Patent Application
20090142377
This application claims priority to European Patent Application Serial No. EP 07120303.8, filed Nov. 8, 2007, the entire contents of which is hereby incorporated herein by this reference.
The invention relates to the fields of cell biology, immunology, vaccinology, adjuvant technology, and medicine.
Vaccines can be divided in two basic groups, i.e., prophylactic vaccines and therapeutic vaccines. Prophylactic vaccines have been made and/or suggested against essentially every known infectious agent (virus, bacterium, yeast, fungi, parasite, mycoplasm, etc.), which has some pathology in man, pets and/or livestock, which is therefore collectively referred to as pathogen. Therapeutic vaccines have been made and/or suggested for infectious agents as well, but also for treatments of cancer and other aberrancies, as well as for inducing immune responses against other self antigens, as widely ranging as, e.g., LHRH for immunocastration of boars, or for use in preventing graft versus host (GvH) and/or transplant rejections.
In vaccines in general there are two vital issues. Vaccines have to be efficacious and vaccines have to be safe. It often seems that the two requirements are mutually exclusive when trying to develop a vaccine. The most efficacious vaccines so far have been modified live infectious agents. These are modified in a manner that their virulence has been reduced (attenuation) to an acceptable level. The vaccine strain of the infectious agent typically does replicate in the host, but at a reduced level, so that the host can mount an adequate immune response, also providing the host with long term immunity against the infectious agent. The downside of attenuated vaccines is that the infectious agents may revert to a more virulent (and thus pathogenic) form.
This may happen in any infectious agent, but is a very serious problem in fast mutating viruses (such as in particular RNA viruses). Another problem with modified live vaccines is that infectious agents often have many different serotypes. It has proven to be difficult in many cases to provide vaccines which elicit an immune response in a host that protects against different serotypes of infectious agents.
Vaccines in which the infectious agent has been killed are often safe, but often their efficacy is mediocre at best, even when the vaccine contains an adjuvant. In general an immune response is enhanced by adding adjuvants (from the Latin adjuvare, meaning “to help”) to the vaccines. The chemical nature of adjuvants, their proposed mode of action and their reactions (side effect) are highly variable. Some of the side effects can be ascribed to an unintentional stimulation of different mechanisms of the immune system whereas others reflect general adverse pharmacological reactions which are more or less expected. There are several types of adjuvants. Today the most common adjuvants for human use are aluminium hydroxide, aluminium phosphate and calcium phosphate. However, there is a number of other adjuvants based on oil emulsions, products from bacteria (their synthetic derivatives as well as liposomes) or gram-negative bacteria, endotoxins, cholesterol, fatty acids, aliphatic amines, paraffinic and vegetable oils. Recently, monophosphoryl lipid A, ISCOMs with Quil-A, and Syntex adjuvant formulations (SAFs) containing the threonyl derivative or muramyl dipeptide have been under consideration for use in human vaccines. Chemically, the adjuvants are a highly heterogenous group of compounds with only one thing in common: their ability to enhance the immune response—their adjuvanticity. They are highly variable in terms of how they affect the immune system and how serious their adverse effects are due to the resultant hyperactivation of the immune system. The choice of any of these adjuvants reflects a compromise between a requirement for adjuvanticity and an acceptable low level of adverse reactions. The term adjuvant has been used for any material that can increase the humoral and/or cellular immune response to an antigen. In the conventional vaccines, adjuvants are used to elicit an early, high and long-lasting immune response. The newly developed purified subunit or synthetic vaccines (see below) using biosynthetic, recombinant and other modern technology are poor immunogens and require adjuvants to evoke the immune response. The use of adjuvants enables the use of less antigen to achieve the desired immune response, and this reduces vaccine production costs. With a few exceptions, adjuvants are foreign to the body and cause adverse reactions.
A type of vaccine that has received a lot of attention since the advent of modern biology is the subunit vaccine. In these vaccines only one or a few elements of the infectious agent are used to elicit an immune response. Typically a subunit vaccine comprises one, two or three proteins (glycoproteins) and/or peptides present in proteins or fragments thereof, of an infectious agent (from one or more serotypes) which have been purified from a pathogen or produced by recombinant means and/or synthetic means. Although these vaccines in theory are the most promising safe and efficacious vaccines, in practice efficacy has proved to be a major hurdle.
Molecular biology has provided more alternative methods to arrive at safe and efficacious vaccines that theoretically should also provide cross-protection against different serotypes of infectious agents. Carbohydrate structures derived from infectious agents have been suggested as specific immune response eliciting components of vaccines, as well as lipopolysaccharide structures, and even nucleic acid complexes have been proposed. Although these component vaccines are generally safe, their efficacy and cross-protection over different serotypes has been generally lacking. Combinations of different kinds of components have been suggested (carbohydrates with peptides/proteins and lipopolysaccharide (LPS) with peptides/proteins, optionally with carriers), but so far the safety vs. efficacy issue remains.
Another approach to provide cross-protection is to make hybrid infectious agents which comprise antigenic components from two or more serotypes of an infectious agent. These can be and have been produced by modern molecular biology techniques. They can be produced as modified live vaccines, or as vaccines with inactivated or killed pathogens, but also as subunit vaccines. Cocktail or combination vaccines comprising antigens from completely different infectious agents are also well known. In many countries children are routinely vaccinated with cocktail vaccines against, e.g., diphtheria, whooping cough, tetanus and polio. Recombinant vaccines comprising antigenic elements from different infectious agents have also been suggested. For instance for poultry a vaccine based on a chicken anemia virus has been suggested to be complemented with antigenic elements of Marek disease virus (MDV), but many more combinations have been suggested and produced.
Another important advantage of modern recombinant vaccines is that they have provided the opportunity to produce marker vaccines. Marker vaccines have been provided with an extra element that is not present in wild type infectious agent, or marker vaccines lack an element that is present in wild type infectious agent. The response of a host to both types of marker vaccines can be distinguished (typically by serological diagnosis) from the response against an infection with wild type.
An efficient way of producing immunogenic compositions, or improving the immunogenicity of immunogenic compositions, has been provided in WO 2007/008070. This patent application discloses that the immunogenicity of a composition which comprises amino acid sequences is enhanced by providing the composition with at least one cross-beta structure. A cross-beta structure is a structural element of peptides and proteins, comprising stacked beta sheets, as will be discussed in more detail below. According to WO 2007/008070, the presence of cross-beta structure enhances the immunogenicity of a composition comprising an amino acid sequence. An immunogenic composition is thus prepared by producing a composition which comprises an amino acid sequence, such as a protein containing composition, and administrating (protein comprising) cross-beta structures to the composition. Additionally, or alternatively, cross-beta structure formation in the composition is induced, for instance by changing the pH, salt concentration, reducing agent concentration, temperature, buffer and/or chaotropic agent concentration, and/or combinations of these parameters.
Provided are improved means and methods for producing and/or improving immunogenic compositions. Further provided are compositions with enhanced immunogenicity for use as vaccines. Also provided are compositions with enhanced immunogenicity for use to obtain vaccines. Further provided are means and methods for producing compositions with enhanced capability of, at least in part, preventing and/or counteracting a pathology and/or a disorder for use as passive vaccines. Further provided are compositions for use as passive vaccines.
Provided herein are improved methods for providing an immunogenic composition comprising providing an amino acid sequence containing composition with at least one cross-beta structure and subsequently testing at least one, preferably at least two, immunogenic properties of the resulting composition. Thus provided is a way for controlling a process for the production of an immunogenic composition, so that immunogenic compositions with preferred immunogenic properties are produced and/or selected.
Provided is a method wherein an immunogenic composition comprising at least one amino acid sequence such as, but not limited to, a peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein, and here collectively referred to as “protein,” is provided with at least one cross-beta structure, where after at least one of the following properties is tested:
Cross-beta structures are present in a subset of misfolded proteins such as for instance amyloid. A misfolded protein is defined herein as a protein with a structure other than a native, non-amyloid, non-cross-beta structure. Hence, a misfolded protein is a protein having a non-native three dimensional structure, and/or a cross-beta structure, and/or an amyloid structure.
Misfolded proteins tend to multimerize and can initiate fibrillization. This can result in the formation of amorphous aggregates that can vary greatly in size. In certain cases misfolded proteins are more regular and fibrillar in nature. The term amyloid has initially been introduced to define the fibrils, which are formed from misfolded proteins, and which are found in organs and tissues of patients with the various known misfolding diseases, collectively termed amyloidoses. Commonly, amyloid appears as fibrils with undefined length and with a mean diameter of 10 nm, is deposited extracellularly, stains with the dyes Congo red and Thioflavin T (ThT), shows characteristic green birefringence under polarized light when Congo red is bound, comprises β-sheet secondary structure, and contains the characteristic cross-beta conformation (see below) as determined by X-ray fiber diffraction analysis. However, since it has been determined that protein misfolding is a more general phenomenon and since many characteristics of misfolded proteins are shared with amyloid, the term amyloid has been used in a broader scope. Now, the term amyloid is also used to define intracellular fibrils and fibrils formed in vitro. Also the terms amyloid-like and amylog are used to indicate misfolded proteins with properties shared with amyloids, but that do not fulfill all criteria for amyloid, as listed above.
In conclusion, misfolded proteins are highly heterogeneous in nature, ranging from monomeric misfolded proteins, to small oligomeric species, sometimes referred to as protofibrils, larger aggregates with amorphous appearance, up to large highly ordered fibrils, all of which appearances can share structural features reminiscent to amyloid. As used herein, the term “misfoldome” encompasses any collection of misfolded proteins.
Amyloid and misfolded proteins that do not fulfill all criteria for being identified as amyloid can share structural and functional features with amyloid and/or with other misfolded proteins. These common features are shared among various misfolded proteins, independent of their varying amino acid sequences. Shared structural features include for example the binding to certain dyes, such as Congo red, ThT, Thioflavin S, accompanied by enhanced fluorescence of the dyes, multimerization, and the binding to certain proteins, such as tissue-type plasminogen activator (tPA), the receptor for advanced glycation end-products (RAGE) and chaperones, such as heat shock proteins, like BiP (grp78 or immunoglobulin heavy chain binding protein). Shared functional activities include the activation of tPA and the induction of cellular responses, such as inflammatory responses and an immune response, and induction of cell toxicity.
A unique hallmark of a subset of misfolded proteins such as for instance amyloid is the presence of the cross-beta conformation or a precursor form of the cross-beta conformation.
A cross-beta structure is a secondary structural element in peptides and proteins. A cross-beta structure (also referred to as a “cross-β,” a “cross-beta” or a “cross-β structure”) is defined as a part of a protein or peptide, or a part of an assembly of peptides and/or proteins, which comprises single beta-strands (stage 1) and a(n ordered) group of beta-strands (stage 2), and typically a group of beta-strands, preferably composed of 5-10 beta-strands, arranged in a beta-sheet (stage 3). A cross-beta structure often comprises in particular a group of stacked beta-sheets (stage 4), also referred to as “amyloid.” Typically, in cross-beta structures the stacked beta sheets comprise flat beta sheets in a sense that the screw axis present in beta sheets of native proteins, is partly or completely absent in the beta sheets of stacked beta sheets. A cross-beta structure is formed following formation of a cross-beta structure precursor form upon protein misfolding like for example denaturation, proteolysis or unfolding of proteins. A cross-beta structure precursor is defined as any protein conformation that precedes the formation of any of the aforementioned structural stages of a cross-beta structure. These structural elements present in cross-beta structure (precursor) are typically absent in globular regions of (native parts of) proteins. The presence of cross-beta structure is for example demonstrated with X-ray fiber diffraction or binding of ThT or binding of Congo red, accompanied by enhanced fluorescence of the dyes.
A typical form of a cross-beta structure precursor is a partially or completely misfolded protein. A typical form of a misfolded protein is a partially or completely unfolded protein, a partially refolded protein, a partially or completely aggregated protein, an oligomerized or multimerized protein, or a partially or completely denatured protein. A cross-beta structure or a cross-beta structure precursor can appear as monomeric molecules, dimeric, trimeric, up to oligomeric assemblies of molecules and can appear as multimeric structures and/or assemblies of molecules.
Cross-beta structure (precursor) in any of the aforementioned states can appear in soluble form in aqueous solutions and/or organic solvents and/or any other solutions. Cross-beta structure (precursor) can also be present as solid state material in solutions, like for example as insoluble aggregates, fibrils, particles, like for example as a suspension or separated in a solid cross-beta structure phase and a solvent phase.
Protein misfolding, formation of cross-beta structure precursor, formation of aggregates or multimers and/or cross-beta structure can occur in any composition comprising peptides with a length of at least 2 amino acids, and/or protein(s). The term “peptide” is intended to include oligopeptides as well as polypeptides, and the term “protein” includes proteinaceous molecules including peptides, with and without post-translational modifications such as for instance glycosylation, citrullination, oxidation, acetylation and glycation. It also includes lipoproteins and complexes comprising a proteinaceous part, such as for instance protein-nucleic acid complexes (RNA and/or DNA), membrane-protein complexes, etc. As used herein, the term “protein” also encompasses proteinaceous molecules, peptides, oligopeptides and polypeptides. Hence, the use of “protein” or “protein and/or peptide” in this application have the same meaning.
A typical form of stacked beta-sheets is in a fibril-like structure in which the beta-strands are oriented in either the direction of the fiber axis or perpendicular to the direction of the fiber axis. The direction of the stacking of the beta-sheets in cross-beta structures is perpendicular to the long fiber axis.
A cross-beta structure conformation is a signal that triggers a cascade of events that induces clearance and breakdown of the obsolete protein or peptide. When clearance is inadequate, unwanted proteins and/or peptides aggregate and form toxic structures ranging from soluble oligomers up to precipitating fibrils and amorphous plaques. Such cross-beta structure conformation comprising aggregates underlie various diseases and disorders, such as for instance, Huntington's disease, amyloidosis type disease, atherosclerosis, cardiovascular disease, diabetes, bleeding, thrombosis, cancer, sepsis and other inflammatory diseases, rheumatoid arthritis, transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease, multiple sclerosis, auto-immune diseases, uveitis, ankylosing spondylitis, diseases associated with loss of memory such as Alzheimer's disease, Parkinson's disease and other neuronal diseases (epilepsy), encephalopathy and systemic amyloidoses.
A cross-beta structure is, for instance, formed during unfolding and refolding of proteins and peptides. Unfolding of peptides and proteins occur regularly within an organism. For instance, peptides and proteins often unfold and refold spontaneously at the end of their life cycle. Moreover, unfolding and/or refolding is induced by environmental factors such as for instance pH, glycation, oxidative stress, citrullination, ischeamia, heat, irradiation, mechanical stress, proteolysis and so on. As used herein, the terms “cross-beta” and “cross-beta structure” also encompasses any cross-beta structure precursor and any misfolded protein, even though a misfolded protein does not necessarily comprise a cross-beta structure. The term “cross-beta binding molecule” or “molecule capable of specifically binding a cross-beta structure” also encompasses a molecule capable of specifically binding any misfolded protein.
The terms unfolding, refolding and misfolding relate to the three-dimensional structure of a protein or peptide. Unfolding means that a protein or peptide loses at least part of its three-dimensional structure. The term refolding relates to the coiling back into some kind of three-dimensional structure. By refolding, a protein or peptide can regain its native configuration, or an incorrect refolding can occur. The term “incorrect refolding” refers to a situation when a three-dimensional structure other than a native configuration is formed. Incorrect refolding is also called misfolding. Unfolding and refolding of proteins and peptides involves the risk of cross-beta structure formation. Formation of cross-beta structures sometimes also occurs directly after protein synthesis, without a correctly folded protein intermediate.
In certain embodiments, a method is provided wherein an immunogenic composition comprising at least one peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein is provided with at least one cross-beta structure. This is performed in various ways. For instance, a peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein is subjected to a cross-beta inducing procedure, preferably a change of pH, salt concentration, temperature, buffer, reducing agent concentration and/or chaotropic agent concentration. These procedures are known to induce and/or enhance cross-beta formation. In certain embodiments, the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein is subjected to a cross-beta inducing procedure before it is used for the preparation of an immunogenic composition. It is, however, also possible to subject the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein to a cross-beta inducing procedure while it is already present in an immunogenic composition.
Additionally, or alternatively, a peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein is provided with a (peptide or protein comprising a) cross-beta structure, either before it is used for the preparation of an immunogenic composition or after it has been used for the preparation of an immunogenic composition.
After an immunogenic composition according to the invention has been provided with cross-beta structures, one or more immunogenic properties of the resulting composition are tested.
In certain embodiments, it is tested whether a binding compound capable of specifically binding an epitope of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein is capable of specifically binding the resulting immunogenic composition. In principle, induction and/or administration of a cross-beta structure into a composition could result in a diminished availability of an epitope of interest. For instance, if a cross-beta structure is induced in a region of a peptide or protein wherein an epitope is present, the epitope is at risk of being shielded. The conformation of the epitope is also at risk of being disturbed. Alternatively, if a peptide sequence of a composition is coupled to a cross-beta containing peptide or protein, the coupling could take place at the site of an epitope of interest, thereby reducing its availability for an animal's immune system. In short, the availability of an epitope of interest for an animal's immune system could be diminished after an immunogenic composition has been provided with cross-beta structures. This is in certain embodiments, tested by determining whether a binding compound which is capable of specifically binding an epitope of interest, such as for instance an antibody or a functional fragment or a functional equivalent thereof, is still capable of binding the immunogenic composition after the composition has been provided with cross-beta structure. If the binding compound is capable of specifically binding the resulting immunogenic composition, it shows that the epitope is still available for an animal's immune system.
In another embodiment it is tested whether the degree of multimerization of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein in the immunogenic composition allows recognition of an epitope of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein by an animal's immune system. Proteins comprising cross-beta structures tend to multimerize. Hence, after an immunogenic composition has been provided with cross-beta structures, multimerization of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein in the immunogenic composition will occur. According to the invention, it is tested whether the degree of multimerization, if occurred at all, is such that an animal's immune system is still capable of recognizing an epitope (of interest). For instance, too much multimerization will result in the formation of a fibril wherein epitopes of interest are shielded from the immune system.
Preferably monomers and/or multimers of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein in the immunogenic composition have dimensions in the range of 0.5 nm to 1000 μm, and more preferably, in the range of 0.5 nm to 100 μm, and even more preferably in the range of 1 nm to 5 μm, and even more preferably in the range of 3-2000 nm. This range of dimensions is determined by the number of proteinaceous molecules per multimer, with a given number of amino acid residues per proteinaceous molecule. Therefore, the dimensions are alternatively or additively expressed in terms of number of proteinaceous molecule monomers per multimer.
In another embodiment, it is tested whether between 4-75% of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein content of the composition is in a conformation comprising cross-beta structures. According to the invention, even though cross-beta structure enhances immunogenicity, the presence of too many cross-beta structures negatively influences immunogenicity. A cross-beta content between (and including) 4 and 75% is preferred. It is possible to determine the ratio between total cross-beta structure and total protein content. In certain embodiments, however, the cross-beta content within single proteins is determined. Preferably, individual proteins have a cross-beta content of between (and including) 4 and 75%, so that at least one epitope remains available for an animal's immune system. Most preferably, at least 70% of the individual proteins each have a cross-beta content of between (and including) 4 and 75%.
In another embodiment, it is tested whether the at least one cross-beta structure comprises a property allowing recognition of an epitope of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein by an animal's immune system. Recognition of a cross-beta structure by a component of an animal's immune system, for instance by a multiligand receptor, results in (the initiation of) an immunogenic reaction against a peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein of an immunogenic composition according to the invention (see for instance
In certain embodiments, at least two of the above mentioned tests are carried out. Of course, any combination of tests is possible. In certain embodiments, at least three of the above mentioned tests are carried out.
The invention thus provides a method for producing an immunogenic composition, the composition comprising at least one peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein, the method comprising providing the composition with at least one cross-beta structure and determining: whether a binding compound capable of specifically binding an epitope of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein is capable of specifically binding the immunogenic composition; whether the degree of multimerization of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein in the composition allows recognition of an epitope of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein by an animal's immune system; whether between 4-75% of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein content of the composition is in a conformation comprising cross-beta structures; and/or whether the at least one cross-beta structure comprises a property allowing recognition of an epitope of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein by an animal's immune system.
In certain embodiments, it is determined whether monomers and/or multimers of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein in the immunogenic composition have dimensions in the range of 0.5 nm to 1000 μm, and more preferably, in the range of 0.5 nm to 100 μm, and even more preferably in the range of 1 nm to 5 μm, and even more preferably in the range of 3-2000 nm. This range of dimensions is determined by the number of proteinaceous molecules per multimer, with a given number of amino acid residues per proteinaceous molecule. Therefore, the dimensions are alternatively or additively expressed in terms of number of proteinaceous molecule monomers per multimer.
An animal comprises any animal having an immune system, preferably a mammal. In certain embodiments, the animal comprises a human individual.
A protein-membrane complex is defined as a compound or composition comprising an amino acid sequence as well as a lipid molecule, and/or a fragment thereof, and/or a derivative thereof, for example assembled in a membrane and/or vesicle and/or liposome type of arrangement.
