MX2007004511A - Methods and compositions for combinatoral-based production of multivalent recombinant antigens - Google Patents

Abstract

The present invention provides methods and compositions for rapidly producing multivalent recombinant vaccines using filamentous fungal heterokaryons. The present invention relies on the use of filamentous fungal heterokaryons that are generated from combinations of two or more parent strains into which recombinant DNA molecules encoding variants of antigens derived from pathogenic organisms have been introduced. The resulting vaccines are multivalent.

Description

METHODS AND COMPOSITIONS FOR COMBINATORY-BASED PRODUCTION OF RECOMBINANT ANTIGENS, MULTIVALENTS FIELD OF THE INVENTION The invention disclosed relates to the field of molecular biology and the production of multivalent vaccines against pathogenic organisms. One embodiment of the invention specifically provides methods and compositions that provide a population of nucleotide sequences encoding antigen in heterokaryotic filamentous fungi that can be used to produce a population of multivalent vaccines.

BACKGROUND OF THE INVENTION Vaccines are currently produced by a variety of methods. Typically, influenza vaccines are produced using fertilized chicken eggs. In the United States of America, the Centers for Disease Control will select three strains of virus that are thought to represent the viruses most likely to attack in a particular flu season. Samples of the selected viruses are provided to manufacturers as concentrations of seed virus that possess the desired antigenic characteristics. Seed viruses are injected into fertilized chicken eggs. The eggs are incubated while the influenza viruses multiply. After a suitable period of time the eggs are opened and the whites are collected. This sample contains the viruses. The viruses are purified from the egg material and inactivated. The individual virus concentrations are then combined to create the vaccine for common influenza, which is typically a trivalent vaccine. There are a variety of problems that can arise which can compromise a total batch of vaccines. For example, problems with sterility led to the decertification of the facility for the production of Chiron vaccines in 2004. This situation illustrates how unreliable methods can be for the production of traditional vaccines. In addition, the methods for the production of current influenza vaccines employ the use of hundreds of millions of chicken eggs each year. Storage, handling, and processing steps are time consuming and labor intensive. Additionally, given the longer production times, if a new strain of influenza virus becomes predominant during a time of flu. Methods for production based on current eggs could take several months for a novel vaccine to be produced.

In view of these limitations, a more flexible and efficient method is urgently needed for the production of antigenic material, such as, for example, an influenza vaccine.

Recombinant fungal protein expression Cloning and expression of heterologous genes in fungi has been used to produce a variety of useful proteins. For example: Lambowitz, U.S. Patent No. 4,486,533, discloses the autonomous replication of DNA vectors for filamentous fungi by mitochondrial plasmid DNA and the introduction and expression of heterologous genes in Neurospora; Yelton et al., U.S. Patent No. 4,816,405, discloses tools and systems that allow the modification of important strains of filamentous ascomycetes to produce and secrete large amounts of desired heterologous proteins; Buxton et al., U.S. Patent No. 4,885,249, discloses the transformation of Aspergillus niger by a DNA vector containing a selectable marker capable of being incorporated into host A. Niger cells; and McKnight et al., U.S. Patent No. 4,935,349, discloses a method for expressing the major eukaryotic genes in Aspergillus which involves promoters capable of directing the expression of a heterologous gene in Aspergillus and other filamentous fungi. Similar techniques have been used to clone the mtr gene involved with the transport of amino acids in Neuroespora crassa ("N. crassa") and to verify the tight junction of the cloned DNA with the genomic markers flanking this gene in vivo. Stuart, W.D. et al., Genome (1988) 30: 198-203; Koo, K. and Stuart, W.D. Genome (1991) 34: 644-651. Filamentous fungi have many characteristics that make them good candidates for use in the production of eukaryotic proteins. Filamentous fungi can secrete complex proteins; correctly folded three-dimensional proteins that include the formation of disulfide bonds; proteins protected proteolytically after translation; and glycosylated proteins using N-linked and O-linked glycosylation reactions. These abilities have made this group of organisms attractive hosts for the production of secreted recombinant proteins. (MacKenzie, D.A. et al., J Gen Microbial (1993) 139: 2295-2307; Peberdy, J.F., Trains in BioTechnology (1994) 12: 50-57). Neuroespora crassa has been used as a host cell for the production of homologous and heterologous recombinant proteins. (Carattoli, A., et al., Proc Nat Acad Sci USA, (1995) 92: 6612-6616, Yamashita, R. A. Et al., Fungal Genetics Newsletter (1995 Suppl.) 42A; Kato. al., Fungal Genetics Newsletter (1995 Suppl.) 42A; Buczynski, S. et al., Fungal Genetics Newsletter (1995 Suppl.) 42A, Nakano, ET et al., Fungal Genetics Newsletter (1995 Suppl.) 40: 540). In addition, Neuroespora crassa has been used as a host cell to express heterodimeric and multimeric recombinant proteins by means of a heterocarion, U.S. Patent No. 5,643,745, July 1997, Stuart 435 / 69.1.

