example, the kit may include a syringe and various vials. The adjuvant and/or vaccine components of the kit may be provided in the form of products prepared by a spray drying method or encapsulated. Additionally, or alternatively, the adjuvant/vaccine components of the kit may be provided in lyophilised form for reconstitution prior to use.
The adjuvants (which comprise any of the sialic acid binding molecules (for example any of the CBM type molecules)) described herein, may be used in any suitable amount. Further, the adjuvants may be formulated for oral, mucosal or parenteral administration—the precise formulation may depend on the formulation of the antigen(s) with which the adjuvant is to be administered. For example a vaccine (comprising one or more antigens) that is to be administered mucosally may require the use of an adjuvant which is also formulated for mucosal administration. This may not always be the case and it is possible that the adjuvant could be administered via a route different to that of the antigen. The amount of adjuvant used may vary and in some cases may comprise an amount from about 0.1 μg to about 100 μg. For example about 0.2 μg, 0.3 μg, 0.4 μg, 0.5 μg, 1 μg, 1.1 μg, 1.2 μg, 1.3 μg, 1.4 μg, 1.5 μg, 2 μg, 5 μg, 10 μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg or 95 μg of the sialic acid binding molecule (CBM) based adjuvant may be used. The amount of adjuvant may be formulated within a suitable volume of a pharmaceutically acceptable excipient, diluent or buffer. The excipient, diluent or buffer may be sterile and/or pyrogen (endotoxin) free. Suitable excipient, diluent or buffer volumes may include, for example, volumes of about 1 μl to about 5 ml. For example, the required amount of sialic acid binding molecule (CBM) based adjuvant may be combined (or formulated) with about 5 μl, 10 μl, 15 μl, 20 μl, 25 μl, 30 μl, 35 μl, 40 μl, 45 μl, 50 μl, 55 μl, 60 μl, 65 μl, 70 μl, 75 μl, 80 μl, 85 μl, 90 μl, 95 μl, 100 μl, 200 μl, 300 μl, 400 μl, 500 μl, 600 μl, 700 μl, 800 μl, 900 μl, 1 ml, 2 ml, 3 ml or 4 ml. For example, when an adjuvant of this disclosure is to be administered intranasally about 1 μg, 1.3 μg, 2 μg or 10 μg (for example) may be used in about 20 μl, 30 μl, 40 μl or 50 μl of a suitable excipient, diluent or buffer. Where the adjuvant is a fusion, the total amount of protein used in any given dose may be about 0.1 μg to about 100 μg. For example about 0.2 μg, 0.3 μg, 0.4 μg, 0.5 μg, 1 μg, 1.1 μg, 1.2 μg, 1.3 μg, 1.4 μg, 1.5 μg, 2 μg, 5 μg, 10 μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg or 95 μg.
In use, the adjuvant may be administered multiple times over a number of days or weeks. For example after an initial (or first) administration, the adjuvant may be administered again at about (+/−1 or 2 days) 3, 4, 5, 6, 7, 17, 21, 28 or 35 days later. On any given day, a specific dose of adjuvant may be administered 1, 2, 3 or more times. Each time, the adjuvant may be administered (by whatever route is considered best) with or without the antigen to which it is hoped an immune response will be raised.
This disclosure further provides an adjuvant which comprising any one or more of the sialic acid binding molecules described herein. The disclosure also provides any one or more of the sialic acid binding molecules described herein for use as an adjuvant.
The disclosure also relates to a composition comprising an antigen and any of the sialic acid binding molecules described herein for use in raising an immune response in a human or animal subject. The raised immune response may be a mucosal immune response. The composition (for use in raising an immune response) may be a vaccine, for example a mucosal vaccine.
Also described herein is a vaccine comprising an antigen and any one or more of the sialic acid binding molecules described herein, wherein the molecule capable of binding sialic acid is used (or included) in the vaccine as an adjuvant.
The disclosure will now be described in detail by reference to the following figures, which show:
Standard ELISAs using immobilised biologics were used to analyse bronchiolar alveolar lavage (BAL) samples collected from a murine study where mCBM40s were given intranasally prior to a lethal influenza challenge. The mice were culled 7 days later.