An immunogenic composition comprising at least one peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein is defined herein as a composition comprising at least one amino acid sequence, which composition is capable of eliciting and/or enhancing an immune response in an animal, preferably a mammal, against at least part of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein after administration of the immunogenic composition to the animal. The immune response preferably comprises a humoral immune response and/or a cellular immune response. The immune response needs not be protective, and/or therapeutic and/or capable of diminishing a consequence of disease. An immunogenic composition according to the invention is preferably capable of inducing and/or enhancing the formation of antibodies, and/or activating B-cells and/or T-cells which are capable of specifically binding an epitope of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein.
In certain embodiments, an antibody, or a functional fragment or a functional equivalent thereof, is used in order to determine whether an epitope of interest is still available for an animal's immune system after an immunogenic composition has been provided with cross-beta structures. Further provided is therefore a method according to the invention, comprising determining whether an antibody or a functional fragment or a functional equivalent thereof, capable of specifically binding an epitope of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein, is capable of specifically binding the immunogenic composition. A functional fragment of an antibody is defined as a fragment which has at least one same property as the antibody in kind, not necessarily in amount. The functional fragment is preferably capable of binding the same antigen as the antibody, albeit not necessarily to the same extent. A functional fragment of an antibody preferably comprises a single domain antibody, a single chain antibody, a Fab fragment or a F(ab′)2 fragment. A functional equivalent of an antibody is defined as a compound which is capable of specifically binding the same antigen as the antibody. A functional equivalent for instance comprises an antibody which has been altered such that the antigen-binding property of the resulting compound is essentially the same in kind, not necessarily in amount. A functional equivalent is provided in many ways, for instance through conservative amino acid substitution, whereby an amino acid residue is substituted by another residue with generally similar properties (size, hydrophobicity, etc), such that the overall functioning is likely not to be seriously affected.
In another embodiment it is determined whether the immunogenic composition and/or cross-beta structure is capable of specifically binding a cross-beta structure binding compound, preferably at least one compound selected from the group consisting of tPA, BiP, factor XII, hepatocyte growth factor activator, fibronectin, at least one finger domain of tPA, at least one finger domain of factor XII, at least one finger domain of fibronectin, at least one finger domain of hepatocyte growth factor activator, Thioflavin T, Thioflavin S, Congo Red, CD14, a multiligand receptor such as RAGE or CD36 or CD40 or LOX-1 or TLR2 or TLR4, a cross-beta-specific antibody, preferably cross-beta-specific IgG and/or cross-beta-specific IgM, IgIV, an enriched fraction of IgIV capable of specifically binding a cross-beta structure, Low density lipoprotein Related Protein (LRP), LRP Cluster II, LRP Cluster IV, Scavenger Receptor B-I (SR-BI), SR-A, chrysamine G, a chaperone, a heat shock protein, HSP70, HSP60, HSP90, gp95, calreticulin, a chaperonin, a chaperokine and a stress protein.
If the immunogenic composition appears to be capable of specifically binding such cross-beta binding compound, it shows that the immunogenic composition comprises a cross-beta structure which is capable of inducing and/or activating an animal's immune system.
Molecular chaperones are a diverse class of proteins comprising heat shock proteins, chaperonins, chaperokines and stress proteins, that are contributing to one of the most important cell defense mechanisms that facilitates protein folding, refolding of partially denatured proteins, protein transport across membranes, cytoskeletal organization, degradation of disabled proteins, and apoptosis, but also act as cytoprotective factors against deleterious environmental stresses. Individual members of the family of these specialized proteins bind non-native states of one or several or whole series or classes of proteins and assist them in reaching a correctly folded and functional conformation. Alternatively, when the native fold cannot be achieved, molecular chaperones contribute to the effective removal of misfolded proteins by directing them to the suitable proteolytic degradation pathways. Chaperones selectively bind to non-natively folded proteins in a stable non-covalent manner. To direct correct folding of a protein from a misfolded form to the required native conformation, mostly several chaperones work together in consecutive steps.
Chaperonins are molecular machines that facilitate protein folding by undergoing energy (ATP)-dependent movements that are coordinated in time and space by complex allosteric regulation. Examples of chaperones that facilitate refolding of proteins from a misfolded conformation to a native form are heat shock protein (hsp) 90, hsp60 and hsp70. Chaperones also participate in the stabilization of unstable protein conformers and in the recovery of proteins from aggregates. Molecular chaperones are mostly heat- or stress-induced proteins (hsps), that perform critical functions in maintaining cell homeostasis, or are transiently present and active in regular protein synthesis. Hsps are among the most abundant intracellular proteins. Chaperones that act in an ATP-independent manner are for example the intracellular small hsps, calreticulin, calnexin and extracellular clusterin. Under stress conditions such as elevated temperature, glucose deprivation and oxidation, small hsps and clusterin efficiently prevent the aggregation of target proteins. Interestingly, both types of hsps can hardly chaperone a misfolded protein to refold back to its native state. In patients with Creutzfeldt-Jakob, Alzheimer's disease and other diseases related to protein misfolding and accumulation of amyloid, increased expression of clusterin and small hsps has been seen. Molecular chaperones are essential components of the quality control machineries present in cells. Due to the fact that they aid in the folding and maintenance of newly translated proteins, as well as in facilitating the degradation of misfolded and destabilized proteins, chaperones are essentially the cellular sensors of protein misfolding and function. Chaperones are therefore the gatekeepers in a first line of defense against deleterious effects of misfolded proteins, by assisting a protein in obtaining its native fold or by directing incorrectly folded proteins to a proteolytic breakdown pathway. Notably, hsps are over-expressed in many human cancers. It has been established that hsps play a role in tumor cell metastasis, proliferation, differentiation, invasion, death, and in triggering the immune system during cancer.
One of the key members of the quality control machinery of the cell is the ubiquitous molecular chaperone hsp90. Hsp90 typically functions as part of large complexes, which include other chaperones and essential cofactors that regulate its function. Different cofactors seem to target hsp90 to different sets of substrates. However, the mechanism of hsp90 function in protein misfolding biology remains poorly understood.
Intracellular pathways that are involved in sensing protein misfolding comprise the unfolded protein response machinery (UPR) in the endoplasmic reticulum (ER). Accumulation of unfolded and/or misfolded proteins in the ER induces ER stress resulting in triggering of the UPR. Environmental factors can transduce the stress response, like for example changes in pH, starvation, reactive oxygen species. During these episodes of cellular stress, intracellular heat shock proteins levels increase to provide cellular protection. Activation of the UPR includes the attenuation of general protein synthesis and the transcriptional activation of the genes encoding ER-resident chaperones and molecules involved in the ER-associated degradation (ERAD) pathway. The UPR reduces ER stress by restoration of the protein-folding capacity of the ER. A key protein acting as a sensor of protein misfolding is the chaperone BiP (also referred to as grp78; Immunoglobulin heavy chain-binding protein/Endoplasmic reticulum luminal Ca2+-binding protein).
After testing of at least one immunogenic property of an immunogenic composition according to the invention, an immunogenic composition with a desired property is preferably selected. If a desired property, such as the availability of an epitope of interest, appears not to be present (anymore) after a composition comprising at least one peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein has been provided with cross-beta structures, another batch of the same kind of composition is preferably provided with cross-beta structures and tested again. If needed, this procedure is repeated until an immunogenic composition with at least one desired property/properties is obtained.
In certain embodiments, an immunogenic composition is selected with a degree of multimerization of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein which allows recognition of an epitope by an animal's immune system. Further provided is therefore a method according to the invention, further comprising selecting an immunogenic composition wherein the degree of multimerization of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein in the composition allows recognition of an epitope of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein by an animal's immune system.
In another embodiment, an immunogenic composition is selected with a cross-beta content of between 4-75% so that the immunogenicity is enhanced, while at least one epitope remains available for an animal's immune system. The term immunogenicity is defined herein as the capability of a compound or a composition to activate an animal's immune system. Of course, if it is intended that an animal's immune system is, at least in part, directed against an epitope of interest, the epitope of interest should be available for the animal's immune system. Further provided is therefore a method according to the invention, further comprising selecting an immunogenic composition wherein between 4-75% of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein content of the composition is in a conformation comprising cross-beta structures.
In yet another embodiment an immunogenic composition is selected which comprises a cross-beta structure having a binding property which allows (the initiation of) an immunogenic reaction against a peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein of an immunogenic composition according to the invention. Further provided is therefore a method according to the invention, further comprising selecting an immunogenic composition which comprises a cross-beta structure which is capable of specifically binding a cross-beta structure binding compound, preferably tPA, BiP, factor XII, fibronectin, hepatocyte growth factor activator, at least one finger domain of tPA, at least one finger domain of factor XII, at least one finger domain of fibronectin, at least one finger domain of hepatocyte growth factor activator, Thioflavin T, Thioflavin S, Congo Red, CD14, a multiligand receptor such as RAGE or CD36 or CD40 or LOX-1 or TLR2 or TLR4, a cross-beta-specific antibody, preferably cross-beta-specific IgG and/or cross-beta-specific IgM, IgIV, an enriched fraction of IgIV capable of specifically binding a cross-beta structure, Low density lipoprotein Related Protein (LRP), LRP Cluster II, LRP Cluster IV, Scavenger Receptor B-I (SR-BI), SR-A, chrysamine G, a chaperone, a heat shock protein, HSP70, HSP60, HSP90, gp95, calreticulin, a chaperonin, a chaperokine and/or a stress protein.
In a particularly preferred embodiment, an immunogenic composition is selected which is capable of specifically binding an antibody, or a functional fragment or a functional equivalent thereof, which is capable of specifically binding an epitope of interest. Preferably, an immunogenic composition is selected which is capable of specifically binding an antibody, or a functional fragment or a functional equivalent thereof, which is capable of specifically binding a functional, native epitope which is exposed on an natively folded antigen molecule. Such immunogenic composition comprises an epitope of interest which is available for an animal's immune system after the immunogenic composition has been provided with cross-beta structures.
Further provided is therefore a method according to the invention, further comprising selecting an immunogenic composition which is capable of specifically binding an antibody or a functional fragment or a functional equivalent thereof which is capable of specifically binding an epitope of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein. The epitope of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein is preferably surface-exposed when the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein is in its native conformation so that, after administration to a suitable host, an immune response against the native form of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein is elicited.
In certain embodiments, for selection of an immunogenic composition having a greater chance of being capable of eliciting a protective prophylactic immune response and/or a therapeutic immune response in vivo, as compared to other immunogenic compositions of a given plurality of immunogenic compositions, antibodies, or functional fragments or equivalents thereof, are used which are capable of at least in part preventing and/or counteracting a pathology and/or a disorder against which an immunogenic composition is produced. The pathology and/or disorder for example being caused by a pathogen, tumor, cardiovascular disease, atherosclerosis, amyloidosis, autoimmune disease, graft-versus-host rejection and/or transplant rejection in the target species for which an immunogenic composition according to the invention is designed. The target species for example comprises a mammal, preferably a human individual. The antibodies, or the B-cells producing these antibodies, are preferably isolated from individuals who successfully combated and/or counteracted the pathology and/or disorder, for example a viral infection. These antibodies, or the B-cells producing these antibodies, are preferably originating from the target species for which the immunogenic composition is designed. Alternatively, these antibodies are originating from a different animal species, and are for example modified to obtain antibodies more closely resembling antibodies of a target species. A non-limiting example of such modified antibody is a humanized antibody from murine origin, which is particularly suitable when the immunogenic composition is designed for human use. Collectively, these aforementioned antibodies are referred to as functional antibodies (in vivo). Non-limiting examples of these functional antibodies are those described in the art against H5N1 influenza virus, tetanospasmin of Clostridium tetani, rabies, Hepatitis B, Hepatitis A, antisera against snake venoms, for example against the poisonous snake venom proteins, and against toxic poisonous insect proteins. For example, these functional antibodies have proven efficacy when applied in passive immunization strategies.
In an alternative embodiment, antibodies are used for selection of an immunogenic composition that is capable of preventing and/or counteracting, at least in part, a disorder against which an immunogenic composition is sought. The antibodies are preferably selected using a passive immunization approach including actively inflicting the pathology and/or disorder against which protection is sought, termed challenge, after treatment of individuals with the antibodies. This approach is termed Reverse Vaccine Development. For the Reverse Vaccine Development approach, firstly antibodies are selected that have the ability to protect and/or cure an individual from an infection and/or disorder upon application of the antibodies to the individual, and/or antibodies are selected that have the ability to modulate the response of an individual to an infection and/or disorder upon application of the antibodies to the individual. Next, these antibodies are used for selection of an immunogenic composition that comprises at least one epitope for these functional antibodies, combined with immunogenic cross-beta adjuvant, preferably in the context of an optimal multimeric size. The term cross-beta adjuvant refers to an amino-acid sequence with an appearance of a cross-beta conformation which is capable, upon introduction of the cross-beta conformation to an animal, of activating the immune system of the receiving animal. As stated before, a capability of activating the immune system of the receiving animal is referred to as immunogenicity. Preferably, antibodies originating from the target species are used for passive immunization of individuals of the same species. Alternatively, antibodies from a different, preferably closely related species, are used for cross-species passive immunizations. For example, murine antibodies for passive immunization of ferrets in an influenza virus challenge model, or murine antibodies in a CSFV challenge model with pigs. Preferably, individuals of the target species for whom the immunogenic composition is meant, are treated with the antibodies or, alternatively, individuals of other species are treated, for example individuals of closely related species such as for example macaques when the target species are humans. Passive immunizations are preferably performed according to methods known to a person skilled in the art for their efficacy. Passive immunization is for example performed by intravenous administration, and/or by intradermal administration, and/or by intramuscular administration. Antibodies used for passive immunizations are preferably selected based on their known ability to modify the response of in vitro (testing) systems. Non-limiting examples of such in vitro experiments are virus neutralization tests, for example for influenza virus or CSFV, hemagglutination inhibition tests, for example for influenza virus, bactericidal activity test, for example for Neisseria meningitides, antibody dependent cell-mediated cytotoxicty (ADCC) and blood coagulation tests, for example for determination of FVIII inhibitors in Haemophilia patient samples. Collectively, these aforementioned antibodies are referred to as functional antibodies (in vitro). Alternatively, other antibodies without (known) functional activity in in vitro (testing) systems that are capable of binding the antigen of choice for incorporation in an immunogenic composition, are used for the described passive immunization approaches. These antibodies are either originating from the animal species for which the immunogenic composition is meant, or are originating from a different species. Following this (cross-species) passive immunization approach, and a subsequent challenge, functional antibodies (in vivo) are selected from the pool of applied antibodies and used for passive vaccination. These functional antibodies (in vivo) are then preferably subsequently used for selection of immunogenic compositions having a greater chance of being capable of eliciting a protective prophylactic immune response and/or a therapeutic immune response in vivo, as compared to other immunogenic compositions of a given plurality of immunogenic compositions.
In certain embodiments, immunogenic compositions selected based on binding of (one or more) antibodies that are proven to be functional antibodies (in vivo), that is to say, the antibodies have the ability to protect, diminish and/or cure an individual from an infection and/or disorder upon passive vaccination (Step 1), are used as active vaccine (product I). In another embodiment these immunogenic compositions, referred to as product I, are used in an immunization approach followed by actively inflicting the pathology and/or disorder against which protection is sought, i.e., a challenge, after immunization of individuals, or followed by a naturally occurring infection or disorder against which protection is sought after the immunization (Step 2-a). When conducting a challenge approach, the pathogen isolate used for the challenge and the antigen in the immunogenic composition are either homologous, or heterologous. Alternatively, these immunogenic compositions, referred to as product I, are used in an immunization approach with individuals who suffer from an infection or disorder, against which an immunogenic composition is sought that diminishes symptoms related to the infection or disorder, and/or that cures an individual from the infection or disorder (Step 2-b). When the individuals that received the immunogenic composition are protected against the infection or disorder, or have diminished symptoms related to the infection or disorder, and/or are cured from an infection or disorder, reconvalescent serum is collected from the individuals (Step 3). Reconvalescent serum is defined as the serum obtained from an individual recovering and/or recovered from a disorder, disease or infection. This serum is analyzed for the presence of antibodies that bind the native antigen and the antigen used in the immunogenic composition, for example using an ELISA with antigen and antigen in the used immunogenic composition immobilized onto an 96-wells plate, which is then incubated with a dilution series of sera, followed by detecting whether antibodies from the sera bound to the antigen, and to which extent (Step 4). In addition, the binding of antibodies in the sera to antigen is compared to the binding of functional antibodies (in vivo), that were initially used for the passive immunization (Step 5). Comparison is for example done in a competition ELISA. In the competition ELISA, for example a fixed amount of functional antibody (in vivo) that gives sub-maximal binding to the immobilized antigen, is mixed with a concentration series of reconvalescent serum, and the extent of binding of the functional antibodies (in vivo) is assessed. When antibodies are present in the reconvalescent serum that bind to the same or similar epitopes as the functional antibodies (in vivo), decreased binding of functional antibodies (in vivo) will be measured with increasing serum concentration. Steps 1-5 is termed Reverse Vaccine Development. In certain embodiments, the above mentioned reconvalescent serum comprising antibodies that bind to the same and/or similar epitopes that are capable of being bound by functional antibodies (in vivo) is subsequently used for passive immunization, followed by a challenge (Step A). Reconvalescent serum is then preferably selected that is capable of at least in part preventing and/or counteracting a pathology and/or a disorder against which an immunogenic composition is sought (Step B). Preferably, this latter reconvalescent serum has an increased capability of at least in part preventing and/or counteracting a pathology and/or a disorder against which an immunogenic composition is sought when compared to the functional antibodies (in vivo) used initially for passive immunizations. Then, this reconvalescent serum with improved capability of at least in part preventing and/or counteracting a pathology and/or a disorder, termed product II, is preferably used in passive immunization strategies (Step C). The improved reconvalescent serum is in another embodiment further refined towards an even more improved reconvalescent serum in an iterative process, by subjecting this improved reconvalescent serum to the aforementioned Steps 1-5, A, B, and, when an acceptable further improved reconvalescent serum is achieved, as product II in Step C. Thus, in Step 1, the functional antibodies (in vivo) are replaced by the improved reconvalescent serum, followed by Step 2 and further. Functional antibodies (in vivo) are isolated from reconvalescent serum using standard affinity based purification procedures, for example by subjecting the serum to an affinity matrix comprising immobilized native antigen, separating the antibodies that bind to the native antigen from the serum, and collecting the antibodies that bound to the native antigen. These purified functional antibodies (in vivo) can be used as product II in Step C, and/or can be subjected to Steps 1-5, A, C for further improvement of the functional antibodies (in vivo). With the functional antibodies (in vivo), which is polyclonal of nature, in the reconvalescent serum, collectively termed functional antibody passive immunization (FAPI) for, for example use in passive immunization strategies, having similar or improved capacities when compared to the functional antibody and/or antibodies (in vivo) originally used in Step 1, FAPI is preferably used for passive immunization purposes. In this way the Reversed Vaccine Development technology provides for two products; product I, an (optimized) immunogenic composition capable of eliciting a protective prophylactic immune response and/or a therapeutic immune response in vivo, and product II, FAPI vaccine with improved capability of at least in part preventing and/or counteracting a pathology and/or a disorder. FAPI is also preferably used coupled to a preferred antigen in immune complex vaccination. The immune complex vaccine products are therefore also herewith provided.
One embodiment therefore provides a reconvalescent serum and/or an antibody capable of at least in part preventing and/or counteracting a pathology and/or a disorder, obtainable by immunizing an animal with an immunogenic composition according to the invention and, subsequently, harvesting reconvalescent serum and/or an antibody from the animal. The reconvalescent serum and/or antibody is preferably used as a passive vaccine. A use of a reconvalescent serum and/or an (improved) antibody according to the invention as a vaccine, or for the preparation of a vaccine, is therefore also herewith provided.
One embodiment provides an (optimized) immune complex vaccine capable of eliciting a protective prophylactic immune response and/or a therapeutic immune response in vivo, obtainable with a method according to the invention. Another embodiment provides a FAPI vaccine with improved capability of at least in part preventing and/or counteracting a pathology and/or a disorder.
Further provided is a method for obtaining a reconvalescent serum and/or an antibody capable of at least in part preventing and/or counteracting a pathology and/or a disorder, the method comprising: producing and/or selecting an immunogenic composition with a method according to the invention, preferably using an antibody, or a functional part thereof, which is capable of specifically binding an epitope of interest of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein present in the immunogenic composition; immunizing an animal with the immunogenic composition; and harvesting reconvalescent serum and/or an antibody from the animal.
In certain embodiments, the animal comprises a non-human animal. It is, however, also possible to use an immunogenic composition according to the invention for vaccination of human individual; and to obtain serum and/or antibodies from the human individual.
Further provided is a use of an immunogenic composition according to the invention for obtaining functional antibodies and/or reconvalescent serum. As described above, the functional antibodies and/or reconvalescent serum, preferably with a higher affinity for an antigen of interest as compared to antibodies which were originally used for the preparation of the immunogenic composition, are particularly suitable for the preparation of an improved composition meant for passive immunization and/or for preparation of immune complexes. A use of the reconvalescent serum and/or functional antibodies, or a functional fragment or functional equivalent thereof, for the preparation of a composition for passive immunization and/or for preparation of immune complexes is also herewith provided. The composition meant for passive immunization and/or for preparation of immune complexes is preferably a vaccine. The reconvalescent serum and/or functional antibodies, or a functional fragment or functional equivalent thereof, is preferably used for the preparation of a prophylactic and/or therapeutic vaccine for the prophylaxis and/or treatment of a disorder caused by a pathogen, tumor, cardiovascular disease, atherosclerosis, amyloidosis, autoimmune disease, graft-versus-host rejection and/or transplant rejection.