SUMMARY OF THE INVENTION The present invention provides heterocarion filamentous fungi that produce multivalent vaccines. The individual heterocarions of the present invention are generated by fusing a first, and a second, and, in the case of trivalent vaccines, a third original fungal strain and, in the case of higher levels of vaccine valencies, an additional original strain for each set of aggregated antigens, each original strain contains the markers necessary to maintain a heterokaryotic state as well as an expression unit that codes for a variant that occurs in the nature of an antigen from a multivalent vaccine, a variant designated rationally from a antigen of a multivalent vaccine, or a variant generated randomly from an antigen of a multivalent vaccine. In this way, in addition to the natural variants, variants generated through the use of chemical, physical or site-directed mutagenesis or other techniques can be produced. The heterocarions of the present invention are useful for selectively producing desired multivalent vaccines of the defined antigens. Based on the foregoing, the present invention provides heterocarions that produce variants of multivalent vaccines.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the nucleotide sequence (SEQ ID N0: 1) of the synthetic hemagglutinin gene 0 (HAO) (A / New Caledonial / 20/1999 / H1N1) and the conceptual translation: for the fungal expression. The start codon is underlined. Figure 2 shows the amino acid sequence (SEQ ID NO: 2) encoded by the synthetic hemagglutinin gene 0 (HAO). The fungal signal sequence is shown in bold. Figure 3 shows the nucleotide sequence (SEQ ID NO: 3) of the synthetic hemagglutinin (HA) gene (A / Vietnam / 119/2004 / H5N1). The start codon is underlined. Figure 4 shows the amino acid sequence (SEQ ID NO: 4) encoded by the synthetic hemagglutinin (HA) gene. The fungal signal sequence is shown in bold. Figure 5 shows the nucleotide sequence (SEQ ID NO: 5) of the synthetic matrix protein Mi (A / Vietnam / 1194/2004 / H5Nl). The start codon is underlined. Figure 6 shows the amino acid sequence (SEQ ID NO: 6) encoded by the MI gene. Figure 7 shows a Western blot detection of synthetic HAO expression in N. crassa. The medium from 2-day shake flask cultures of the strains HA05 and HA08, as well as the untransformed control (c), and 50 and 200 ng of control HA (A / New Caldonia 20/99, Protein Sciences ), were divided by SDS PAGE and transferred to nitrocellulose. Hemagglutinin was detected using a goat polyclonal anti-HA (H1N1) antibody (BioDesign), followed by anti-charge and colorimetric Ig-alkaline phosphatase conjugate. Figure 8 shows a gel stained with Coomassie brilliant blue from the medium from 2-day shake flask cultures of HA05 and HA08. The secreted protein relatively little was produced for 2 days in shake flasks under these conditions. Figure 9 shows the expression of static cultures of HA05. The Western transfer of the medium comes from a static 6-day culture of HA05. The HA proteins were detected as in the previous Figure 7. In this experiment, the highest detected band is at ~ 57 kDa (the predicted size based on amino acid composition), against 72 kDa for the control HA protein (C).

DETAILED DESCRIPTION OF THE INVENTION A "heterocarion" (or a heterocarotytic cell) is a cell formed from the fusion of two (or more) filamentous fungal stem strains, each heterokaryotic cell thus containing two (or more) genetically different nuclei. . Heterocarions contain nuclei from stem strains that are generally homozygous for all heterocarion compatibility alleles (except for the matching allele when the tol gene is present). At least ten chromosomal loci have been identified for heterocarion incompatibility: het-c, het-d, het-e, het-i, het-5, het-6, het-7, het-8, het-9 and het-10, and it has been deduced that there are more Peris et al., " Chromosomal Loci of Neurospora Crassa ", Microbiological Reviews (1982) 46: 426-570 through 478. The present invention anticipates the work of what is disclosed in U.S. Patent Nos. 5,643,745, 5,683,899 and 6,268,140 by providing the methods and compositions for producing a population of multivalent vaccines using heterokaryotic filamentous fungi. These methods and compositions are useful in the discovery and production of multivalent vaccines, such as, for example, antiviral vaccines, antibacterial vaccines and antifungal vaccines.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention provides novel methods and compositions for generating a population of multivalent vaccines. Multivalent vaccines are generated from an ordered or random combination of defined antigens. To obtain a multivalent vaccine, a heterocarion is obtained in a method comprising the steps of introducing a first, a second, and sometimes a third or more populations of DNA molecules that encode defined antigens of a multivalent vaccine in first, second and sometimes third or more foster parents, forming heterokaryotic fungal strains using the first, second and sometimes third or more host host parents, and then, if appropriate, cultivate the resulting heterokaryon under conditions in which the subunit coding for the DNAs are expressed and further selected for the resulting heterokaryons for the production of a multivalent vaccine having the desired properties. Each of the elements, namely the fungal parents, the DNA molecules and the fusion methods are described in detail below.