In one study, mCBM40s were shown to elicit a strong mucosal IgA response in a dose-dependent manner (see
In another mouse study a low (1 μg) repeated dose of mCBM40 was administered up to a month before a IFV-challenge, with mice being culled 7 days later. Mouse tissues were analysed for evidence of IFV HA-specific, and mCBM40-specific antibodies from BAL, lung extracts and sera. We observed both anti-IFV HA IgA and IgG antibodies in BAL and lung extracts from mice that were treated with mCBM40s and that the levels of antibodies were increased in some of these mice compared to mice that were given virus only (see
Despite mice being exposed to virus for only a week before culling, the data suggests that the mCBM40s, even at low doses, appear to enhance the level of antiviral antibodies in lung extracts. A similar murine study where mCBM40, Vc2CBMTD, was given to mice up to 1 week before a lethal IFV A/California/pdm09 challenge, and with mice culled approximately 2 weeks later, also showed the presence of both mCBM40 and antiviral IgA and IgG antibodies in BAL and lung homogenates (see
The difference in antiviral HA antibodies between treated and untreated infected mice were statistically significant; however, some mice from the untreated, infected group were culled just before the designated cull date due to greater than 25% weight loss (around Days 9-11 post infection). Despite this, mCBM40s have the potential to be potent non-toxic, mucosal adjuvants for antigens or vaccines of interest, particularly for respiratory pathogens.
When mCBM40s are administered in mice, they have been shown to be non-toxic, and capable of generating protective immune responses against respiratory pathogens, such as an influenza virus (IFV), that target cell surface sialic acid-receptors during infection (PNAS (2014) 111, 6401; AAC (2015) 59, 1495). Analyses of mucosal tissue from mice infected with IFV indicate that mCBM40s are also potent immunogens and appear to enhance an IFV antigen-specific antibody response when mCBM40s are directed to the same tissue. Based on these findings, we investigated whether the immune response of a test antigen is enhanced through the use of a CBM-based adjuvant.
Intranasal immunization of mice. All CBM40 proteins were prepared as described in PNAS (2014) 111, 6401. Female BALB/c mice (6-week old) were used for the study. All intranasal inoculations and mice bleeds were performed under isofluorane anaesthesia. The immunization schedule and bleeds were performed as outlined in
Antibody Analysis.
Immune samples (BAL, lungs and sera) were analysed for the presence of anti-GFP antibodies and anti-mCBM40 antibodies using the ELISA technique as described in PNAS (2014) 111, 6401. For this, antigens (1 μg per well) were immobilized on 96-well plates (Corning). Tissue samples were diluted in blocking buffer as followed: BAL (1:4), homogenized lung tissue (1:4) and sera (1:50). Samples were added to wells followed by goat anti-mouse IgG, IgA, or IgM HRP-conjugate antibodies (Santa Cruz, 1:5000 dilution), and the presence of antibodies was detected using TMB (Sigma). Absorbance was measured at the 450-nm wavelength (620-nm wavelength used as reference).
Statistical Analysis.
Pairwise comparisons were made using one-way ANOVA and Tukey's multiple comparisons test. GraphPad Prism 7 (GraphPad Software) was used for all analysis. Tests with p<0.05 were deemed statistically significant. Unless otherwise stated, all values are presented as mean±SD, with statistical results presented as: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.
1. Conjugation of the test antigen (GFP) to Sp2CBMTD, generated both local and systemic anti-GFP antibodies from 7 days post intranasal administration.
2. The conjugation method using Sp2CBMTD was more efficient in generating anti-GFP antibodies compared to either GFP alone or co-administration with Sp2CBMTD or Vc2CBMTD (
3. Mucosal IgA and IgG responses of both GFP and mCBM40 (Sp2CBMTD, Vc2CBMTD) in lung tissue and BAL were also observed after 35 days.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1616007.9 | Sep 2016 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2017/052805 | 9/20/2017 | WO | 00 |