In certain embodiments, functional antibodies are used directly for selection of immunogenic compositions that have a greater chance, as compared to other immunogenic compositions of a given plurality of immunogenic compositions, for at least in part preventing, diminishing and/or counteracting the pathology and/or disorder against which an immunogenic composition is sought, thereby not pre-selecting the functional antibodies (in vitro) in order to obtain functional antibodies (in vivo).
Alternatively, antibodies that are not functional antibodies and for which functional activity in in vitro disorder models is not known, and that are capable of binding an antigen of choice for incorporation in an immunogenic composition, are used for selection of immunogenic compositions having a greater chance of being capable of eliciting a protective prophylactic immune response and/or a therapeutic immune response in vivo, as compared to other immunogenic compositions of a given plurality of immunogenic compositions.
In all of the above approaches, preferably monoclonal antibodies or combinations of antibodies are used. Combinations of antibodies for example comprise combined monoclonal antibodies, and/or sera or plasma, and/or polyclonal antibodies isolated from serum or plasma, preferably sera or plasma from mammals known to have developed an immune response, preferably an effective immune response, i.e., reconvalescent serum/plasma.
In all of the above approaches either antibodies of a single class or compositions of antibodies of plural classes are used. For example, for selection of immunogenic compositions, in certain embodiments, only antibodies of the IgG, or IgA, or IgM, or IgD class are used, or combinations of these classes of antibodies, either separately for each class, or in mixtures of antibodies of combined classes. For example, in certain embodiments, combined IgG and IgM antibodies are used. When IgGs are considered, in certain embodiments, IgGs of a single isotype are used, or IgGs of plural isotypes are used, either separately, or in combined compositions of IgGs. For example, murine IgG1, or IgG2a is used separately, or murine immune serum comprising all IgG isotypes is used.
In all of the above descriptions, the term antibody refers to any molecule comprising an affinity region, originating from any species. An affinity region influences the affinity with which a protein or peptide binds to an epitope and is herein defined as at least part of an antibody that is capable of specifically binding to an epitope. The affinity region for instance comprises at least part of an immunoglobulin, at least part of a monoclonal antibody and/or at least part of a humanized antibody. The affinity region preferably comprises at least part of a heavy chain and/or at least part of a light chain of an antibody. In certain embodiments, the affinity region comprises a double F(ab′)2 or single form Fab fragment. Non-limiting examples of molecules with affinity regions are mouse monoclonal antibodies, human immune serum comprising a collection of immunoglobulins, and llama, camel, alpaca or camelid antibodies, also referred to as nanobodies.
In preferred embodiments, either one kind of monoclonal antibody, or a combination of antibodies, or a series of individual monoclonal antibodies, or a series of combinations of antibodies, or a combined series of individual monoclonal antibodies and combinations of antibodies, is used for selection of immunogenic compositions having a greater chance of being capable of eliciting a protective prophylactic immune response and/or a therapeutic immune response in vivo, as compared to other immunogenic compositions of a given plurality of immunogenic compositions. Preferably, 1 to 15 monoclonal antibodies and/or combinations of antibodies are used for these screenings, and even more preferably, 3 to 10 monoclonal antibodies and/or combinations of antibodies are used for the selections. These multiple antibodies preferably have varying affinity for, for example identical and/or similar and/or overlapping epitopes on the antigen. These multiple antibodies preferably bind to distinct epitopes on the antigen.
A method according to the invention is particularly suitable for selecting, from a plurality of immunogenic compositions, one or more immunogenic compositions having a greater chance of being capable of eliciting a protective prophylactic immune response and/or a therapeutic immune response in vivo, as compared to the other immunogenic compositions of the plurality of immunogenic compositions. One or more immunogenic compositions are selected which appear to have a desired property in any of the aforementioned tests. Further provided is therefore an in vitro method for selecting, from a plurality of immunogenic compositions comprising at least one peptide and/or polypeptide and/or protein and/or glycoprotein and/or lipoprotein and/or protein-DNA complex and/or protein-membrane complex with a cross-beta structure, one or more immunogenic compositions having a greater chance of being capable of eliciting a protective prophylactic immune response and/or a therapeutic immune response in vivo, as compared to the other immunogenic compositions of the plurality of immunogenic compositions, the method comprising: selecting, from the plurality of immunogenic compositions, an immunogenic composition: which is capable of specifically binding an antibody or a functional fragment or a functional equivalent thereof which is capable of specifically binding an epitope of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein; wherein the degree of multimerization of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein in the composition allows recognition of an epitope of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein by an animal's immune system; wherein between 4-75% of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein content of the composition is in a conformation comprising cross-beta structures; and/or which comprises a cross-beta structure which is capable of specifically binding a cross-beta structure binding compound, preferably tPA, BiP, factor XII, fibronectin, hepatocyte growth factor activator, at least one finger domain of tPA, at least one finger domain of factor XII, at least one finger domain of fibronectin, at least one finger domain of hepatocyte growth factor activator, Thioflavin T, Thioflavin S, Congo Red, CD14, a multiligand receptor such as RAGE or CD36 or CD40 or LOX-1 or TLR2 or TLR4, a cross-beta-specific antibody, preferably cross-beta-specific IgG and/or cross-beta-specific IgM, IgIV, an enriched fraction of IgIV capable of specifically binding a cross-beta structure, Low density lipoprotein Related Protein (LRP), LRP Cluster II, LRP Cluster IV, Scavenger Receptor B-I (SR-BI), SR-A, chrysamine G, a chaperone, a heat shock protein, HSP70, HSP60, HSP90, gp95, calreticulin, a chaperonin, a chaperokine and/or a stress protein.
In a particularly preferred embodiment, a method according to the invention is performed wherein an immunogenic composition is selected which is capable of specifically binding at least two antibodies, or functional fragments or functional equivalents thereof, which are capable of specifically binding at least two different epitopes, and/or which are capable of specifically binding the same epitope although with varying affinities, of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein. Such immunogenic composition comprises at least two different epitopes which are available for an animal's immune system and, therefore, is particularly immunogenic. In a more preferred embodiment an immunogenic composition is selected which is capable of specifically binding at least three antibodies, or functional fragments or functional equivalents thereof, which are capable of specifically binding at least three different epitopes, and/or which are capable of specifically binding the same epitope although with varying affinities, of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein. The at least two or three different epitopes may be partially overlapping.
As already described above, a method according to the invention preferably comprises selecting an immunogenic composition which is capable of specifically binding at least one antibody, or a functional fragment or functional equivalent thereof, which is capable of providing a protective prophylactic and/or a therapeutic immune response in vivo.
A composition comprising at least one peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein is provided with at least one cross-beta structure in various ways. In certain embodiments, the cross-beta structure is induced in at least part of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein. Various methods for inducing a cross-beta structure are known in the art. For instance, the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein is at least in part misfolded. In certain embodiments, an immunogenic composition comprising at least one peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein is subjected to a cross-beta inducing procedure. The cross-beta inducing procedure preferably comprises a change of pH, salt concentration, temperature, buffer, reducing agent concentration and/or chaotropic agent concentration. A method according to the invention, wherein at least one peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein is subjected to a cross-beta inducing procedure, preferably a change of pH, salt concentration, reducing agent concentration, temperature, buffer and/or chaotropic agent concentration, is therefore also provided. Non-limiting examples of cross-beta inducing procedures are heating, chemical treatments with, e.g., high salts, acid or alkaline materials, pressure and other physical treatments. A preferred manner of introducing cross-beta structures in an antigen is by one or more treatments, either in combined fashion or sequentially, of heating, freezing, reduction, oxidation, glycation pegylation, sulphatation, exposure to a chaotropic agent (the chaotropic agent preferably being urea or guanidinium-HCl), phosphorylation, partial proteolysis, chemical lysis, preferably with HCl or cyanogenbromide, sonication, dissolving in organic solutions, preferably 1,1,1,3,3,3-hexafluoro-2-propanol and trifluoroacetic acid, or a combination thereof.
In a particularly preferred embodiment, the immunogenic composition comprising at least one peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein is coupled to a cross-beta comprising compound. For instance, the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein is linked to a peptide or protein comprising a cross-beta structure. It is, however, also possible to administer a cross-beta comprising compound to a composition according to the invention, without linking the cross-beta comprising compound to the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein. Preferably the cross-beta comprising compound is an otherwise inert compound. Inert is defined as not eliciting an unwanted immune response or another unwanted biochemical reaction in a host, at least not to an unacceptable degree, preferably only to a negligible degree.
A cross-beta structure comprising compound may be added to a composition by itself, but it is also useful to use the cross-beta structure comprising compound as a carrier to which elements of the infectious agent(s) and/or antigen(s) of an immunogenic composition according to the invention are linked. This linkage can be provided through chemical linking (direct or indirect) or, for instance, by expression of the relevant antigen(s) and the cross-beta comprising compound as a fusion protein. In both cases linkers between the two may be present. In both cases dimers, trimers and/or multimers of the antigen (or one or more epitopes of a relevant antigen) may be coupled to a cross-beta comprising compound. However, normal carriers comprising relevant epitopes or antigens coupled to them may also be used. The simple addition of a cross-beta comprising compound will enhance the immunogenicity of such a complex. This is more or less generally true. An immunogenic composition according to the invention may typically comprise a number or all of the normal constituents of an immunogenic composition (in particular a vaccine), supplemented with a cross-beta structure (conformation) comprising compound.
In certain embodiments, the cross-beta structure comprising compound is itself a vaccine component, also referred to in this text as cross-beta antigen (i.e., derived from an infectious agent and/or antigen against which an immune response is desired).
An immunogenic composition according to the invention is preferably used for the preparation of a vaccine. A method according to the invention, further comprising producing a vaccine comprising the selected immunogenic composition, is therefore also herewith provided. Preferably a prophylactic and/or therapeutic vaccine is produced. In certain embodiments, a subunit vaccine is produced.
In certain embodiments, an immunogenic composition which is produced and/or selected with a method according to the invention is used as a vaccine. No other carriers, adjuvants and/or diluents are necessary because of the presence of cross-beta structures. However, if desired, such carriers, adjuvants and/or diluents may be administered to the vaccine composition at will. Further provided is therefore a use of an immunogenic composition produced and/or selected with a method according to the invention as a vaccine, preferably as a prophylactic and/or therapeutic vaccine. In certain embodiments, the vaccine comprises a subunit vaccine.
The invention further provides an immunogenic composition selected and/or produced with a method according to the invention. The immunogenic composition preferably comprises a vaccine, more preferably a prophylactic and/or therapeutic vaccine. An immunogenic composition according to the invention is particularly suitable for the preparation of a vaccine for the prophylaxis and/or treatment of a disorder caused by a pathogen, tumor, cardiovascular disease, atherosclerosis, amyloidosis, autoimmune disease, graft-versus-host rejection and/or transplant rejection. A use of an immunogenic composition according to the invention for the preparation of a vaccine for the prophylaxis and/or treatment of a disorder caused by a pathogen, tumor, cardiovascular disease, atherosclerosis, amyloidosis, autoimmune disease, graft-versus-host rejection and/or transplant rejection is therefore also herewith provided.
Further provided are uses of such immunogenic compositions for at least in part preventing and/or counteracting such disorders. One embodiment provides a method for at least in part preventing and/or counteracting a disorder caused by a pathogen, tumor, cardiovascular disease, atherosclerosis, amyloidosis, autoimmune disease, graft-versus-host rejection and/or transplant rejection, comprising administering to a subject in need thereof a therapeutically effective amount of an immunogenic composition according to the invention. The animal may be a human individual.
A method according to the invention is particularly suitable for producing and/or selecting an immunogenic composition with desired, preferably improved, immunogenic properties. It is, however, also possible to perform a method according to the invention for improving existing immunogenic compositions. Further provided is therefore a method for improving an immunogenic composition, the composition comprising at least one peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein, the method comprising providing the composition with at least one cross-beta structure and selecting an immunogenic composition: which is capable of specifically binding an antibody or a functional fragment or a functional equivalent thereof which is capable of specifically binding an epitope of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein; wherein the degree of multimerization of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein in the composition allows recognition of an epitope of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein by an animal's immune system; wherein between 4-75% of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein content of the composition is in a conformation comprising cross-beta structures; and/or which is capable of specifically binding a cross-beta structure binding compound, preferably tPA, BiP, factor XII, fibronectin, hepatocyte growth factor activator, at least one finger domain of tPA, at least one finger domain of factor XII, at least one finger domain of fibronectin, at least one finger domain of hepatocyte growth factor activator, Thioflavin T, Thioflavin S, Congo Red, CD14, a multiligand receptor such as RAGE or CD36 or CD40 or LOX-1 or TLR2 or TLR4, a cross-beta-specific antibody, preferably cross-beta-specific IgG and/or cross-beta-specific IgM, IgIV, an enriched fraction of IgIV capable of specifically binding a cross-beta structure, Low density lipoprotein Related Protein (LRP), LRP Cluster II, LRP Cluster IV, Scavenger Receptor B-I (SR-BI), SR-A, chrysamine G, a chaperone, a heat shock protein, HSP70, HSP60, HSP90, gp95, calreticulin, a chaperonin, a chaperokine and/or a stress protein.
A method according to the invention is particularly suitable for producing and/or selecting an immunogenic composition which is capable of eliciting an immune response in an animal. It is, however, also possible to use the teaching of the invention in order to avoid the use of immunogenic compounds. For instance, if a composition comprising at least one peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein is used for a non-immunogenic purpose, for instance as a medicament, immunological reactions after administration of the composition to an animal are undesired. In such cases, it is not intended to induce cross-beta structures in the composition comprising at least one peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein. However, cross-beta structures may form anyway. Therefore, in order to test such compositions for non-immunogenic use, a composition comprising at least one peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein is subjected to any of the tests described hereinbefore. If a composition appears to have become too immunogenic, it is not used. Instead, another batch of the same kind of composition is preferably tested with a method according to the invention. If needed, this procedure is repeated until a composition with no, or an acceptable, immunogenic property has been obtained. For applications wherein compositions comprising at least one peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein are tested, reference is for instance made to WO 2007/008069 (quality control of medicaments) and WO 2007/008071 (quality control of other kinds of compositions), the contents of each of which are incorporated herein by this reference.
One embodiment therefore provides a method according to the invention, comprising selecting an immunogenic composition which is not, or to an acceptable extent, capable of specifically binding an antibody or a functional fragment or a functional equivalent thereof which is capable of specifically binding an epitope of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein.
Another embodiment provides a method according to the invention, comprising selecting an immunogenic composition wherein the degree of multimerization of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein in the composition does not, or to an acceptable extent, allow recognition of an epitope of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein by an animal's immune system.
Another embodiment provides a method according to the invention, comprising selecting an immunogenic composition wherein less than 4% of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein content of the composition is in a conformation comprising cross-beta structures.
Another embodiment provides a method according to the invention, comprising selecting an immunogenic composition which is not, or to an acceptable extent, capable of specifically binding a cross-beta structure binding compound, preferably tPA, BiP, factor XII, fibronectin, hepatocyte growth factor activator, at least one finger domain of tPA, at least one finger domain of factor XII, at least one finger domain of fibronectin, at least one finger domain of hepatocyte growth factor activator, Thioflavin T, Thioflavin S, Congo Red, CD14, a multiligand receptor such as RAGE or CD36 or CD40 or LOX-1 or TLR2 or TLR4, a cross-beta-specific antibody, preferably cross-beta-specific IgG and/or cross-beta-specific IgM, IgIV, an enriched fraction of IgIV capable of specifically binding a cross-beta structure, Low density lipoprotein Related Protein (LRP), LRP Cluster II, LRP Cluster IV, Scavenger Receptor B-I (SR-BI), SR-A, chrysamine G, a chaperone, a heat shock protein, HSP70, HSP60, HSP90, gp95, calreticulin, a chaperonin, a chaperokine and/or a stress protein.
A method according to the invention is particularly suitable for producing and/or selecting an immunogenic composition which is capable of eliciting a humoral and/or cellular immune response. For a schematic overview of a humoral and cellular immune response, reference is made to
In order to produce and/or select an immunogenic composition which is specifically adapted for avoiding a humoral immune response, a method according to the invention preferably comprises the following steps: selecting, from a plurality of immunogenic compositions, an immunogenic composition: which is not capable of specifically binding an antibody or a functional fragment or a functional equivalent thereof which is capable of specifically binding an epitope of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein; wherein the degree of multimerization of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein in the composition does not allow recognition of an epitope of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein by an animal's immune system; wherein less than 4% of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein content of the composition is in a conformation comprising cross-beta structures; and/or which comprises a cross-beta structure which is not capable of specifically binding a cross-beta structure binding compound, preferably tPA, BiP, factor XII, fibronectin, at least one finger domain of tPA, at least one finger domain of factor XII, hepatocyte growth factor activator, at least one finger domain of fibronectin, at least one finger domain of hepatocyte growth factor activator, Thioflavin T, Thioflavin S, Congo Red, CD14, a multiligand receptor such as RAGE or CD36 or CD40 or LOX-1 or TLR2 or TLR4, a cross-beta-specific antibody, preferably cross-beta-specific IgG and/or cross-beta-specific IgM, IgIV, an enriched fraction of IgIV capable of specifically binding a cross-beta structure, Low density lipoprotein Related Protein (LRP), LRP Cluster II, LRP Cluster IV, Scavenger Receptor B-I (SR-BI), SR-A, chrysamine G, a chaperone, a heat shock protein, HSP70, HSP60, HSP90, gp95, calreticulin, a chaperonin, a chaperokine and/or a stress protein, at a detectable level.
In order to produce and/or select an immunogenic composition which is suitable for activating T-cells and/or a T-cell response, a method according to the invention preferably comprises the following steps: determining whether a peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein comprises a T-cell epitope motif; selecting a peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein comprising a T-cell epitope motif; providing a composition comprising the selected peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein; and providing the composition with at least one cross-beta structure.
In certain embodiments, a method according to the invention also comprises the production of an immunogenic composition which is capable of activating T-cells and/or a T-cell response, the composition comprising at least one peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein comprising a T-cell epitope and/or a T-cell epitope motif, the method comprising providing the composition with at least one cross-beta structure and determining: whether the degree of multimerization of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein in the composition allows recognition, binding, excision, processing and/or presentation of a T-cell epitope of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein by an animal's immune system; whether between 4-75% of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein content of the composition is in a conformation comprising cross-beta structures; whether the at least one cross-beta structure comprises a property allowing recognition, binding, excision, processing and/or presentation of a T-cell epitope of the peptide, polypeptide, protein, glycoprotein and/or lipoprotein by an animal's immune system; and/or whether a compound capable of specifically recognizing, binding, excising, processing and/or presenting a T-cell epitope of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein is capable of specifically recognizing, binding, excising, processing and/or presenting the T-cell epitope. The compound capable of specifically recognizing, binding, excising, processing and/or presenting a T-cell epitope preferably comprises a T-cell receptor (TCR), an MHC complex, and/or a component of the MHC antigen processing pathway.
In certain embodiments, it is determined whether a component of the MHC antigen processing pathway is capable of recognizing, binding, excising, processing and/or presenting a T-cell epitope of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein.
In order to produce and/or select an immunogenic composition which is suitable for activating T-cells and/or a T-cell response, an immunogenic composition whereby a component of the MHC antigen processing pathway is capable of recognizing, binding, excising, processing and/or presenting a T-cell epitope of the peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein is preferably selected.
The invention is further explained in the following examples. These examples do not limit the scope of the invention, but merely serve to clarify the invention.
abbreviations: ADCC, antibody dependent cell-mediated cytotoxicty; AFM, atomic force microscopy; ANS, 1-anilino-8-naphthalene sulfonate; aPMSF, 4-Amidino-Phenyl)-Methane-Sulfonyl Fluoride; BCA, bicinchoninic acid; bis-ANS, 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid; CD, circular dichroism; CR, Congo red; CSFV, Classical Swine Fever Virus; DLS, dynamic light scattering; DNA, Deoxyribonucleic acid; dOVA, misfolded ovalbumin comprising cross-beta; ELISA, enzyme linked immuno sorbent assay; ESI-MS, electron spray ionization mass spectrometry; FPLC, fast protein liquid chromatography; FVIII, coagulation factor VIII; g6p, glucose-6-phosphate; GAHAP, alkaline-phosphatase labeled goat anti-human immunoglobulin antibody; h, hour(s); H#, hemagglutinin protein of influenza virus, number #; HBS, HEPES buffered saline; HCV, hepatitis C virus; HGFA, Hepatocyte growth factor activator; HK, Hong kong; HPLC, high performance, or high-pressure liquid chromatography; HRP, horseradish peroxidase; hrs, hours; Ig, immunoglobulin; IgG, immunoglobulin of the class ′G; IgIM, immunoglobulins intramuscular; IgIV, immunoglobulins intravenous; kDa, kilo Dalton; LAL, Limulus Amoebocyte Lysate; MDa, mega Dalton; NMR, nuclear magnetic resonance; OVA, ovalbumin; PBS, phosphate buffered saline; Plg, plasminogen; RAGE, receptor for advanced glycation end-products; RAMPO, peroxidase labeled rabbit anti-mouse immunoglobulins antibody; RNA, ribonucleic acid; RSV, respiratory syncytial virus; RT, room temperature; SDS-PAGE, sodium-dodecyl sulphate-polyacryl amide gel electrophoresis; SEC, size exclusion chromatography; SWARPO, peroxidase labeled swine anti-rabbit immunoglobulins antibody; TEM, transmission electron microscopy; ThS, Thioflavin S; ThT, Thioflavin T; tPA, tissue type plasminogen activator; VN, Vietnam; W, tryptophan.