Nature of filamentous fungi and background requirements for the formation of heterokaryons Fungi may occur in individual mononuclear cells that provide filamentous multinuclear strains, yeast cells, bodies in fruit production with various spores, and / or cells that differ sexually. They can also exist in multinucleated forms. The main element of the growth form of a fungus as a mold is the hypha, a tubular branching structure, of approximately 2μ ?? - 10μG? diameter. Hyphae grow by lengthening their tips (apical growth) and by producing lateral branches. In this way, as a colony grows, its hyphae form a mass of interlaced chains.

Some hyphae penetrate into the culture medium in which the fungus is developing to absorb nutrients, while those hyphae that project above the surface of the medium constitute an "aerial mycelium". Most colonies develop on the surface of liquid or solid media such as irregular, dry, filamentous mats. In most species, hyphae are divided by transverse walls called "septa". However, these septa have fine central pores. In this way, even the septated hyphae have nuclei that are embedded in a continuous mass of cytoplasm and, indeed, contain a multiplicity of nuclei in a transportable cytoplasm. The term "filamentous fungi" refers to those fungi that can form a mycelium through a mass of branched, interlaced filaments and, although interrupted by transverse walls, allow passage of the cytoplasm between the compartments due to perforations in the transverse walls. Many of these fungi form meiotic spores inside a sac when they spread sexually. However, with adequate stimulation, the mechanism of them has not been fully understood, reproduction can occur asexually. In this form of reproduction, the spores known as "conidia" behave externally at the tips of the grafted projections, formed at various locations along the filaments. The mushrooms The filamentous materials used to generate the heterocarion panels of the present invention are generally Phycomycetes, Ascomycetes, Basidiomycetes, and Deuteromycetes. Phycomycetes include all fungi without septation, as well as some filamentous septates. Their asexual spores are of various types and include sporangioespores contained within bags formed at the end of specialized peduncles. Different species have different sexual cycles. The Ascomycetes are distinguished from other fungi by the asea, a sac-like structure that contains sexual spores, known as ascospores. Ascospores are the final product of sexual union, the fusion of nuclei in male and female, two meiotic divisions, and usually a final mitotic division. The Basidiomycetes are distinguished by sexual spores that form on the surface of a specialized structure. The Deuteromycetes are often referred to as "imperfect fungi" because no sexual phase has been observed. Their hyphae are separated, and the conidial forms are similar to those of the Ascomycetes. The preferred filamentous fungus is from the group Ascomycetes, more preferably, of the genus Neurospora, Aspergillus, Fusarium, Trichoderma, Chrysosporium, and Penicillium. Particularly useful species from Neurospora include N. intermedia, N. crassa, N. sitopula, and N. tetraspora, of which the most preferred species is N. crassa. Useful species of Aspergillus include A. nidulans, A. niger, A. terreus, and A. fumegatus. The vegetative development of filamentous fungi implies nuclear division with cell division (mitosis). This type of cell division consists of an asexual reproduction, that is, the formation of a new clone without the intervention of gametes and without nuclear fusion by means of conidia. For example, the Neurospora species contains in its nuclei seven different chromosomes, each having an individual copy, that is, the haploid vegetative organism. This haploid state is typically maintained during mycelial development and during asexual reproduction through the formation of conidia. Sexual reproduction can also occur, and then two haploid cells (hyphae or conidia) of different types of mating merge to form a dikaryotic cell that contains two different nuclei. The two haploid nuclei of this form coexist in the same cytoplasm and, for some time, they divide more or less in synchrony. However, if a cell initiates the formation of ascospores, the two different haploid nuclei can actually merge to form a diploid nucleus, which contains pairs of homologous chromosomes. This diploid cell then begins meiosis. A "heterocarion" (or heterocarion cell) is a cell with two (or more) genetically different nuclei. The heterocarions of the invention should contain nuclei from cells that are homozygous for all alleles of heterocarionic compatibility (except for the mating-type allele when the tol gene is present). In a Neurospora for example, at least ten chromosomal loci have been identified for heterocarionic incompatibility: het-c, het-d, het-e, het-i, het-5, het-6, het-7, het-8 , het-9 and het-1 0, and it follows that most exist. Perkins et al., "Chromosomal Loci of Neurospora Crassa", Microbiological Reviews (1982) 46: 426-570, to 478. If two strains carry different alleles at one or more locus het, they are unable to form stable heterocarions. Protoplasmic extermination occurs after the fusion of different hyphae or after the microinjection of cytoplasm or extracts into different strains. When the duplications (partial diploids) are heterozygous for one or more alleles, the development is inhibited and is quite abnormal. Several of the heterocarionic incompatibility loci (specifically, het-c, -d, -e, and -i) were first defined by heterocarionic tests. Locus het-5 was detected up to -10 by the use of duplications, as differences in the locus het are common in natural populations. Id. The mating type alleles "A" and "a" also act as het genes in N. crassa, although some decrease in the development of heterokaryotes may occur. Microinjection experiments have involved proteins in the extermination reaction. In this way, the opposite mating types are also generally important for complex events associated with the proliferation of heterokaryotic toiletgenous hyphae. Id. At 436 and 478. However, if the tol gene is present, the vegetative incompatibility (heterocarion) associated with the A and a alleles of the opposite mating type is suppressed without affecting sexual compatibility. In this way, heterocarions (tol; A + a; a) can be fully compatible and stable if the other locus het are of the same allele (or conallic) and the A / a duplications develop normally when the tol gene is present. If hyphae from two different strains are provided that are conallic for the compatibility loci, they can be fused when the development is in the same medium, particularly when the fusion is forced as will be described later. The resulting culture will then contain the nuclei from both strains that circulate in the shared cytoplasm of a common mycelial mating.