Cross-Beta Detection Assays
Congo red fluorescence. Congo red is a relatively small molecule (chemical name: C32H22N6Na2O6S2) that is commonly used as histological dye for detection of amyloid. The specificity of this staining results from Congo red's affinity for binding to fibrillar proteins enriched in beta-sheet conformation and comprising cross-beta. Congo red is also used to selectively stain protein aggregates with amyloid properties that do not necessarily form fibrils. Congo red is also used in a fluorescence enhancement assay to identify proteins with cross-beta in solution. This assay, also termed Congo red fluorescence measurement, is for example performed as described in patent application WO2007008072, paragraph [101], the contents of which are incorporated herein by this reference. Fluorescence can be read on various readers, for example fluorescence is read on a Gemini XPS microplate reader (Molecular Devices).
Thioflavin T fluorescence. Thioflavin T, like Congo red, is also used by pathologists to visualize plaques composed of amyloid. It also binds to beta sheets, such as those in amyloid oligomers. The dye undergoes a characteristic 115 nm red shift of its excitation spectrum that may be selectively excited at 442 nm, resulting in a fluorescence signal at 482 nm. This red shift is selectively observed if structures of amyloid fibrillar nature are present. It will not undergo this red shift upon binding to precursor monomers or small oligomers, or if there is a high beta sheet content in a non-amyloid context. If no amyloid fibrils are present in solution, excitation and emission occur at 342 and 430 nm respectively. Thioflavin T is often used to detect cross-beta in solutions. For example, the Thioflavin T fluorescence enhancement assay, also termed ThT fluorescence measurement, is performed as described in patent application WO2007008072, paragraph [101]. Fluoresence can de read on various readers, for example fluorescence is read on a Gemini XPS microplate reader (Molecular Devices).
Thioflavin S fluorescence. Thioflavin S, is a dye similar to Thioflavin T and the fluorescence assay is performed essentially similar to ThT and CR fluorescence measurements.
tPA binding ELISA. tPA binding ELISA with immobilized misfolded proteins; is performed as described in patent application WO2007008070, paragraph [35-36]. One of our first discoveries was that tPA binds specifically to misfolded proteins comprising cross-beta. Binding of tPA to misfolded proteins is mediated by its finger domain. Other finger domains and proteins comprising homologous finger domains are also applicable in a similar ELISA setup (see below).
BiP binding ELISA. BiP binding ELISA with immobilized misfolded proteins; is performed as described in patent application WO2007108675, section “Binding of BiP to misfolded proteins with cross-beta structure,” the contents of which are incorporated herein by this reference, with the modification that BiP purified from cell culture medium using Ni2+ based affinity chromatography, is used in the ELISAs. It has been demonstrated previously that chaperones like for example BiP bind specifically to misfolded proteins comprising cross-beta. Other heat shock proteins, such as hsp70, hsp90 are also applicable in a similar ELISA setup.
IgIV binding ELISA. Immunoglobulins intravenous (IgIV) binding ELISA with immobilized misfolded proteins; is performed as described in patent application WO2007094668, paragraph [0115-0117], the contents of the entirety of which are incorporated herein by this reference. Alternatively, IgIV that is enriched using an affinity matrix with immobilized protein(s) comprising cross-beta, is used for the binding ELISA with immobilized misfolded proteins (see patent application WO2007094668, paragraph [0143]). It has been demonstrated previously that a subset of immunoglobulins in IgIV bind selectively and specifically to misfolded proteins comprising cross-beta. Other antibodies directed against misfolded proteins are also applicable in a similar ELISA setup.
Finger binding ELISA using fibronectin finger domains. Fibronectin finger 4-5 binding ELISA with immobilized misfolded proteins; is performed as described in patent application WO2007008072. It has been demonstrated previously that finger domains of fibronectin selectively and specifically bind to misfolded proteins comprising cross-beta. In addition to, or alternative to finger domains of fibronectin, finger domains of tPA and/or factor XII and/or hepatocyte growth factor activator are used.
Factor XII activation assay. Factor XII/prekallikrein activation assay is performed as described in patent application WO2007008070, paragraph [31-34]. It has been demonstrated previously that factor XII selectively and specifically bind to misfolded proteins comprising cross-beta, resulting in its activation.
tPA/plasminogen activation assay. Enhancement of tPA/plasminogen activity upon exposure of the two serine proteases to misfolded proteins was determined using a standardized chromogenic assay (see for example patent application WO2006101387, paragraph [0195], patent application WO2007008070, paragraph [31-34], and [Kranenburg et al., 2002, Curr. Biology 12(22), pp. 1833)]. Both tPA and plasminogen act in the Cross-beta Pathway. Enhancement of the activity of the cross-beta binding proteases is a measure for the presence of misfolded proteins comprising cross-beta structure. 4-Amidinophenylmethanesulfonyl fluoride hydrochloride (aPMSF, Sigma, A6664) was added to protein solutions to a final concentration of 1.25 mM from a 5 mM stock. Protein solutions with added aPMSF were kept at 4° C. for 16 h before use in a tPA/plasminogen activation assay. In this way, proteases that are putatively present in protein solutions to be analyzed, and that may act on tPA, plasminogen, plasmin and/or the chromogenic substrate for plasmin, are inactivated, to prevent interference in the assay.
Binding assays. Apart from the above described binding assays using cross-beta binding compounds, additional cross-beta binding compounds are used in binding assays for determination of the presence and extent of cross-beta in a sample of a peptide, polypeptide, protein, glycoprotein, protein-DNA complex, protein-membrane complex and/or lipoprotein. In general, cross-beta binding compounds useful for these determinations are tPA, BiP, factor XII, fibronectin, hepatocyte growth factor activator, at least one finger domain of tPA, at least one finger domain of factor XII, at least one finger domain of fibronectin, at least one finger domain of hepatocyte growth factor activator, Thioflavin T, Thioflavin S, Congo Red, CD14, a multiligand receptor such as RAGE or CD36 or CD40 or LOX-1 or TLR2 or TLR4, a cross-beta-specific antibody, preferably cross-beta-specific IgG and/or cross-beta-specific IgM, IgIV, an enriched fraction of IgIV capable of specifically binding a cross-beta structure, Low density lipoprotein Related Protein (LRP), LRP Cluster II, LRP Cluster IV, Scavenger Receptor B-I (SR-BI), SR-A, chrysamine G, a chaperone, a heat shock protein, HSP70, HSP60, HSP90, gp95, calreticulin, a chaperonin, a chaperokine and/or a stress protein. In addition, as disclosed previously in patent application WO2007008072, cross-beta binding compounds for use for the aforementioned determinations are 2-(4′-(methylamino)phenyl)-6-methylbenzothiaziole, styryl dyes, BTA-1, Poly(thiophene acetic acid), conjugated polyelectrolyte, PTAA-Li, Dehydro-glaucine, Ammophedrine, isoboldine, Thaliporphine, thalicmidine, Haematein, ellagic acid, Ammophedrine HBr, corynanthine, Orcein.
Turbidity of protein solutions. With turbidity measurements the diffraction of light scattered by protein particles in the sample is detected. Light is scattered by the solid particles and absorbed by dissolved protein. In a turbidity measurement the amount of insoluble particles in a solution is determined. This aspect is used to determine the amount of insoluble protein in samples of protein that is subjected to misfolding conditions, compared to the fraction of insoluble protein in the non-treated reference sample.
Recording changes in binding characteristics of binding partners for a protein. Antibodies specific for a protein in a certain conformation are used to measure the amount of this protein present in this specific state. Upon treatment of the protein using misfolding conditions, binding of antibodies is inhibited or diminished, which is used as a measure for the progress and extent of misfolding. In addition or alternatively, antibodies are used that are specific for certain conformations and/or post-translational modifications, for example glycation, oxidation, citrullination (gain of binding to the protein subjected to misfolding conditions). When for example glycation and/or oxidation and/or citrullination procedures is/are part of the misfolding procedure, the effect of the treatment with respect to the occurrence of modified amino-acid residues is recorded by determining the relative binding of the antibodies, compared to the non-treated reference protein. Alternatively or in addition to the use of antibodies, any binding partner and/or ligand of the non-treated protein is used similarly, and/or any binding partner and/or ligand other than antibodies, of the misfolded protein is used. When a protein changes conformation ligands or binding partners express altered binding characteristics, which is used as a measure for the extent of protein modification and/or extent of misfolding. This binding of antibodies, ligands and/or binding partners is measured using various techniques, such as direct and/or indirect ELISA, surface plasmon resonance, affinity chromatography and immuno-precipitation approaches.
Differential scanning calorimetry/micro DSC for detecting changes in protein conformation. Differential scanning calorimetry (DSC) is a thermo-analytical technique in which the difference in the amount of heat required to increase the temperature of a sample and a reference is measured as a function of temperature. The temperature is linearly increased over time. When the protein in the sample changes its conformation, more or less heat (depending on if it is an endo- or exothermic reaction) will be required to increase the temperature at the same rate as the reference sample. In this way the conformational changes as a result of an increase in temperature can be measured.
Particle analyzer. A particle analyzer measures the diffraction of a laser beam when targeted at a sample. The resulting data is transformed by a Fourier transformation and gives information about particle size and shape. When applied to protein solutions, putatively present protein aggregates are detected, when larger than the lower detection limit of the apparatus, for example in the sub-micron range.
Direct light microscope. With a regular direct-light microscope with a preferable magnification range of 10×-100×, one can determine visually if there are any protein aggregates present in a sample.
Photon correlation spectroscopy (dynamic light scattering spectroscopy). Photon correlation spectroscopy can be used to measure particle size distribution in a sample in the nm-μm range.
Nuclear magnetic resonance spectroscopy. Nuclear Magnetic Resonance Spectroscopy (NMR) can be used to assess the electromagnetic properties of certain nuclei in proteins. With this technique the resonance frequency and energy absorption of protons in a molecule are measured. From this data structural information about the protein, like angles of certain chemical bonds, the lengths of these bonds and which parts of the protein are internally buried, can be obtained. This information can then be used to calculate the complete three dimensional structure of a protein. This method however is normally restricted to relatively small molecules. However with special techniques like incorporation of specific isotopes and transverse relaxation optimized spectroscopy, much larger proteins can now be studied with NMR.
X-ray diffraction. In X-ray diffraction with protein crystals, the elastic scattering of X-rays from a crystallized protein is measured. In this way the arrangement of the atoms in the protein can be determined, resulting in a three-dimensional structural model of the protein. First a protein is crystallized and then a diffraction pattern is measured by irradiating the crystallized protein with an X-ray beam. This diffraction pattern is a representation of how the X-ray beam is scattered from the electrons in the crystal. By gradually rotating the crystal in the X-ray beam, the different atomic positions in the crystal can be determined. This results in an electron density map, with which a complete three-dimensional atomic model of the crystallized protein can be calculated, regularly at the 1-3 Å scale. In this model it can be deduced whether protein molecules underwent conformational changes upon treatment with misfolding conditions, when compared to the structural model of the non-treated protein. In addition, modifications of amino-acid residues become apparent in the structural model, as well as whether the protein molecule forms ordered multimers of a defined size, like for example in the range of dimers-octamers.
Determination of the presence of cross-beta in fibers comprising crystallites, and/or in other appearances of protein aggregates comprising at least a fraction of the protein molecules in a crystalline ordering, can be assessed using X-ray fiber diffraction, as for example shown in [Bouma et al., J. Biol. Chem. V278, No. 43:41810-41819, 2003, “Glycation Induces Formation of Amyloid Cross-beta Structure in Albumin”].
Fourier Transform infrared spectroscopy. Detection of protein secondary structure in Fourier Transform Infrared Spectroscopy (FTIR), an infrared beam is split in two separate beams. One beam is reflected on a fixed mirror, the second on a moving mirror. These two beams together generate an interferogram which consists of every infrared frequency in the spectrum. When transmitted through a sample specific functional groups in the protein adsorb infrared of a specific wavelength. The resulting interferogram must be Fourier transformed, before it can be interpreted. This Fourier transformed interferogram gives a plot of al the different frequencies plotted against their adsorption. This interferogram is specific for the structure of a protein, like a “molecular fingerprint,” and provides information on types of atomic bonds present in the molecule, as well as the spatial arrangement of atoms in for example alpha-helices or beta-sheets.
8-Anilino-1-naphthalenesulfonic acid fluorescence enhancement assay. 8-Anilino-1-naphthalenesulfonic acid (ANS) fluorescence enhancement assay, or ANS fluorescence measurement; was performed as described in patent application WO2007094668. Modification: fluorescence is read on a Gemini XPS microplate reader (Molecular Devices).
ANS is a chemical binds to hydrophobic surfaces of a protein and its fluorescence spectrum shifts upon binding. When proteins are in an unfolded state, they generally display more hydrophobic sites, resulting in an increased ANS shift compared to the protein in its native more globular state. ANS can therefore be used to measure protein unfolding.
bis-ANS fluorescence enhancement assay. 4,4′ dianilio-1,1′ binaphthyl-5,5′ disulfonic acid di-potassium salt (Bis-ANS) fluorescence enhancement assay; is performed as described in patent application WO2007094668. Essentially, bis-ANS has characteristics comparable to ANS, and bis-ANS is also used to probe for differences in solvent exposure of hydrophobic patches of proteins, when measuring bis-ANS binding with a reference protein samples, and with a protein sample subjected to a misfolding procedure.
Gel electrophoresis. Gel electrophoresis using sodium dodecyl-sulphate polyacryl amide gels (SDS-PAGE) and Coomassie stain, with various gels with resolutions between for example 100 Da up to several thousands of kDa, provides information on the occurrence of protein modifications and on the occurrence of multimers. Multimers that are not covalently coupled may also appear as monomers upon the assay conditions applied, i.e., heating protein samples in assay buffer comprising SDS. Samples are heated in the presence or absence of a reducing agent like for example dithiothreitol (DTT), when the protein amino-acid sequence comprises cysteines, that can form disulphide bonds upon subjecting the protein to misfolding conditions.
Western blot. When antibodies are available that bind to epitopes on the protein under the denaturing conditions as applied during SDS-PAGE, Western blotting is performed with the same protein samples as applied for SDS-PAGE with Coomassie stain, using the same molecular weight cut-off gels, and using the same protein sample handling approaches.
Centrifugation. Centrifugation and subsequent comparing the protein concentration in the supernatant with respect to the concentration before centrifugation provides insight into the presence of insoluble precipitates in a protein sample. Upon applying increasing g-forces for a constant time, and/or upon applying fixed or increasing g-forces for an increasing time frame, to a protein solution, with analyzing the protein content in between each step, information is gathered about the presence of insoluble multimers. For example, protein solutions are subjected for 10 minutes to 16,000*g, or for 60 minutes to 100,000*g. The first approach is commonly used to prepare protein solutions for, for example use on FPLC columns or in biological assays, with the aim of pelleting insoluble protein aggregates and using the supernatant with soluble protein. It is generally accepted that after applying 100,000*g for 60 minutes to a protein solution, only soluble multimers are left in the supernatant. As multimers ranging from monomers up to huge multimers comprising thousands of protein monomers may all have a density equal to the density of the buffer solution, applying these g-forces to protein solutions does not separate exclusively on size, but on density differences between the solution and the protein multimers.
Electron spray ionization mass spectrometry. Electron spray ionization mass spectrometry (ESI-MS) with protein solutions provides information on the multimer size distribution when sizes range from tens of Da up to the MDa range.
Ultrasonic spectrometry. Ultrasonic spectroscopy analysis, for example using an Ichos-II (Process Analysis and Automation, Ltd), provides insight into protein conformation and changes in tertiary structure are measured. In addition the technique can provide information on particle size of protein assemblies, and allows for monitoring protein concentration.
Dialysis (membranes with increasing molecular weight cut-off). Using one or a series of dialysis membranes with varying molecular weight cut-offs, size distribution/multimer distribution of protein can be assessed at the sub-oligomer scale, depending on the molecular weight of the monomer. Protein concentration analysis between each dialysis step with gradually increasing pore size (suitable for molecular weight ranges between approximately 1000-50000 Da). Protein concentration is for example monitored using BCA or Coomassie+determinations (Pierce), and/or absorbance measurements at 280 nm, using for example the nanodrop technology (Attana).
Filtration (filters with increasing molecular weight cut-off). Filtration using a series of filters with gradually increasing MW cut-offs, ranging from the monomer size of the protein under investigation up to the largest MW cut-off available, reveals information on the distribution and presence of protein molecules in multimers in the range from monomers, lower-order multimers and large multimers comprising several hundreds of monomers. For example, filters with a MW cut-off of I kDa up to filters with a cut-off of 5 μm (MWs for example 1/3/10/30/50/100 kDa, completed with filters with cut-offs of for example 200/400/1000/5000 nm). In between each subsequent filtration step, protein concentration is assessed using for example the BCA or Coomassie+method (Pierce), and/or visualization on SDS-PA gel stained with Coomassie.
Transmission electron microscopy. Transmission electron microscopy (TEM) is a imaging technique that provides structural information of proteins at a nm to μm scale. With this resolution it is possible to identify the occurrence of protein assemblies ranging from monomers up to multimers of several thousands molecules, depending on the molecular weight of the parent protein molecule. Furthermore, TEM imaging provides insight into the structural appearance of protein multimers. For example, protein multimers appear as rods, globular structures, strings of globular structures, amorphous assemblies, unbranched fibers, commonly termed fibrils, branched fibrils, and/or combinations thereof.
In the current studies, TEM images were collected using a Jeol 1200 EX transmission electron microscope (Jeol Ltd., Tokyo, Japan) at an excitation voltage of 80 kV. For each sample, the formvar and carbon-coated side of a 100-mesh copper or nickel grid was positioned on a 5 μl drop of protein solution for 5 minutes. Afterwards, it was positioned on a 100 μl drop of PBS for 2 minutes, followed by three 2-minute incubations with a 100 μl drop of distilled water. The grids were then stained for 2 minutes with a 100 μl drop of 2% (m/v) methylcellulose with 0.4% uranyl acetate pH 4. Excess fluid was removed by streaking the side of the grids over filter paper, and the grids were subsequently dried under a lamp. Samples were analyzed at a magnification of 10 K.
Atomic force microscopy. Similar to TEM imaging, atomic force microscopy provides insights into the structural appearance of protein molecules at the protein monomer level up to the macroscopic level of large multimers of protein molecules.
Size exclusion chromatography, or gel filtration chromatography. With size exclusion chromatography (SEC) using HPLC and/or FPLC, a qualitative and quantitative insight is obtained about the distribution of protein molecules over monomers up to multimers, with a detectable size limit of the multimers restricted by the type of SEC column that is used. SEC columns are available with the ability to separate molecular sizes in the sub kDa range up to in the MDa range. The type of column is selected based on the molecular weight of the analyzed protein, and on any indicative information at forehand about the expected range of multimeric sizes. Preferably, a reference non-treated protein is compared to a protein that is subjected to misfolding procedures.
Tryptophan fluorescence. Assessment of differences in tryptophan (W) fluorescence intensity between two appearances of the same protein provides information on the occurrence of protein folding differences. In general, in globular proteins W residues are mostly buried in the interior of the globular fold. Upon unfolding, refolding, misfolding, W residues tend to become more solvent exposed, which is recorded in the W fluorescence measurement as a change in fluorescent intensity compared to the protein with a more native fold.
Dynamic Light Scattering. With the Dynamic Light Scattering (DLS) technique, particle size and particle size distribution is assessed. When protein solutions are considered distribution of proteins over a range of multimers ranging from monomers up to multimers is measured, with the upper limit of detected multimer size limited by the resolution of the DLS technique.
Circular dichroism spectropolarimetry. With circular dichroism spectropolarimetry (CD) the relative presence of protein secondary structural elements is determined. Therefore, this technique allows for the comparison of the relative occurrence of alpha-helix, beta-sheet and random coil between a reference protein that is non-treated, and the protein that is subjected to misfolding conditions. An example of a CD experiment for assessment of conformational changes in proteins upon treatment with misfolding conditions is given in [Bouma et al., J. Biol. Chem. V278, No. 43:41810-41819, 2003, “Glycation Induces Formation of Amyloid Cross-beta Structure in Albumin”].
Native gel electrophoresis. Distribution over multimers in the range of approximately monomers up to 100-mers is assessed by applying native gel electrophoresis. For this purpose a reference non-treated protein sample is compared to a protein sample which is subjected to a misfolding procedure. When misfolding procedures are applied that introduce modifications on amino-acid residues, like for example but not limited to, glycation or oxidation or citrullination, these changes are becoming apparent on native gels, as well.
Examples of Proteins that are Used for Preparation of Immunogenic Compositions
Envelope protein E2 of Classical Swine Fever Virus. The envelope protein E2 of Classical Swine Fever Virus (CSFV) strain Brescia 456610 is used as a prototype subunit vaccine candidate for examples described below. Currently, a subunit vaccine that provides protection in pigs against CSF comprises recombinantly produced E2 antigen in cell culture medium, adjuvated with a double emulsion of water-in-oil-in-water, comprising PBS, Marcol 52, Montanide 80. The vaccine comprises at least 32 μg E2/dose of 2 ml, and is injected intramuscularly.
E2 was recombinantly produced in insect Sf9 cells (Animal Sciences Group, Lelystad, The Netherlands) or in human embryonic kidney 293 cells (293) (ABC-Protein Expression facility, University of Utrecht, The Netherlands), as described in patent application WO2007008070. E2 produced in Sf9 cells and lacking any tags is in PBS after dialysis of cell culture medium (storage of aliquots at −20° C. or at −80° C.), or in cell culture medium (storage at −20° C.). Cell culture medium is SF900 II medium with 0.2% pluronic (serum free). After culturing of cells, the cell culture medium is micro-filtrated. Virus is inactivated with 8-12 mM 2-bromo-ethyl-ammonium bromide. The E2 produced in 293 cells comprises a C-terminal FLAG-tag followed by a His-tag, and is purified using Ni2+-based affinity chromatography. Concentration and purity of E2 from both sources is determined as follows. Quantification of the total protein concentration is performed with the BCA method (Pierce) or with the Coomassie+method (Pierce). E2 specific bands on a Western blot are visualized using anti-FLAG antibody (mouse antibody, M2, peroxidase conjugate; Sigma, A-8592) for the E2-FLAG-His construct, and a 1:1:1 mixture of three horseradish peroxidase (HRP) tagged mouse monoclonal anti-E2 antibodies (CediCon CSFV 21.2, 39.5 and 44.3; Prionics Lelystad) for the E2-FLAG-His construct and the E2 construct from Sf9 cells. The purity of E2 batches was determined by densitometry with a Coomassie stained sodium dodecyl sulphate-polyacryl amide (SDS-PA) gel after electrophoresis.
In
Before use in misfolding procedures, cross-beta analyses, multimer analyses and/or immunization, non-treated E2 solution was warmed to 37° C. for 10-30 minutes, left on a roller device for 10-30 minutes, at room temperature, warmed again at 37° C. for 0-30 minutes and left again on a roller device for 0-30 minutes. Alternatively, non-treated E2 solutions were quickly thawed at 37° C. and directly kept on wet ice until further use.
ovalbumin. Ovalbumin is incorporated as a candidate ingredient of immunogenic compositions comprising cross-beta structure. The ovalbumin is either serving as the antigen itself, to which an immune response should be directed, or ovalbumin is used as the cross-beta adjuvant part in immunogenic compositions, comprising a target antigen with a different amino-acid sequence. For this latter use, ovalbumin comprising cross-beta is combined with the target antigen, to which an immune response is desired. Cross-beta adjuvated ovalbumin is for example covalently coupled to the antigen of choice, using coupling techniques known to a person skilled in the art. When ovalbumin is the target antigen itself, non-treated ovalbumin and cross-beta-adjuvated ovalbumin are used in a similar way, in immunogenic composition preparations.
Lyophilized ovalbumin, or chicken egg-white albumin (OVA, Sigma, A5503 or A7641) is dissolved as follows. OVA is gently dissolved at indicated concentration in phosphate buffered saline (PBS; 140 mM sodium chloride, 2.7 mM potassium chloride, 10 mM disodium hydrogen phosphate, 1.8 mM potassium dihydrogen phosphate, pH 7.3; local pharmacy), avoiding any foam formation, stirring, vortexing or the like. OVA is dissolved by gently swirling, 10 minutes rolling on a roller device, 10 minutes warming in a 37° C.-water bath, followed by 10 minutes rolling on a roller device. Aliquots in Eppendorf tubes are frozen at −80° C. Before use, OVA solution is either prepared freshly, or thawed from −80° C. to 0° C., or after thawing kept at 37° C. for 30 minutes. Furthermore, an OVA solution is applied to an endotoxin affinity matrix for removal of endotoxins present in the OVA preparation. Before and after applying OVA to the matrix, endotoxin levels are determined using an Endosafe apparatus (Charles River), and/or using a chromogenic assay for determining endotoxin levels (Cambrex), both using Limulus Amoebocyte Lysate (LAL). Misfolded OVA, termed dOVA, is prepared as indicated below (see Section “Protocols for introducing cross-beta in proteins”).
Hemagglutinin 5 protein of H5N1 virus strain A/Hong kong/156/97. Hemagglutinin 5 protein (H5) of H5N1 virus strain A/Hong kong/156/97 (A/HK/156/97) is expressed in 293 cells with a C-terminal FLAG tag and His tag, and purified using Ni2+-based affinity chromatography as described in patent application WO/2007/008070, the contents of which are incorporated herein by this reference. In addition, the recombinantly produced H5-FLAG-His construct is purified using affinity chromatography with the anti-FLAG antibody M2 immobilized on a matrix (Sigma, A2220), according to the manufacturer's recommendations and using FLAG peptide (Sigma, F3290) for elution of H5-FLAG-His from the matrix. Protein solutions are stored at −80° C. for a long term and after micro filtration at 4° C., for a short term. In this example, upon purification using anti-FLAG antibody based affinity chromatography, two batches of H5 were obtained. One batch of H5-FLAG-His is termed non-treated H5, batch 2 (“nH5-2,” concentration 30 μg/ml). A second batch of H5-FLAG-His was subsequently subjected to size-exclusion chromatography (SEC) using a HiLoad 26/60 Superdex 200 column on an Äkta Explorer (GE Healthcare; used at the ABC-protein expression facilities of the University of Utrecht, Dr R. Romijn & Dr. W. Hemrika). For this purpose, H5-FLAG-His solution in PBS is concentrated on Macrosep Centrifugal Devices 10K Omega (Pall Life Sciences) or CENTRIPREP Centrifugal Filter Devices YM-300 (Amicon). Running buffer was PBS. The H5 batch after the SEC run, termed non-treated H5, batch 1 (“nH5-1”), was stored at 4° C. after micro filtration (concentration 400 μg/ml, as determined with the BCA method). This batch nH5-1 is used for misfolding procedures described below.
H5 of H5N1 strain A/Vietnam/1203/04. H5 of H5N1 strain A/Vietnam/1203/04 (A/VN/1203/04) is purchased from Protein Sciences, and consists mainly of HA2, with relatively lower amounts of HA1 and HA0. Purity is 90%, as determined with densitometry, according to the manufacturer's information. Buffer and excipients are 10 mM sodium phosphate, 150 mM NaCl, 0.005% Tween80, pH 7.2. The H5 concentration is 922 μg/ml (lot 45-05034-2) or 83 μg/ml (lot 45-05034RA-2). This non-treated H5 is termed “nH5” and stored at 4° C. or at −80° C.
Factor VIII. Factor V111 (FVIII) of human plasma origin or recombinantly produced based on cDNA coding for human FVIII is used. Examples of suitable FVIII preparations are Helixate (Nexgen), Kogenate (Bayer), Advate (Baxter), Recombinate (Baxter), ReFacto (FVIII in which the B-domain is deleted; Wyeth), which are all recombinantly produced, and AAfact (Sanquin) and Haemate P (Aventis Behring), which are purified from blood. FVIII preparations are dissolved according to the manufacturer's recommendations. For the examples disclosed below, Helixate (NexGen 250 IE/vial, lot 80A0777, exp. date: 03.2007) is used, termed non-treated FVIII and designated as “FVIII.”
The proteins described above are used for preparation of immunogenic compositions. However, the disclosed technologies are by no means restricted to the generation of immunogenic compositions comprising OVA, FVIII, H5 of A/VN/1203/04 or A/HK/156/97, or E2. Examples that further disclose the described technologies and their applications are also generated using other and/or additional peptides, polypeptides, proteins, glycoproteins, protein-DNA complexes, protein-membrane complexes and/or lipoproteins as a basis for immunogenic compositions. These peptides, polypeptides, proteins, glycoproteins, protein-DNA complexes, protein-membrane complexes and/or lipoproteins are the antigen component, the cross-beta-adjuvated component or both the antigen component and the cross-beta-adjuvated component of immunogenic compositions. The peptides, polypeptides, proteins, glycoproteins, protein-DNA complexes, protein-membrane complexes and/or lipoproteins are for instance originating from amino-acid sequences unrelated to pathogens and/or diseases, when used as the cross-beta-adjuvated ingredient of an immunogenic composition, or are for instance originating from amino-acid sequences that are related to and/or involved in and/or are part of pathogens, tumors, cardiovascular diseases, atherosclerosis, amyloidosis, autoimmune diseases, graft-versus-host rejection and/or transplant rejection, when they are part of the target antigen and/or are the cross-beta-adjuvated ingredient of an immunogenic composition. In fact, the disclosed technologies are applicable to any amino-acid sequence, either of the antigen, or of the cross-beta-adjuvant.
Non-limiting examples of peptides, polypeptides, proteins, glycoproteins, protein-DNA complexes, protein-membrane complexes and/or lipoproteins that are used as antigen and/or as cross-beta-adjuvant are for example virus surface proteins, bacterial surface proteins, pathogen surface exposed proteins, gp120 of HIV, proteins of human papilloma virus, any of the neuramidase proteins or hemagglutinin proteins or any of the other proteins of any influenza strain, surface proteins of blue tongue virus, proteins of foot- and mouth disease virus, bacterial membrane proteins, like for example PorA of Neisseria meningitides, oxidized low density lipoprotein, tumor antigens, tumor specific antigens, amyloid-beta, antigens related to rheumatoid arthritis, B-cell surface proteins CD19, CD20, CD21, CD22, proteins suitable for serving as target for immunocastration, proteins of hepatitis C virus (HCV), proteins of respiratory syncytial virus (RSV), proteins specific for non small cell lung carcinoma, malaria antigens, proteins of hepatitis B virus.
Peptides, polypeptides, proteins, glycoproteins, protein-DNA complexes, protein-membrane complexes and/or lipoproteins, in summary referred to as “protein” throughout this section, are misfolded with the occurrence of cross-beta structure after subjecting them to various cross-beta-inducing procedures. Below, a summary is given of a non-limiting series of those procedures, which are preferably applied to the proteins used in immunogenic compositions.
Misfolding of proteins with the occurrence of cross-beta is induced using selected combinations of several parameters. The following parameters settings are applied for proteins:
Furthermore, protein misfolding is induced for example by, but not limited to, post-translational modifications like for example glycation, using for example carbohydrates, oxidation, using for example CuSO4, citrullination, using for example using peptidylarginine deiminases, acetylation, sulfatation, (partial) de-sulfatation, (partial) de-glycosylation, enzymatic cleavage, polymerization, exposure to chaotropic agents like urea (for example 0.1-8 M) or guanidinium-HCl (for example 0.1-7 M).
Misfolding of proteins with appearance of cross-beta is also achieved upon subjecting proteins to exposure to adjuvants currently in use or under investigation for future use in immunogenic compositions. Proteins are exposed to adjuvants only, or the exposure to adjuvants is part of a multi-parameter misfolding procedure, designed based on the aforementioned parameters and conditions. Non-limiting examples of adjuvants that are implemented in protocols for preparation of immunogenic compositions comprising cross-beta are alum (aluminium-hydroxide and/or aluminium-phosphate), MF59, QS21, ISCOM matrix, ISCOM, saponin, QS27, CpG-ODN, flagellin, virus like particles, IMO, ISS, lipopolysaccharides, lipid A and lipid A derivatives, complete Freund's adjuvant, incomplete Freund's adjuvant, calcium-phosphate, Specol.
A typical method for induction of cross-beta conformation in a protein is designed as follows in a matrix format, from which preferably subsets of parameter settings are selected.
Subsets of selected parameter settings are for example as follows.
Misfolding of E2. E2 protein is misfolded accompanied by introduction of cross-beta, by applying various parameter ranges, selected from described parameters a-f (see above). For example, E2 concentration ranges from 50 μg/ml to 2 mg/ml; selected pH is 2, 7.0-7.4 and 12; selected NaCl concentration is 0-500 mM, for example 0/50/150/500 mM; selected buffer is PBS or HBS or no buffer (H2O); selected temperature gradient is for example as described for OVA, below. For example, E2 at approximately 300 μg/ml in PBS, heated in PCR cups in a PTC-200 thermal cycler (MJ Research, Inc.): 25° C. for 20 seconds and subsequently heated (0.1° C./second) from 25° C. to 85° C. followed by cooling to 4° C. for 2 minutes. This cycle is for example repeated twice (total number of cycles is 3). For example, E2 is subsequently stored at −20° C.
For the examples described below, non-treated E2 (nE2) at approximately 280 μg/ml in PBS was incubated at 25° C. for 20 seconds and was subsequently gradiently heated (0.1° C./second) from 25° C. to 85° C. followed by cooling at 4° C. for 2 minutes. This cycle was repeated twice and then, the E2 solution, referred to as cross-beta E2 (cE2) was stored at −20° C.
Structural differences and differences in cross-beta content between nE2 and cE2 were assessed using ThT fluorescence measurement, tPA/Plg activation analysis and TEM imaging. See
Misfolding of OVA. OVA is for example misfolded with introduction of cross-beta using the following misfolding procedures:
OVA was subjected to the following misfolding procedure for inducing cross-beta conformation. OVA was dissolved in PBS to a concentration of 1.0 mg/ml. The solution was put on a roller device for 10 minutes at room temperature (RT), than 10 minutes at 37° C. in a water bath and subsequently again for 10 minutes on the roller device (RT). Then, 200 μl aliquots of OVA solution was heat-treated in a PTC-200 PCR machine (MJ Research) as follows: five cycles of heating from 30° C. to 85° C. at 5° C./minute; cooling back to 30° C. After five cycles misfolded OVA, termed dOVA, was cooled to 4° C. and subsequently stored at −80° C. This preparation of dOVA is used as a standard reference, termed “standard,” with cross-beta content that results in a maximal signal (arbitrarily set to 100%) in indicated cross-beta detecting assays, at a given concentration.
Cross-beta analyses are performed with dOVA standard at a regular basis in our laboratories. For example in
Misfolding of H5 of H5N1 strain A/HK/156/97. The H5-FLAG-His batch nH5-1, obtained after anti-FLAG antibody affinity chromatography and size exclusion chromatography, was subjected to two misfolding procedures.
For structure analyses and for formulation of vaccine candidate solution, before use the nH5-1 and nH5-2 were centrifuged for 10 minutes at 16,000*g at room temperature. CH5-A and CH5-B were used without the centrifugation step.
The nH5-1 and CH5-B samples were analyzed on an analytical SEC column (U-Express Proteins, Utrecht, The Netherlands). For this purpose, approximately 80 μl of the 400 μg/ml stocks was applied to a Superdex200 10/30 column, connected to an Äkta Explorer (GE Healthcare). Running buffer was PBS. Samples were centrifuged for 20 minutes at 13,000*g before loading onto the column. The samples were run at a flow rate of 0.2 ml/minute and elution of protein was recorded by measuring absorbance at 280 nm.
The nH5-1 and nH5-2 preparations appear on SDS-PA gel and Western blot as multimers ranging from monomer up till aggregates that do not enter the gel (
The nH5-1 and nH5-2 preparations comprise a considerable amount of cross-beta conformation, as depicted in
Misfolding of H5 of H5N1 strain A/VN/1203/04. H5 of H5N1 strain A/VN/1203/04, as obtained from Protein Sciences, was subjected to four misfolding procedures, as indicated below.
1. nH5
For comparison, NaCl from a 5 M stock was added to non-treated H5 stock (922 μg/ml, 4° C., 150 mM NaCl), to a final concentration of 171 mM NaCl, and subsequently aliquoted in Eppendorf cups and stored at −20° C. Endotoxin level: <0.05 EU/10 μg/ml solution nH5 (determined using an Endosafe pts apparatus (Charles River). The solution was clear and colorless. For structure analyses and for formulation of vaccine candidate solution, before use the nH5 was centrifuged for 10 minutes at 16,000*g at room temperature.
2. CH5-1
Aliquots of nH5 in PCR strips (Roche) were incubated at 25° C. for 20 seconds and subsequently gradiently heated (0.1° C./second) from 25° C. to 85° C. followed by cooling back to 4° C., and kept at 4° C. for 2 minutes. This heat cycle was repeated twice. Then, aliquots in Eppendorf 500 μL cups were stored at −20° C. Code: “CH5-1.” The preparation CH5-1 was slightly turbid with some visible precipitates after heat treatment.
3. CH5-2
The pH of the nH5 stock kept at 4° C., was lowered to pH 2 by adding HCl from a 15% v/v stock. Then, aliquots of 100 μL/cup in PCR strips were heated in a PTC-200 thermal cycler, as follows. The samples were incubated at 25° C. for 20 seconds and subsequently gradiently heated (0.1° C./second) from 25° C. to 85° C. followed by cooling back to 4° C., and kept at 4° C. for 2 minutes. This heat cycle was repeated twice. Subsequently, the pH was adjusted to pH 7 by adding a volume NaOH solution from a 5 M stock. Then, aliquots in Eppendorf 500 μL cups were stored at −20° C. Code: “CH5-2.” The solution was clear and colorless.
4. CH5-3
The pH of nH5 kept at 4° C., was elevated to pH 12 by adding a volume NaOH solution from a 5 M stock. Then, aliquots of 100 μL/cup in PCR strips were treated as follows in a PTC-200 thermal cycler. The samples were incubated at 25° C. for 20 seconds and subsequently gradiently heated (0.1° C./second) from 25° C. to 85° C. followed by cooling back to 4° C., and kept at 4° C. for 2 minutes. This heat cycle was repeated twice. Subsequently the pH was adjusted to pH 7 by adding a volume HCl solution from a 5 M stock. Then, aliquots in Eppendorf 500 μL cups were stored at −20° C. Code: “CH5-3.” The solution was clear and colorless.
5. CH5-4
D-Glucose-6-phosphate disodium salt hydrate (g6p, Sigma; G7250) was added from a 2 M stock in PBS to nH5 to a final concentration of 100 mM g6p (20-fold dilution). Then it was incubated for 67 h at 80° C. The solution was intensively dialyzed against PBS, aliquoted in Eppendorf 500 μL cups, and stored at −20° C. The solution was light brown with white precipitates, visible by eye.
For structure analyses and for formulation of vaccine candidate solution, before use the nH5 was centrifuged for 10 minutes at 16,000*g at room temperature. CH5-1 to 4 were used without the centrifugation step.
The nH5 protein, as purchased from Protein Sciences, appears predominantly as the approximately 25 kDa HA2 fragment, with a smaller content of HA0 (full-length H5) and HA1 (molecular weight approximately 50 kDa) on reducing and non-reducing SDS-PA gels, stained with Coomassie (
The nH5 appears on a TEM image as amorphous multimers which are relatively small in size and which tend to aggregate into clusters, as seen in the supernatant after 10 minutes centrifugation at 16,000*g (
ThT fluorescence is enhanced with CH5-1 to 3, when compared to nH5 (
Misfolding of FVIII. FVIII is for example misfolded by using from the above listed spectrum of misfolding procedures parameter combinations as follows. Helixate sterile stock solution is preferably prepared according to the manufacturer's recommendations (100 IE/ml) and is subsequently used directly as freshly dissolved ingredient for immunogenic compositions, termed “FVIII” and numbered “9,” and used as non-treated FVIII.
For preparation of immunogenic compositions FVIII was subjected to the following procedures:
1) FVIII kept at 4° C. for 20 hours, in the dark, followed by storage at −80° C.→referred to as cross-beta FVIII-1 (cFVIII-1), or 1
2) FVIII kept at room temperature for 20 hours, in the dark, followed by storage at −80° C.→cFVIII-2, or 2
3) FVIII kept at 37° C. for 20 hours, in the dark, followed by storage at −80° C.→cFVIII-3, or 3
4) FVIII kept at 65° C. for 20 hours, in the dark, followed by storage at −80° C.→cFVIII-4, or 4
5) FVIII kept at 95° C. for 5 minutes, in the dark, followed by storage at −80° C.→cFVIII-5, or 5
6) FVIII with a pH lowered to pH 2, using a 5 M HCl stock, and kept at 65° C. for 20 hours, in the dark; the pH is raised to 7 afterwards by adding NaOH solution from a 5 M stock, followed by storage at −80° C.→cFVIII-6, or 6
7) FVIII with a pH raised to pH 12, using a 5 M NaOH stock, and kept at 65° C. for 20 hours, in the dark; the pH is lowered to 7 afterwards by adding HCl solution from a 5 M stock, followed by storage at −80° C.→cFVIII-7, or 7
8) FVIII dissolved freshly and subsequently stored at 4° C. for indicated times→cFVIII-8, or 8
9) freshly dissolved FVIII, used and analyzed within 8 hours after dissolving lyophilized sample→FVIII, or 9
Based on the aforementioned set of parameters a-f that parameters are preferably chosen for design of additional protein misfolding procedures, FVIII is for example misfolded in a selection of alternative ways. For example, FVIII is misfolded using prolonged incubation of FVIII at 4° C. and/or room temperature and/or 37° C., preferably in the dark. Alternatively, FVIII is for example subjected to exposure to 1-100 mM CuCl2 for 1-16 hours at room temperature or 37° C., followed by dialysis against PBS.
FVIII subjected to the misfolding conditions 1-8, giving FVIII variants cFVIII-1 to 8, were subsequently analyzed for the presence and extent of cross-beta conformation. For this purpose, ThT fluorescence enhancement, Congo red fluorescence enhancement and tPA/plasminogen activation were determined using two-fold diluted samples in the assay. See
The FVIII samples 1-9 all appeared as clear and colorless solutions. In order to investigate whether soluble oligomers are present in the preparations, the FVIII solutions 4-6 and 8 were subjected to ultracentrifugation for 1 h at 100,000*g. As the, protein that remains in the supernatant after applying these g-forces to the solution is considered as “soluble oligomers,” including soluble monomers. After ultracentrifugation, ThT fluorescence was measured with two-fold dilutions of FVIII samples (
To further analyze the structural aspects with respect to cross-beta formation and multimer size distribution, FVIII samples are preferably subjected to TEM imaging, ThS fluorescence analysis, bis-ANS fluorescence analysis, tPA binding ELISA, BiP binding ELISA, fibronectin finger 4-5 binding ELISA, IgIV binding ELISA, SDS-PAGE followed by Western blotting and/or Coomassie stain, circular dichroism analysis, analysis under a direct light microscope with 10-100× magnification, dynamic light scattering analysis, particle analysis in solution, and SEC analysis.
For example, for selection of immunogenic compositions having a greater chance of being capable of eliciting a protective prophylactic immune response against infection with CSFV, for example strain Brescia 456610, in animals, for example in mice and/or in pigs, the following mouse monoclonal antibodies are implicated in the screenings.
purchased from Prionics-Lelystad, and which neutralize CSFV in vitro (information from the manufacturer). The antibodies are more preferably subjected to passive immunizations of animals, for example mice and/or pigs, followed by a challenge infection with CSFV, for example strain Brescia 456610. Then, antibodies that provide at least in part protection against the challenge viral infection are selected for selection of immunogenic compositions.
For example, for selection of immunogenic compositions having a greater chance of being capable of eliciting an immune response against a protein, for example OVA, in animals, for example in mice and/or in rabbits, the following mouse monoclonal antibodies and polyclonal antibodies are implicated in the screenings.
For example, for selection of immunogenic compositions having a greater chance of being capable of eliciting a protective prophylactic immune response against infection with influenza virus H5N1 strain A/VN/1203/04 or strain A/HK/156/97 in mice and/or in ferrets, the following mouse monoclonal antibodies, that are affinity purified, are implicated in the screenings.
The anti-H5 antibodies purchased from Rockland (a-e) inhibit hemagglutination and neutralize H5N1 A/VN/1203/04 virus, according to the supplied datasheets. Antibodies purchased from HyTest (f-h) inhibit hemagglutination when H5N1 of the strains A/VN/1203/04 or A/HK/156/97 is used, according to information from the manufacturer. The antibodies are more preferably subjected to passive immunizations of animals, for example mice and/or ferrets, followed by a challenge infection with influenza virus, for example an H5N1 strain, most preferably the A/HK/156/97 strain and/or the A/VN/1203/04 strain. Then, antibodies that provide at least in part protection against the challenge viral infection are selected for selection of immunogenic compositions.
For example, for selection of FVIII compositions having a smaller chance of being capable of eliciting an undesired immune response against FVIII upon, for example intravenous, introduction to an animal, for example a human individual, antibodies which inhibit FVIII in coagulation assays are implicated in the screenings of FVIII compositions. These antibodies are for example monoclonal antibodies, for example from human or murine origin, and most preferably these monoclonal antibodies are of human origin when FVIII compositions are sought for human use. Alternatively, polyclonal antibodies are implicated in the selection. For example polyclonal antibodies in immune serum and/or plasma, for example of murine origin, and most preferably from human origin, are used when FVIII compositions are sought for human use. Antibodies which inhibit FVIII in coagulation assays are routinely determined in human plasma samples of Haemophilia patients, using for example the Bethesda assay, known to a person skilled in the art. For selection of FVIII compositions having a smaller chance of being capable of eliciting an undesired immune response against FVIII, those FVIII compositions are selected that show lowest or preferably no binding of the FVIII inhibiting antibodies, when cross-beta adjuvant is detected in the composition. Of course, most preferably, no cross-beta conformation is detected in FVIII compositions meant for therapeutic use, at all, thereby having at forehand a smaller chance of the FVIII composition being capable of eliciting an undesired immune response against FVIII, due to the absence of cross-beta adjuvant.
For the detection of antibody binding, for example an ELISA setup is used. For example, the cross-beta antigen is preferably coated and subsequent the binding of the antibody is detected. Alternatively, the native protein is coated and detected with the antibody, and the ability of cross-beta antigens or immunogenic compositions comprising cross-beta conformation and epitopes for antibodies, to compete with this binding is tested. Preferably, in this setup such amount of antibody is used that results in approximately half-maximal binding. For example such analyses are performed as described in more detail below. In both ways for selecting immunogenic compositions, those cross-beta antigens or immunogenic compositions comprising cross-beta conformation and epitopes for antibodies are selected which have either lost certain amount of epitopes for the antibody or which have remained their epitopes.
E2
For analysis of relative binding of three mouse monoclonal horseradish peroxidase-labeled anti-E2 antibodies CediCon CSFV 21.2, CediCon CSFV 39.5 and Cedicon CSFV 44.3 (Prionics-Lelystad, The Netherlands) to non-treated E2 (nE2) and misfolded E2 comprising increased content of cross-beta (cE2), ELISAs were conducted. For this purpose, nE2, cE2 and nE2-FLAG-His were coated to Microlon high-binding plates (Greiner) and overlayed with dilution series of the three antibodies. For control purposes, non-coated wells were overlayed with the antibody dilutions as well, and E2-coated wells were overlayed with binding buffer only. See
To analyze whether cE2 is inducing CSFV neutralizing antibodies in mice, the following immunization trial was conducted.
Start of the study: day −1. Four groups of five female BalbC mice were incorporated in the study. Blood was drawn at day −1 and 7, and is drawn at day 14, 21, 28 for preparation of serum. At day 28, the study is terminated and mice are sacrificed (final blood draw by heart puncture under anesthesia). Mice were immunized at day 0 with: group 1, placebo; group 2, 100% nE2, 3 μg/mouse; group 3, 100% cE2, 3 μg/mouse; group 4, 50% nE2+50% cE2, 1.5 μg nE2/mouse+1.5 μg cE2/mouse. Dose: 500 μl, 6 μg E2/ml in PBS, or PBS (placebo). At day 0 mice were immunized subcutaneously (s.c.) in the neck. At day 14, mice are immunized for a second time, using the same doses. Now, mice are immunized s.c. in the left flank. When the immunization is terminated after 29 days, most preferably the following analyses are conducted with the sera or plasma. Total IgG/IgM titers against nE2-FLAG-His are assessed for sera or plasma of each individual mouse and for pooled sera or plasma for each of the four groups. In addition, for each individual serum and for pooled serum for each group, IgG1 and IgG2a titers are determined as a measure for the occurrence of a humoral response and/or a cellular response. Virus neutralization titers using CSFV strain Brescia 456610 are also conducted to analyze the relative virus neutralizing capacity amongst in sera or plasma of the four groups of mice. Finally, the ability of the dilution series of the sera or plasma to compete for binding of the antibodies CediCon CSFV 21.2, CediCon CSFV 39.5 and Cedicon CSFV 44.3 to nE2-FLAG-His immobilized on an ELISA plate is assessed. In this way information is gathered on whether antibodies induced in mice upon immunization with nE2 and cE2 recognize the same or similar epitopes compared to those epitopes recognized by CediCon CSFV 21.2, CediCon CSFV 39.5 and Cedicon CSFV 44.3, and to which relative extent nE2 and cE2 induce antibodies. In this way, data is collected that is compared to the data obtained with the series of cross-beta measurements and to the data obtained with the series of multimer size and distribution measurements.
A typical challenge experiment with CSFV in pigs, after immunization with immunogenic compositions comprising cross-beta adjuvant and exposed epitopes for functional antibodies, is for example conducted as follows. For example, a vaccination-challenge experiment is conducted with five groups of for example 3-9 pigs, and preferably 5-6 pigs, for example approximately 6 weeks of age at the start of the experiment. Blood is drawn at day −1, for collection of pre-immune serum. All pigs are clinically observed each day, throughout the whole study period. For control purposes, E2 vaccine is prepared according to the procedures applied to E2 to obtain the commercially available water-in-oil-in-water CSFV vaccine. At day 0, pigs are immunized intramuscularly. Typical immunogenic compositions consist of: group 1, placebo; group 2, non-treated E2; group 3, cross-beta-adjuvated E2 with exposed epitopes for antibodies; group 4, cross-beta-adjuvated E2 lacking exposed epitopes for antibodies; group 5, non-treated E2+cross-beta-adjuvated E2 with exposed epitopes for antibodies. Blood is drawn for serum preparation at day 7, 14, 21, 28, 25, 42. A second immunization is performed at day 21. Virus neutralization tests are performed with serum collected at day −1/7/14/21/28/35, and CSFV strain Brescia 456610. Rectal temperature is measured from day 40 on, at each day of the remaining period of the study. Furthermore, signs of CSF like anorexia and paresis are noted. At day 42, all pigs are challenged intranasally with CSFV strain Brescia 456610. From day 42 on, for 15 days up till day 56 of the study, the pigs are monitored daily with respect to the following parameters: leucopenia test, thrombocytopenia test. In addition, virus secretion is measured with samples collected for example at day 42/44/47/49/51/54/56. Pigs are euthanized in case of critical illness. Virus content of white blood cells is for example assessed with samples collected at day 42/44/47/49/51/54/56.
OVA
The series of OVA variants obtained by subjecting OVA to the misfolding procedures outlined before are analyzed for their type and relative content of cross-beta appearance, their multimeric size and multimer distribution, and their relative ability to bind the antibodies as described above. Based on combinations of cross-beta appearance and content, and multimer size, cross-beta dOVA variants are subjected to analyses for binding of monoclonal and/or polyclonal antibodies. Based on these analyses, OVA variants are selected that combine the occurrence of cross-beta in the context of a multimer size of preferably the size of a monomer, up to the size of multimers with dimensions of for example in the range of 1-10 μm, and more preferably a multimer size of monomers up to 1000-mers, with the binding of antibodies or with inhibited antibody binding or the lack of antibody binding. This selected series of OVA variants is then used as immunogenic composition in immunization trials in animals, preferably in mice. Subsequently, in sera or plasma the presence of anti-OVA antibodies is analyzed. In addition, the ability of the antibodies in the sera or plasma to compete for binding of the monoclonal antibodies that only bind native OVA and not denatured OVA, to native OVA is assessed. For this purpose, the monoclonal antibodies are preferably tagged or labeled, for example with biotin, peroxidase or alkaline phosphatase. Most preferably, a series of OVA variants is selected for the immunizations, that span the parameter windows to a large extent. For example, OVA variants with no or extreme large cross-beta content are selected. For example, OVA monomers up to large aggregates visible by eye are selected, with OVA variants comprising various multimer sizes in between. For example, OVA variants that display as high-affinity binding partners for the antibodies are incorporated in the immunization studies, as well as OVA variants that expose antibody epitopes to an intermediate extent, and as well as OVA variants that do not expose antibody epitopes at all.
H5 of H5N1 strain A/HK/156/97
The four variant of H5 of H5N1 virus strain A/HK/156/97 comprise varying cross-beta contents and multimer size distributions. The nH5-1, nH5-2, CH5-A and CH5-B variants are subjected to antibody binding analyses in ELISAs, using four mouse monoclonal antibodies 200-301-975 to -978 (Rockland). These antibodies are raised against H5N1 A/VN/1203/04, neutralize virus of this strain, and inhibit hemagglutination induced by this virus. In
Nine groups of 8 female Balb/c mice are included in the experiment. Pre-immune serum is collected before the first immunization, and serum is collected four times more between one week after the first immunization and the day of the viral challenge (day 42). Mice are immunized subcutaneously at day 0 and day 21 with doses of 500 μl/mouse, according to the following scheme of test items per group:
In the period before the challenge, mice are clinically observed daily, and putative occurrence of injection site reactions is monitored twice to thrice a week. At day 42, mice are inoculated with H5N1 virus of strain A/HK/156/97. From day 41 till the end of the study at day 56, mice are clinically observed for clinical signs of influenza, and body weight is measured daily. Serum is analyzed for the presence of virus neutralizing antibodies, using H5N1 A/HK/156/97, and hemagglutination inhibition titers are determined. Total IgG/IgM titers are determined using ELISA with non-treated H5 of H5N1 A/HK/156/97 and/or using H5 of H5N1 A/VN/1203/04. In addition, IgG1 and IgG2a titers are determined. Finally, the capacity of the anti-H5 antibodies in the sera or plasma to compete for binding of the series of monoclonal anti-H5 antibodies listed above is assessed in competition ELISAs. These listed antibodies neutralize H5N1 and inhibit hemagglutination by H5N1. Most preferably, antibodies that provide protection against H5N1 infection upon passive vaccination, are used for the ELISAs. For the ELISAs, biotinylated mouse monoclonal antibodies are used. Serum dilution series are prepared with biotinylated anti-H5 antibodies incorporated in the dilution series at a concentration that gives sub-optimal binding when assessed in the absence of immune serum. In the ELISA, binding of biotinylated anti-H5 antibody is determined using Streptavidin.
H5 of H5N1 strain A/VN/1203/04
As described above, non-treated H5 of H5N1 strain A/VN/1203/04 comprises various appearances upon subjecting nH5 to four different misfolding procedures. Cross-beta parameters differ amongst CH5-1 to 4, as well as the size and shape of multimers, as seen on TEM images (
Non-treated H5 of H5N1 A/VN/1203/04 and misfolded variants that comprise cross-beta structure and exposed epitopes for functional antibodies, in the context of a multimer size suitable for immunizations, are for example implicated in vaccination trials followed by viral challenge in, for example, ferrets and/or mice. Such a vaccination trial is for example performed similarly to the protocol described for H5 of H5N1 A/HK/156/97, above. Similar parameters are analyzed.
Factor VIII
FVIII ELISA with Haemophilia Patient Plasma
Certain Haemophilia patients suffer from a qualitative shortens and/or a quantitative shortens of functional FVIII, resulting in a mild to severe bleeding tendency. As a therapeutic approach, patients receive intravenous injections with recombinant and/or plasma-derived human FVIII and/or FVIII derivatives, like for example FVIII lacking the B-domain. A drawback of this treatment approach is the induction of anti-FVIII (auto-)antibodies, also referred to as inhibitor formation, which occurs in approximately 5-30% of the patients, and which hampers effective further treatment of the underlying disease. In patent applications US2007015206, the contents of which are incorporated herein by this reference, and WO2007008070 we disclosed that proteins comprising cross-beta structure elicit an immune response due to the adjuvating properties of cross-beta conformation. Furthermore, in patent application US2007015206 we disclosed that a series of biopharmaceuticals, including FVIII, comprise protein with cross-beta conformation to various extents. In patent applications US2007015206 and WO2007008070 we demonstrated that proteins comprising cross-beta structure, being it either biopharmaceuticals, or viral proteins used as vaccine candidates, in fact induce an immune response directed to natively folded counterparts of the proteins comprising cross-beta. This demonstrates that protein formulations that comprise cross-beta harbor the risk for eliciting antibody titers directed against the native, functional protein molecules. That is to say, misfolded FVIII protein molecules in FVIII formulation meant for therapeutic use are contributing to the observed built up of an immune response against FVIII in haemophilia patients.
In the current example we demonstrate that a series of misfolded forms of human FVIII comprise molecules with cross-beta conformation and in addition harbor epitopes for anti-FVIII antibodies present in plasma from Haemophilia patients suffering from FVIII inhibiting anti-FVIII antibodies.
Protocol for Anti-FVIII Titer Determination in Haemophilia Patient Plasma, Using ELISA
Anti-FVIII titers were determined in a fourfold dilution series starting from 1:16, to 1:65536, of plasma from seven haemophilia patients (kind gift of the University Medical Center Utrecht, Utrecht, The Netherlands). Patients A-D had tested positive in a Bethesda type of assay for anti-FVIII antibodies that inhibit FVIII, whereas patients E-G had tested negative. Plasma of one healthy donor was incorporated in the ELISAs as an additional negative control. Helixate FVIII was used as the coated antigen in the ELISAs, and was coated at 10 IE/ml, in 100 mM NaHCO3, pH 9.6, on Microlon high-binding 96-wells plates (Greiner). Wash buffer was 50 mM Tris, 150 mM NaCl, 0.1% v/v Tween20, pH 7.0-7.4. Binding buffer for the plasma dilutions and secondary antibody was PBS with 0.1% v/v Tween20. FVIII was coated at room temperature, for 1 h, with agitation (50 μl/well). After washing, 200 μl/well Blocking Reagent (Roche) was incubated for 1 h at 4° C., with agitation. After 3 washes, the fourfold plasma dilutions series of the eight indicated plasma's (patients A-G, control donor) was incubated for 1 h at 4° C., with agitation, with 50 μl/well. After 3 washes, 50 μl/well of 1:3000 diluted Goat-anti human IgG (GAHAP-IgG; Biosource Int., catalogue number AHI0305) was incubated for 30 minutes at 4° C., with agitation. After 5 washes and subsequently two more washes with PBS only, bound GAHAP-IgG was visualized using 100 μl/well DEA-NPP-substrate (p-nitrophenyl phosphate (600 μg/ml) in DEA buffer pH 9.8 (10% v/v diethanolamine in H2O, with 240 μM MgCl2.6H2O, pH adjusted with HCl)) for 1.0 minutes at room temperature, before adding 50 μl/well 2.4 M NaOH to stop the reaction. Absorbance was read at 405 nm on a Spectramax Plus384 Microplate Reader (Molecular Devices). See
Comparison of Anti-FVIII Antibody Binding from Hemophilia Patient Plasmas that Tested Positive for the Presence of FVIII Inhibiting Antibodies, to Non-Treated FVIII and Various Forms of FVIII Subjected to Misfolding Conditions
See for the nine different forms of FVIII that were included in these examples the section above: Misfolding of FVIII, and
Binding of anti-FVIII antibodies from Hemophilia patient plasma with FVIII inhibiting antibodies, to the nine FVIII variants was assessed using ELISA. The ELISA was essentially performed as described above. Now, all nine variants were immobilized on ELISA plates, and overlayed with 1:100 or 1:200 diluted plasma. See
In subsequent studies, for example FVIII preparations are produced with alternative appearances of cross-beta conformation combined with exposed epitopes for FVIII inhibiting antibodies, upon subjecting FVIII to various additional misfolding procedures, like for example prolonged incubation of FVIII at 4° C., at room temperature and at 37° C. In time, samples of these FVIII incubations are subjected to various cross-beta assays and structure determinations aiming at providing insight in multimer size and distribution. In addition, binding of patient antibodies is monitored in time. Based on these analyses, it is depicted which molecules with varying combinations of cross-beta, multimer size and antibody binding capacity are selected for immunization trials. Preferably, FVIII variants are included in the immunization trials that comprise combinations of cross-beta conformation or not, that is incorporated in monomers up to for example 1000-mers, and that expose or do not expose epitopes for FVIII inhibiting antibodies. For example, mice are immunized, for example transgenic mice with human FVIII, preferably mice deficient for murine FVIII. A typical example of an immunization experiment is depicted below:
Ten mice/group. Pre-immune serum collection at day −2. Intravenous injections of 200 μl doses. Dose of 1 IE/mouse. Injections at day 0/14/28/42. Additional blood draws at day 14/28/42/49 for collection of serum. Groups: 1, placebo; 2, FVIII; 3, cross-beta FVIII variant A; 4, cross-beta FVIII variant B; 5, 50% FVIII+50% cross-beta FVIII variant A; 6, 50% FVIII+50% cross-beta FVIII variant B, with cross-beta FVIII variant A comprising exposed epitopes for inhibiting anti-FVIII antibodies, and comprising soluble oligomers, and with cross-beta FVIII variant B lacking epitopes for FVIII inhibiting antibodies to a relatively large extent, and comprising insoluble oligomers to a large extent. For example, cross-beta FVIII variant A is cFVIII-4 or 5, and cross-beta FVIII variant B is cFVIII-6 or 7. The sera or plasma are analyzed for the presence of FVIII inhibiting antibodies, for example in a Bethesda assay. Furthermore, the sera or plasma are analyzed for their capacity to compete for binding to FVIII with the patient sera or plasma A-D, which comprise FVIII inhibiting antibodies. In this way, information is obtained about the contribution of various parameter ratios with respect to exposure of epitopes for FVIII neutralizing antibodies, cross-beta content and appearance, and multimer size and multimer size distribution, to the ability to induce anti-FVIII antibodies that inhibit FVIII.
With this example it is demonstrated that the combination of certain cross-beta structures in H5 protein and a certain amount of exposed epitopes for functional antibodies is required for inducing a protecting immune response in mice.
The average van der Waals radius of the 20 amino acids is approximately 0.3 nm, or 3 Å. The approximate average volume of an amino acid is 110 Å3. The approximate average surface of an amino acid residue is 28 Å2, or 0.28 nm2. The approximate average mass of an amino acid residue is 120 Da. From these numbers it is estimated that using the 1.000 kDa MW cut-off filter, at maximum protein assemblies comprising approximately 8500 amino acid residues flow through the filter. This maximum size corresponds to a maximum protein surface on for example a TEM image, of 2400 nm2. Assuming a spherical or squaric arrangement of the protein multimer, this corresponds to protein structures with a radius of approximately 27 nm, or 50×50 nm squares, respectively, on TEM images. With H5 appearing on the SEC column and on SDS-PA gel as amongst others, 33 kDa and 75 kDa molecules, multimers of up to 30 or 13 H5 monomers will flow through the 1.000 kDa filter, at maximum. By approximation, on average, 1 nm2 corresponds to 3.6 amino acid residues or 430 Da, and 1 kDa corresponds to 2.3 nm2.
With this approximate numbers it is possible to calculate the number of H5 monomers that appear in multimers, as seen for example under the direct light microscope, in SEC fractions, on TEM images and on SDS-PA gels. These considerations also apply for any other molecular assembly of one or more protein molecules, like for example ovalbumin, E2 and factor VIII.
Endotoxin measurement. The endotoxin content of H5 as supplied by Protein Sciences was measured at 25 μg/ml (diluted in sterile PBS), the concentration of H5 at which vaccination will occur. The Endosafe cartridge had a sensitivity of 5-0.05 EU/ml (Sanbio, The Netherlands).
The endotoxin level is 0.152 EU/ml. The endotoxin level of the dilution buffer PBS is <0.050 EU/ml.
Methods for Preparing Structural Variants of H5 which Comprise Cross-Beta
Recombinantly produced heamagglutinin 5 (H5) protein of H5N1 strain A/Vietnam/1203/04 (A/VN/1203/04) was purchased from Protein Sciences. The stock concentration was 1 mg/ml (determined with the BCA method (Pierce)) in 10 mM sodium phosphate, pH 7.1, 171 mM NaCl, 0.005% Tween20. H5 is stored at 4° C. The H5 stock as supplied is referred to as cross-beta H5-0, or dH5-0, i.e., H5 that comprises cross-beta structure of arbitrarily chosen type 0. Handlings with H5 solutions are performed under sterile conditions in a flow cabinet. When dH5-0 is ultracentrifuged for 1 h at 100,000*g (4° C.), 62% of the H5 remains in the supernatant; 38% is pelleted. Therefore, 62% of the dH5-0 is designated as soluble H5, 38% as insoluble protein.
The dH5-0 protein solution is analyzed as supplied and in addition after applying a routine centrifugation step, i.e., 10 minutes centrifugation at 16,000-18,000*g, at 4° C., in a rotor with fixed angle. The dH5-0 after this standard centrifugation step is referred to as cdH5-0, cross-beta H5 after centrifugation. For analysis and vaccination trials, the supernatant of cdH5-0 is used. After the centrifugation run a white pellet becomes visible, indicative for the present of insoluble H5 aggregates. An aliquot of 175 μl of the dH5-0 is subjected to size exclusion chromatography on an analytical superdex75 10/30 column (GE Healthcare) by Roland Romijn (U-ProteinExpress, Utrecht, The Netherlands), using an Äkta explorer (GE Healthcare). In
Additionally, for several analyses dH5-0 and other misfolded H5 samples comprising cross-beta structure are ultracentrifuged for 1 h at 100,000*g, at 4° C., using a rotor with swing-out buckets. The supernatants of these ultracentrifuged H5 samples are used for analyses and are referred to as ucdH5-0 or udH5-0, and ucdH5-I/II/III or udH5-I/II/III.
Ultrafiltrated dH5-0, referred to as fdH5-0, is obtained by filtering cdH5-0 for 10 minutes at 16,000*g through a Vivaspin 500 PrNo VS0161, 1×106 Da MW cut-off filter, at 4° C. The flow-through of the filter is used for subsequent analyses and immunizations, and comprises H5 monomers/oligomers with a molecular weight of approximately ≦1.000 kDa. The fraction of dH5-0 that is poured through the filter, i.e., fdH5-0, is 80% of the starting material, as determined with the BCA method after three consecutive filtrations. Therefore, the dH5-0 comprises approximately 20% protein multimers with a molecular mass of >1.000 kDa.
Preparation of Misfolded dH5-I Comprising Cross-Beta Structure
dH5-I (heat cycling at pH 7) is produced from dH5-0 supernatant after centrifugation for 10 minutes at 16,000*g (4° C.), i.e., cdH5-0. The H5 concentration is 1 mg/ml. From a 5 M NaCl stock an amount is added to cdH5-0 in order to adjust the NaCl concentration to that of dH5-II (see below). The cdH5-0 is divided in 100 μL aliquots in a 200-μl PCR plate (BioRad, 96 well, cat nr 2239441) and placed in a thermal cycler (Biorad, MyIQ). The cdH5-0 is incubated at 25° C. for 20 seconds and subsequently heated from 25° C. to 85° C., ramp 0.1° C./s, followed by a 20 s incubation at 85° C. This cycle is repeated twice (total cycles is three). The program finishes with cooling at 4° C. for 2 minutes. The dH5-I aliquots are combined and again divided into aliquots in Eppendorf 500 μL cups. Aliquots of 50 μg dH5-I/vial are stored at −20° C.
Before misfolding the protein solution looks clear, after heat denaturation the sample appears white turbid. After freezing-thawing and subsequent centrifugation a pellet is visible. After ultracentrifugation for 1 h at 100,000*g (4° C.), 37% of the H5 remains in the supernatant.
Preparation of Misfolded dH5-II Comprising Cross-Beta Structure
dH5-II (heat cycling at pH 2) is produced from dH5-0 supernatant after centrifugation for 10 minutes at 16,000*g (4° C.), i.e., cdH5-0. The H5 concentration is 1 mg/ml. The pH of cdH5-0 is lowered to pH 2 by addition of HCl from a 15% (v/v) stock in H2O. Then it is divided into 100 μL per cup in PCR strips (BioRad, 96 well, cat nr 2239441) and placed in a MyIQ RT-PCR cycler (Biorad). The misfolding program is the same as used for preparing dH5-I (see above). Subsequently, dH5-II aliquots are combined and the pH is adjusted back to pH 7 by addition of NaOH solution from a 5 M stock. Then, dH5-II is aliquoted again and stored at −20° C.
Before misfolding the cdH5-0 solution at pH 2 appears clear, after heat denaturation and adjusting the pH back to 7, the dH5-II sample appears slightly turbid. After freezing-thawing and subsequent centrifugation a pellet is visible. After ultracentrifugation for 1 h at 100,000*g (4° C.), 41% of the H5 remains in the supernatant.
Preparation of Misfolded dH5-III Comprising Cross-Beta Structure
dH5-III (prolonged incubation at 5° C. below the melting temperature of dH5-0) is produced from cdH5-0. The H5 concentration is 1 mg/ml. For this, the melting temperature of cdH5-0 at 1 mg/ml was determined using the MyiQ cycler. 0.7 μl Sypro Orange 5000× stock (Sigma) is added to 70 μl cdH5-0 and the sample is heated from 25° C. to 85° C. The ramp rate is set to 0.1° C./min. At each temperature increment of 0.5° C. the Sypro Orange fluorescence is measured at 490 nm (excitation) and 575 nm (emission). The melting temperature was 52.5° C. (See
In Table 1 the results of the visual inspection of the six H5 forms is summarized.
Transmission electron microscopy imaging with H5 forms with/without ultracentrifugation. The various H5 forms are subjected to TEM analysis. The dH5-0, dH5-I, dH5-II and dH5-III forms are analyzed directly, and their supernatants after ultracentrifugation for 1 h at 100,000*g (4° C.) are imaged. PBS served as a negative control and gave an empty image, as expected. The dH5-0 appeared with a background of many non-uniformly shaped protein assemblies of approximately 25×25 nm to 100×100 nm, corresponding to molecular H5 assemblies of approximately 270-4300 kDa (approximately 4-57H5 monomers of 75 kDa). Also large, branched aggregates with strings of protein assemblies are seen. The branches are approximately 100 to 400 nm thick and approximately 2 to 5 μm in length. Upon ultracentrifugation of dH5-0, many string-like protein assemblies are seen, with bead-like subunits. Many have dimensions of approximately 25×50 nm, a few are approximately 100×100 nm up to 400×800 nm. The cdH5-0 appears very similar to udH5-0, with the exception that also larger protein assemblies are seen with dimensions of approximately 1500×1500 nm. The fdH5-0 appears with a background of uniformly shaped relatively tiny protein structures with undefined, though relatively small size and shape. A few relatively large protein structures are seen, which are composed of strings of protein assemblies. These structures have tree-like appearances with branches, and are approximately 400×4000 nm in size. The dH5-I comprises relatively a few but large and dense protein assemblies composed of spherical protein building blocks. The building blocks are connected in branched strings with approximate dimensions of 500×5000 nm. Hardly any H5 is seen in structures apart from the large branched strings. Upon ultracentrifugation, an empty image is obtained, indicated that all dH5-I structures seen before ultracentrifugation are insoluble and pelleted. The dH5-II is seen as amorphous and large protein assemblies with approximate sizes of 3×3 μm. The protein assemblies appear as loosely connected structures. The structures are composed of smaller non-uniformly shaped low-density protein assemblies, which are also seen freely. These building blocks are approximately 50×50 to 100×100 rum in size. Upon ultracentrifugation, the supernatant is fully clear on the TEM image. This shows that H5 multimers are insoluble and pelleted upon ultracentrifugation. The dH5-III is presented on the TEM image as a relatively high number of two types of protein assemblies with a relatively small size of approximately 25×25 nm and approximately 50×50 nm. Upon ultracentrifugation, again many small protein assemblies are seen in the supernatant, on the TEM image. The approximate sizes of the multimers are mostly 20×20 nm with a few protein assemblies of approximately 100×100 nm in size. Apparently, the protein assemblies are soluble and are not pelleted upon ultracentrifugation.
The six H5 structural variants were analyzed on an SDS-PA gel, both with and without a pretreatment in the presence of reducing agent DTT. See
SDS-PAGE with H5 Samples Before/after Ultracentrifugation
The dH5-0, dH5-I, dH5-II and dH5-III are subjected to ultracentrifugation for 1 h at 100,000*g (4° C.). This ultracentrifugation is accepted as a procedure for separation of insoluble protein molecules from the soluble fraction that will remain in the supernatant. Together with starting material and cdH5-0, these ultracentrifuged samples are analyzed on an SDS-PA gel. See
Thioflavin T fluorescence. Binding of Thioflavin T and subsequent enhancement of its fluorescence intensity upon binding to a protein is a measure for the presence of cross-beta structure which comprises stacked beta sheets. For measuring the enhancement of Thioflavin T fluorescence, H5 samples were tested at 100 μg/ml final dilution. Dilution buffer was PBS. Negative control was PBS, positive control was 100 μg/ml standard misfolded protein solution, i.e., dOVA standard. dOVA standard is obtained by cyclic heating from 25 to 85° C. (6° C./minute) of a 1 mg/ml ovalbumin (Albumin from chicken egg white Grade VII, A7641-1 G, Lot 066K7020, Sigma) solution in PBS. The H5 samples cdH5-0, dH5-I, dH5-II and dH5-III are also tested after 1 h centrifugation at 100,000*g, at 4° C. Supernatant is analyzed for its protein concentration using the BCA method. Subsequently, adjusted volumes in order to test identical protein concentrations, are used in the Thioflavin T fluorescence enhancement assay. Ultracentrifuged samples are indicated with a “u.” See
Enhancement of Sypro Orange fluorescence. Sypro Orange is a probe that fluoresces upon binding to misfolded proteins. As a measure for the relative content of misfolded proteins, enhancement of Sypro Orange fluorescence is tested with H5 samples at 25 μg/ml final dilution. Dilution buffer was PBS. Negative control was PBS, positive control was 100 μg/ml dOVA standard. The H5 samples cdH5-0, dH5-I, dH5-II and dH5-III are also tested after 1 h centrifugation at 100,000*g, at 4° C. Supernatant is analyzed for its protein concentration using the BCA method. Subsequently, adjusted volumes in order to test identical protein concentrations, are used in the Sypro Orange fluorescence enhancement assay. Ultracentrifuged samples are indicated with a “u.” See
Finger domains of tPA, factor XII, hepatocyte growth factor activator and fibronectin bind to cross-beta structure in protein, when the free finger domains are contacted with proteins comprising cross-beta structure, as well as when the finger domains are part of the full-length or truncated proteins. We now assessed the binding of the fourth and fifth finger domain of fibronectin (Fn F4-5) to the various H5 forms, as depicted in
In
tPA/Plg Activation by H5 Samples Comprising Cross-Beta Structure.
The six H5 samples were tested for their tPA mediated plasminogen activation potency at a concentration of 50 μg/ml. The results are shown in
Epitope Scanning with Nine Functional Monoclonal Anti-H5 Antibodies
As outlined above previously, nine monoclonal mouse anti-H5 antibodies that neutralize H5N1 virus of strain A/VN/1203/04 and that inhibit hemagglutination by the virus, are used to determine whether the epitopes for these functional antibodies are exposed on the various structural H5 variants. In
In Table 5, the structural data as described above, and the epitope scanning data regarding the presence and nature of binding sites for functional antibodies, is summarized. Based on the analyses, by approximation the six H5 structural variants can be divided in two structural/functional groups. Based on, by estimation, similar parameters, Group I comprises dH5-0, cdH5-0 and fdH5-0. Based on, by estimation, similar appearances and parameters, Group II comprises dH5-I, dH5-II and dH5-III. These H5 forms in group I comprise cross-beta structures that at least in part appear as relatively smaller multimers, and that expose a relatively high number of tPA finger and Fn finger binding sites, with relatively high affinity. In addition, the cross-beta structures of group I H5 variants enhance ThT and Sypro orange fluorescence, although to a lesser extent than the H5 forms in group II. In group II, far less Fn F4-5 and tPA binding sites are present. Multimers appear to be larger, accompanied by increased ThT fluorescence and Sypro orange fluorescence. On average, by approximation the relative number of binding sites for functional antibodies and the relative affinity of functional antibodies for H5 variants in group I is higher than for H5 variants in group II.
Immunization of Mice with Six H5 Variants, Followed by a Challenge with H5N1 Virus
As outlined above, Balb/c mice are immunized twice, at day 0 and day 21, with a dose of 5 μg of the six H5 forms. Group 2, dH5-0; group 3, cdH5-0; group 4, fdH5-0; group 5, dH5-I; group 6, dH5-II; group 7, dH5-III. Controls are group 1, placebo (PBS), group 8, 5 μg cdH5-0 mixed with 40 times diluted alum (Adjuphos, Brenntag), and group 9, commercially available H5N2 killed virus vaccine adjuvated with oil in water emulsion (Nobilis flu, Intervet). None of the vaccine formulations induced a visible reaction in the mice, except for the Nobilis flu vaccine, which induced palpable reactions on the flanks of the mice. At day 33 blood is drawn for titer determination (See Table 6). The total anti-H5 antibody titer of IgG and IgM isotypes is determined, in an ELISA using immobilized cdH5-0 and dilution series of the individual mouse sera. At day 42 blood is drawn for serum collection, and mice are challenged with a lethal dose of H5N1 virus of strain A/VN/1194/04. The virus dose per mouse was approximately 50 μl with a titre 9.7 log TCID50/ml. During 14 days the weight of the mice was measured and the mice were clinically examined, daily. At day 56, blood is drawn from mice that survived the viral challenge, for serum collection. Presence of total anti-H5 antibodies and presence of functional anti-H5 antibodies is assessed.
In Table 5, Table 6 and
In Table 5, the immunization and challenge data are summarized and compared for the H5 forms in group I and the H5 forms in group II. When the titer data, weight loss data and survival data are considered with respect to the cross-beta structure data and the exposed functional epitopes data, it is clear that the dH5-0, cdH5-0 and fdH5-0 are provided with a combination of i) type of cross-beta structure, ii) relative amount of cross-beta structure, iii) relative multimeric molecular distribution, iv) relative fraction of soluble molecules, and v) relative number of exposed epitopes for functional antibodies, with relative high affinity binding sites, that are beneficial for inducing protection against H5N1 infection, when compared to the combined data obtained with H5 forms dH5-I, dH5-II and dH5-III. These latter three forms induced less protection against H5N1 infection, and structural and functional parameters differed from those seen with dH5-0, cdH5-0 and fdH5-0.
Immunization of Pigs with Various E2 Cross-Beta Structural Variants, Followed by a Challenge with Classical Swine Fever Virus (CSFV).
See the example text above for a general outline of the experimental approach.
E2 purification. E2 in cell culture supernatant was obtained frozen at −20° C. from Central Veterinary Institute (CVI, Lelystad, the Netherlands), and labeled by CVI as follows: CGF E2 marker vaccine, Batch: E20-98-A001, Datum 23-2-98. The volume is ˜300 ml. Purification has been performed by R. Romijn (U-ProteinExpress, Utrecht, NL). Thawed supernatant was centrifuged for 10 minutes at 5500*g, at 4° C., and subsequently dialyzed against PBS (Gibco, 20012; 1.54 mM KH2PO4, 155.2 mM NaCl, 2.7 mM Na2HPO4-7H2O, pH 7.2). The endotoxin level of undialyzed supernatant was assessed using an Endosafe PTS apparatus, and was 0.296 EU/ml. Two 149.5 ml aliquots were dialyzed against 800 ml PBS at 4° C. After 5 hours the PBS was replaced by fresh PBS and dialysis was continued overnight at 4° C.
First, an affinity purification has been performed using an anti-E2 antibody column. For this purpose, monoclonal anti-E2 antibody V3 (Prionics, The Netherlands) was coupled to CNBr-activated Sepharose 4 Fast Flow (GE Healthcare), according to the manufacturers protocol. Approximately 20 mg V3 was coupled to 13.5 ml Sepharose. Antibody 39.5 is V3 labeled with horse raddish peroxidase (Prionics, The Netherlands), and is used as outlined below. The running buffer was PBS and after loading the dialyzed supernatant, bound E2 was eluted with 0.1 M glycine pH 2.5. Fractions of 2 ml were collected in 2 ml Eppendorf cups containing 100 μl 1 M Tris (pH not adjusted).
After affinity purification, cross-beta E2, referred to as cE2, is obtained. The cE2 is dialyzed against PBS and appeared as an approximately 100% pure protein on a Coomassie stained polyacryl-amide gel. The 8.3 mg cE2 was subsequently concentrated to 7.9 mg/ml using a Vivaspin20 10 kDa filter (4° C., 4800*g; Sartorius). A fraction of the cE2 was aliquoted and stored at −20° C. Another fraction of the cE2 was applied to a preparative size exclusion chromatography (SEC) column (Superdex200 16/600; GE Healthcare) and fractionated using an Äkta purifier (GE Healthcare). The running buffer was PBS. See
Misfolding procedures applied to cross-beta E2 form SEC-E2. After affinity purification of E2 using the anti-E2 antibody V3 column and subsequently the SEC column, SEC-E2 is used in two misfolding procedures to prepare alternative misfolded forms of E2 comprising cross-beta structure: cE2-A and cE2-B.
cE2-A preparation. For preparation of cE2-A, SEC-E2 was divided in 100 μL aliquots in PCR cups and placed in a thermal cycler (Biorad, MyIQ). The SEC-E2 was incubated at 25° C. for 20 seconds and subsequently heated from 25° C. to 85° C., ramp 0.1° C./s, followed by a 20 s incubation at 85° C. This cycle is repeated twice (total cycles is three). The program finishes with cooling at 4° C. for 2 minutes. The cE2-A aliquots are combined and again divided into aliquots in Eppendorf cups. Aliquots are stored at −20° C.
cE2-B preparation. For preparation of cE2-B, SEC-E2 was divided over five 1.5 ml Eppendorf cups; 1.3 ml/cup. The SEC-E2 was heated for 1 h at 95° C. in a thermo block. After heating, aliquots were recombined and mixed. Then, the cE2-B was again aliquoted in Eppendorf cups and stored at −20° C.
PTS LAL Assay. The endotoxin levels of the four E2 samples were determined with the PTS Endosafe (Sanbio, The Netherlands). The E2 samples were diluted to indicated concentrations and the endotoxin level was calculated for the final formulation at 16 μg/ml E2, which is used during the immunizations of pigs that are enrolled in the CSFV challenge experiment. The results are shown in Table 7.
Analysis of various structural forms of E2 comprising cross-beta structure. The various structural forms of E2 were analyzed in
TEM imaging. TEM images were taken with the four E2 samples cE2, SEC-E2, cE2-A, cE2-B and PBS negative control. No protein structural features were seen on the negative control image. The cE2 appeared as large amorphous aggregates with dimensions of approximately 50×50 nm up to approximately 500×500 nm. No smaller protein structures are observed. In cross-beta E2 form SEC-E2, relatively a few particulate like aggregates are seen, that seem dense in nature and have dimensions of approximately 25×25 nm. In addition, it appears that numerous smaller protein structures are present in SEC-E2, that cover the full image. Dimensions are approximately 20×20 nm or 20×100 nm. Apparently resulted the formulation and storage procedure in the reappearance of E2 aggregates, because initially SEC-E2 comprised E2 monomers and dimers which eluted from the SEC column (See
Direct light microscopy. No aggregates were visible under the direct light microscope for any of the samples.
SDS-PAGE analysis under reducing and non-reducing conditions. An SDS-PAGE analysis was performed with the four cross-beta comprising E2 samples cE2, SEC-E2, cE2-A and cE2-B, and samples were analyzed after heating in reducing and non-reducing sample buffer. The results after Coomassie stain are shown in
ThT fluorescence. ThT fluorescence enhancement was determined with the various cross-beta comprising E2 forms at 50 μg/ml. The results are shown in
The results show that upon SEC purification (SEC-E2), the ThT fluorescence enhancement is lowered compared to cE2, from which SEC-E2 was obtained after SEC. Furthermore, the ThT fluorescence enhancement is increased upon applying heat induced misfolding procedures to SEC-E2. Heat induced misfolding for 1 h at 95° C. (cE2-B) results in higher ThT signals than cyclic heating from 25° C. to 85° C. (cE2-A).
Sypro Orange fluorescence enhancement with E2 forms. The fluorescence enhancement of Sypro Orange was determined with the four cross-beta comprising E2 forms at 25 μg/ml E2, in PBS. The results in
tPA/plasminogen activation assay. tPA mediated plasminogen activation was determined with the tPA/plasminogen assay using a chromogenic substrate for plasmin. The four E2 samples are tested for their potency to activate tPA/plasminogen with their cross-beta structure present in the molecules. E2 is tested at 50 μg/ml final concentration.
The results in
Fn F4-5 ELISA. Binding of Fn F4-5 to the four forms of E2 was assessed in an ELISA experiment. The E2 samples were coated onto ELISA plates and overlayed with a concentration series of Fn F4-5, which comprises a C-terminal FLAG-tag. Binding of Fn F4-5 is monitored upon binding of HRP-tagged anti-FLAG antibody, followed by TMB stain. The results of one out of two experiments are shown in
tPA and K2P-tPA ELISA. Binding of tPA (Actilyse, Boehringer-Ingelheim) and K2P tPA (Reteplase, Boehringer-Ingelheim) to the four E2 forms was determined in an ELISA set-up. The E2 forms were immobilized on an ELISA plate and overlayed with a concentration series of tPA or K2P tPA in PBS with 0.1% Tween20 and the lysine/arginine analogue 10 μM i-amino caproic acid (εACA). The εACA is added to direct binding of tPA to cross-beta and to avoid additional binding of tPA or K2P tPA to lysine/arginine residues via the Kringle2 domain. The results of one out of two experiments are shown in
Epitope Scanning ELISA with Three HRP Labeled CSFV Neutralizing Monoclonal Anti-E2 Antibodies.
Analysis of exposure of functional epitopes on E2 forms. In an ELISA lay-out, it is assessed whether the various cross-beta comprising forms of E2 expose binding sites for CSFV neutralizing antibodies 21.1, 39.5 and 44.4, and for immune serum obtained from pigs that were immunized with various forms of E2 (See FIG. 31A.-G.). The immune sera were obtained during an immunization/CSFV challenge trial as outlined in patent application WO2007008070. Pigs immunized with placebo did not survive a challenge infection with CSFV; pigs immunized with E2-DOE all six survived the challenge infection. Pigs immunized with a different batch of cE2 which was covalently coupled to ovalbumin and subsequently applied to cross-beta inducing procedures, survived the CSFV challenge, as did the pigs that were immunized with cE2 adjuvated with double oil in water emulsion according to a commercialized protocol (CVI). The cE2 used for this previously disclosed immunization/challenge trial was from a different lot than the cE2 used for the currently disclosed experiments. It is seen that cE2-B hardly exposes epitopes for the functional monoclonals, neither for the anti-E2 antibodies in immune serum. Epitopes are similarly in number exposed in SEC-E2 and cE2-A, whereas somewhat less epitopes are accessible for the functional antibodies in cE2.
Subsequently, binding of virus neutralizing mouse monoclonal antibodies 39.5 and 44.3 to nE2 under influence of a dilution series of pooled pig serum obtained after immunization with placebo/PBS or with cE2 adjuvated with double oil in water emulsion according to a commercialized protocol, was assessed in an ELISA lay-out. The cE2 was coated and the two monoclonal antibodies 39.5 and 44.3 were contacted with the cE2 at a concentration that gave approximately half-maximum binding, as determined in the antibody binding experiment outlined above. A dilution series of immune serum obtained from pigs that were immunized with either placebo (buffer, PBS) or E2 in double oil in water adjuvant, was added to the half-maximum binding concentration of the functional monoclonals. The immune sera were again obtained from a previous immunization/challenge trial as outlined in patent application WO2007008070. Binding of the monoclonals 39.5 and 44.3 was assessed. See
With this information we now know that pigs that survive a challenge with CSFV have antibodies that compete for binding sites on cE2 with virus neutralizing monoclonal antibodies. Therefore, the monoclonal functional antibodies are used for selection of E2 forms that expose the epitopes for the functional antibodies, and thus the epitopes that are bound by antibodies in immune serum of pigs that survive a CSFV challenge infection.
Immunization of Pigs with Various Cross-Beta Comprising Structural Forms of E2, and Subsequent Challenge with Classical Swine Fever Virus
Five groups of six pigs and one group of five pigs (group 2) were immunized with 32 μg recombinant E2/animal or with placebo (PBS, Test group T01). For the vaccination, antigens were applied as depicted in Table 8. Thirty-six male pigs were used, at first, but the sixth animal in group 2 died before the start of the study, at day −2, and could not be replaced anymore. Pigs were housed at the facilities of CVI. The pigs were approximately 6 weeks old at vaccination, and were free of antibodies against CSFV. Pigs were randomly allotted to a vaccine group or control group. The animals were fed, and could drink water ad libitum. At day 0 and 21 the pigs were immunized intramuscular with 2.0 ml test sample, once on the left and once on the right, approximately 2-5 cm behind the ear. Antigens were prepared and formulated by Cross-beta Biosciences, except for test item 3, used for group 3, i.e., E2 adjuvated with DOE. This test item was formulated freshly at the day of the vaccinations, by personel of CVI, according to an internal SOP.
Challenge with CSFV strain Brescia 456610. On day 42 the 35 pigs were inoculated intranasally with a dose of 200 LD50 of the highly virulent CSFV strain Brescia 456610.
Evaluation and examination. Anal temperature was measured starting 4 days before the first immunization and during the challenge until the end of the experiment (day 56) (see
During the course of the whole study the animals were monitored once each day, which is outlined in
Pigs in positive group 3 did not suffer from clinical symptoms and all six survived the CSFV challenge. The pigs in placebo group 1 suffered on average from 6 clinical symptoms, when still surviving. Pigs died at day 8 (2), 9 (1), 12 (1) and 13 (2). Comparing groups 2, 4-6, immunized with various forms of cross-beta E2, reveals that on average pigs in groups 2 and 6 suffered from less clinical symptoms than pigs in groups 4 and 5. Analyzing survival reveals a somewhat different picture. Pigs did not die in group 2, pigs did die at day 10 (1), day 14 (1) in group 4, with four survivors, at day 7 (1), 8 (1), 9 (1), 12 (1), 13 (1) in group 5, with one survivor, at day 6 (1), day 11 (1) and day 12 (1) in group 6, with three survivors. In terms of survival upon challenge protection was provided according to cE2 (5/5)>cE2-A (4/6)>SEC-E2 (3/6)>cE2-B (1/6).
Blood samples for serum collection were taken at regular intervals including day 0, 7, 14, 21, 28, 35, 42 (challenge), 49 and 56 (end of challenge period). Sera was subsequently obtained after centrifugation and stored frozen.
Anti E2 antibody titers were assessed by CV1, using the Ceditest CSFV kit (Prionics, the Netherlands). Results in
Virus isolation from leucocytes and from oropharyngal swabs was performed by CV1, according to standard procedures at CV1. For the virus isolation from leucocytes, first the presence of virus was assessed, followed by a titration experiment with positive samples. In
During the post-challenge period, white blood cells and thrombocytes in blood are counted for all surviving pigs at day 0, 2, 4, 7, 9, 11 and 14 (end of challenge period). See
Based on the cross-beta structural data and on the exposure of epitopes for virus neutralizing antibodies, it was expected that cE2-B would provide pigs with relatively less protection against CSFV challenge, compared to other cross-beta comprising E2 forms (See
Factor VIII structural variants with varying cross-beta content and varying number of exposed epitopes for factor VIII inhibiting antibodies induce factor VIII inhibiting antibodies in mice to various extent.
As described above, a series of factor VIII structural variants comprising cross-beta structure, referred to as cross-beta factor VIII forms, are prepared from Helixate recombinant human factor VIII. Factor VIII monomer has a molecular mass of approximately 280 kDa, comprising 2332 amino acid residues, with eight disulfide bonds and 22 (potential) N-linked carbohydrates.
For immunizations, a modified version of cross-beta factor VIII form 3 is prepared; cross-beta factor VIII form 12, incubated prolonged for 1 week, instead of for 20 h, at 37° C. after dissolving, followed by storage at 4° C. This cross-beta form 12 is compared with cross-beta forms 1 and 5 in the ThT fluorescence enhancement assay (
In addition to the structural data for cross-beta fVIII forms as outlined above, TEM images are taken for cross-beta forms 1, 3 (preparation comparable to cross-beta form 12) and 5, as well as negative control PBS (See
In
Furthermore, the cross-beta factor VIII forms 1, 5 and 12 are compared for their relative exposure of epitopes for factor VIII inhibiting antibodies in human haemophilia patient plasma (
See the general outline of an immunization trial with mice and various cross-beta forms of factor VIII. According to the general experimental outline, four groups of five mice were immunized intravenously for four times. Antigens used were as depicted in the legend to Table 10. The selection of these three cross-beta forms of factor VIII is based on the following criteria. In
Plasma of the mice was collected at day 56 after the first immunization (immunizations at day 0, 14, 26 and 42). Titers against freshly dissolved factor VIII are determined and given in Table 10. At day 97, 55 days after the final immunization, plasma was again collected for analysis of the presence of factor VIII inhibiting antibodies. In a Bethesda assay that is applicable for the use with mouse plasma (developed at Good Biomarker Sciences, Leiden, the Netherlands), the presence and relative amount of antibodies in the mouse immune plasmas that inhibit factor VIII in human plasma, was assessed and given as Bethesda units per ml plasma (BU/ml). Values are given in Table 10. From Table 10 it is clearly seen that cross-beta factor VIII form 1, which comprises cross-beta structure and relatively the most epitopes for factor VIII inhibiting antibodies present in human haemophilia patient plasmas, induces antibody titers in five out of five mice, that inhibit human factor VIII. Cross-beta Factor VIII form 12, comprising relatively more cross-beta structure and a comparable number of epitopes for factor VIII inhibiting antibodies, induces anti-fVIII titers in two out of five mice (a titer of 16 is considered as negative, because one mouse in the placebo PBS group is presented with a titer of 16), which titers are comprising factor VIII inhibiting antibodies. Cross-beta Factor VIII form 5, comprising relatively the most cross-beta structures in on average the largest molecular assemblies which in part are insoluble, and comprising far less epitopes at the molecular surface, if any, for factor VIII inhibiting antibodies, induces titers in four out of five mice, but which titers are not comprising human factor VIII inhibiting antibodies.
From these data it is concluded that the combination of cross-beta structure in factor VIII and exposed epitopes for factor VIII inhibiting antibodies, as in cross-beta factor VIII form 1 and in form 12, is required for eliciting factor VIII inhibiting antibodies in an animal. Cross-beta Factor VIII form 5 comprises immunogenic cross-beta structures, as expressed by the anti-fVIII titers, but comprises hardly any exposed epitopes for factor VIII inhibiting antibodies. Indeed, cross-beta factor VIII form 5 induces an antibody response but these antibodies turn out not to be functional antibodies, i.e., factor VIII inhibiting antibodies, in accordance with the strongly reduced exposure of epitopes in the factor VIII antigen used for immunizations. This demonstrated the necessity of a combination of immunogenic cross-beta structure and exposed and available epitopes for functional antibodies, in an immunogenic composition for induction of functional antibody titers. Based on the molecular size distribution, multimers of up to factor VIII 12-mers are capable of eliciting an immune response.
This example illustrates the ability to generate and select immunogenic compounds comprising a cross-beta structure and epitopes for antibodies capable of inducing an humoral response.
Study design. Ovalbumin was used as test protein and antigen. Cross-beta structure was induced in OVA in three different ways. Exposure of epitopes for a series of anti-OVA antibodies was scanned and compared. Mice were immunized with OVA, comprising relatively low cross-beta structure content (nOVA) or with three cross-beta OVA forms comprising increased numbers of cross-beta structure. In sera the antibody titer against nOVA was determined.
Preparation of Cross-Beta Variants of Ova. Four Different Forms of Ova comprising cross-beta structure, termed nOVA, dOVA-1, dOVA-2 and dOVA-3, were prepared according to examples of procedures to induce cross-beta structure described in this application and described below, and were compared in this example.
Cross-beta nOVA. OVA was dissolved in PBS to a concentration of 1.0 mg/mL. The solution was kept for 20 min at 37° C. in a water bath and subsequently for 10 min on the roller device (at room temperature). Aliquots were stored at −80° C. This cross-beta OVA form is referred to as nOVA, cross-beta nOVA or nOVA standard.
Method for inducing cross-beta structure: dOVA-1. OVA was dissolved at 5.2 mg/ml in HBS buffer (20 mM Hepes, 137 mM NaCl, 4 mM KCl). To dissolve OVA the solution was incubated for 20 min in a water bath at 37° C. and 10 min on a roller device at RT. The solution appeared clear. 5 M HCl is added to 2% of the total volume. The solution was mixed by swirling. The solution was incubated for 40 minutes at 37° C. (water bath). The solution appeared white/turbid. 5 M NaOH stock (2% of the volume) was added to neutralize the solution. The solution was mixed by swirling. The visual appearance of the solution remained turbid. Samples were aliquoted and stored at −80° C.
Method for inducing cross-beta structure: dOVA-2. OVA was dissolved in PBS to a concentration of 1.0 mg/mL. The solution was kept for 20 min at 37° C. in a water bath and subsequently for 10 min on the roller device (at room temperature). 200 μl aliquots in PCR cups were heat-treated in a PCR machine (MJ Research, PTC-200) (from 30° C. to 85° C. in steps of 5° C. per min). This cycle was repeated 4 times (in total 5 cycles). The samples were subsequently cooled to 4° C. The solutions were pooled, divided in 100 μL aliquots and stored at −80° C.
Method for inducing cross-beta structure: dOVA-3. OVA was dissolved in PBS to a concentration of 1 mg/ml and subsequently incubated for ten minutes at 37° C. followed by ten minutes RT incubation on a roller device. 200 μL aliquots were incubated in PCR strips (total 5.5 mL) at 75° C. in MyiQ real time PCR, BIORAD ΔT=one minute at 25° C., 25° C. to 75° C., ramp rate 0.1° C./second, incubation time approximately 16 h at 75° C., without cooling.
Endotoxin measurement. The endotoxin content of OVA was measured at 20 μg/mL (diluted in sterile PBS). The Endosafe cartridge had a sensitivity of 5-0.05 EU/mL (Sanbio, The Netherlands). The endotoxin levels are shown in table 11. The endotoxin level of the dilution buffer PBS is checked regularly and is below 0.050 EU/mL. Mice were immunized with 5 μg of cross-beta OVAs per mouse. The amount of endotoxins in 5 μg is calculated from the endotoxin level determined at 20 μg/mL.
Visual inspection by eye and under a microscope, of various OVA forms. Table 12 describes the appearance of nOVA and the three dOVAs by eye. It is observed that dOVA-1 and dOVA-3 comprise insoluble OVA multimers as the solution is no longer clear upon treatment.
Transmission electron microscopy imaging (TEM) with OVA forms. The various OVA forms are subjected to TEM analysis. Table 13 summarizes the analysis. It is seen that multimeric OVA structures are induced by all three treatments. Aggregates are observed that vary in size in all dOVA variants, indicating the presence of cross-beta structure. In nOVA no aggregates are visible on the TEM image.
SDS-PAGE analysis of the OVA samples.
Enhancement of Thioflavin T fluorescence under influence of various OVA forms. Binding of Thioflavin T and subsequent enhancement of its fluorescence intensity upon binding to a protein is a measure for the presence of cross-beta structure which comprises stacked beta sheets. For measuring the enhancement of Thioflavin T fluorescence, OVA samples were tested at 50 μg/ml final dilution. Dilution buffer was PBS. Negative control was PBS, positive control was 100 U/ml standard (reference) misfolded protein solution, i.e., dOVA standard. dOVA standard is obtained by cyclic heating from 30 to 85° C. in increments of 5° C./minute a 1 mg/ml OVA (ovalbumin from chicken egg white Grade VII, A7641-1G, Lot 066K7020, Sigma) solution in PBS.
Enhancement of Sypro Orange fluorescence. Sypro Orange is a probe that fluoresces upon binding to misfolded proteins. As a measure for the relative content of proteins comprising cross-beta structure, enhancement of Sypro Orange fluorescence is tested with OVA samples at 50 μg/ml final dilution. Dilution buffer was PBS. Negative control was PBS, positive control was 100 μg/ml dOVA standard. The results are shown in
Stimulation of tPA-mediated plasminogen activation by OVA samples. The OVA samples were tested for their tPA mediated plasminogen activation potency at a concentration of 25 and 10 μg/ml. The results are shown in
Binding of Fn F4-5 to various forms of OVA, as determined in an ELISA with immobilized forms of OVA.
Binding of monoclonal antibodies to various forms of OVA, as determined in an ELISA with immobilized forms of OVA. Tables 18 and 19 show the results (Bmax and kD) of binding analysis by ELISA of several antibodies to nOVA and the dOVA samples.
Immune activating potential of various forms of cross-beta OVA in vivo. The immune-activating potential of cross-beta structural variants of OVA were determined in vivo. Therefore, groups of 13 mice were immunized subcutaneously 4 times with 5 μg OVA/100 μl at weekly intervals. Four days after the last immunization anti-OVA antibody titers were determined. The secondary antibody used binds to both IgG and IgM. Table 20 shows the OVA samples that were used to immunize each different group. Group 1 did not receive an OVA sample, but only buffer (placebo group).
Humoral response. Total anti-OVA IgG and IgM titers present in the serum on day 25 was highest in the groups immunized with dOVA forms and comparable to the levels observed after immunization in the presence of complete Freund's adjuvant (CFA,
IgG/IgM ELISA. Antibody titers were determined for each individual serum against OVA using enzyme-linked immunosorbent assay (ELISA). Briefly, OVA was coated on 96-well plates (655092, Greiner Microlon) at a concentration of 1 μg/ml in 0.1 M Sodium Carbonate, pH 9.5. All incubations were performed for one hour at room temperature (RT) intermitted with five repeated washes with PBS/0.1% Tween20. The wells were blocked with 200 μl of blocking buffer (Roche Block) washed and subsequently incubated with dilutions of the sera. As positive controls, monoclonal anti-OVA IgG (A6075, Sigma) was included in each plate. Total IgG was determined using rabbit-anti-mouse peroxidase labeled-conjugate (PO260, DakoCytomation) followed by incubation with TMB substrate (tebu Bio laboratories). Reaction was stopped using 2 M H2SO4. Final titers were determined after subtraction of the no-coat controls. The titer was determined as the reciprocal of the dilution factor that resulted in a signal above the mean signal plus 2 times the standard deviation of the placebo group.
Based on the varying cross-beta structural data, the relative exposure of epitopes for anti-OVA antibodies and the measured anti-OVA antibody titers in mouse sera upon immunization with cross-beta nOVA, cross-beta dOVA-1, cross-beta dOVA-2 and cross-beta dOVA-3, the following conclusions are drawn. Cross-beta nOVA comprises relatively less cross-beta structures which appear as invisible OVA molecular assemblies, compared to the three other cross-beta OVA variants. There is no data showing that nOVA comprises multimers, except for dimers seen on SDS-PA gel. The other three cross-beta dOVA variants 1-3 comprise various amounts of cross-beta structure and comprise multimers as seen on SDS-PA gel and TEM images. All four cross-beta forms of OVA comprise exposed epitopes for a series of anti-OVA antibodies. Upon immunization of mice, the three dOVA forms 1-3 are far more potent in inducing an humoral response than the cross-beta form nOVA, which comprises a relatively low content of cross-beta structure, which appears in relatively low molecular weight OVA assemblies.
†group 2 started with 5 pigs at day 0 (one pig died at day −1)
| Number | Date | Country | Kind |
|---|---|---|---|
| 07120303.8 | Nov 2007 | EP | regional |