Construction of the expression units that code for a mixed population of defined antigens To describe the invention, the following terminology will be used according to the definitions shown below. The invention involves the production of "heterologous multivalent vaccines" in the filamentous fungal heterocarion. In this context, "heterologous" means that the protein is not normally produced by the fungus. "Multivalent" means that the final vaccine product consists of at least two antigens or antigen variants. The product can be a heteromultivalent vaccine, which consists of totally different antigens or can be homomultivalent, consisting of variants of an individual subunit. Examples of multivalent vaccines include, but are not limited to: mixtures of recombinant antigens from cell surfaces, proteins with viral coatings, specific pathogenic protein antigens, and the like. A "nucleotide sequence encoding an antigen" is that portion of a sequence for which the transcript is translated into a polypeptide when functionally linked to the appropriate control sequences. The limits of the coding sequence are determined by a start codon at the 5 'terminus (amino) and a translation terminator codon at the 3' terminus (carboxy). That coding sequence can be derived from, for example, prokaryotic genes, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic DNA (such as, for example, fungal), or it can include synthetic DNA. A polyadenylation signal and a sequence for transcription termination will usually be located at the 3 'end of the coding sequence. A coding sequence is "functionally linked to" control sequence when the control sequences carry out the expression of the coding sequence in the appropriate host cell. An "expression unit" is a DNA molecule that contains a coding sequence functionally linked to a "sequence or control region" that directs the transcription and translation of the functionally linked sequence in a suitable host organism under suitable conditions. A cell has been "transformed" by exogenous DNA when this exogenous DNA has been introduced into the membrane of the host cell. For prokaryotes such as, for example, bacteria, the exogenous DNA can be maintained on an episomal element such as, for example, a plasmid. Because filamentous fungi have nuclei (they are eukaryotic), most stably transformed fungal host cells contain the exogenous DNA integrated into a chromosome, such that it is inherited by daughter cells through chromosomal replication. A "recombinant host" refers to cells that have been transformed, or will be transformed with the DNA sequences prepared by recombinant techniques, and includes the originally transformed cell and the cultures and progeny thereof. A variety of methods can be employed to generate a population of DNA molecules that encode 1) for variants that occur in the nature of an antigen subunit of a multivalent vaccine, 2) for variants generated or randomly selected from an antigen of a multivalent vaccine, or 3) for variants designated or rationally selected from a multivalent vaccine antigen. In the following, an influenza vaccine is used as an illustrative example. An expert can readily use the methods outlined below, or an equivalent method known in the art, to generate the population of DNA molecules that code for a subunit. A population of DNA molecules that code for variants that occur in the nature of an antigen having natural heterogeneity can be produced using standard cDNA generation / cloning techniques. In general, a population of viral genomic mRNA or RNA is first isolated from the pathogen, or for example, in the case of influenza vaccines, directly from the genomic RNA of the virus itself. The isolated population of RNA molecules is then used as a template for the generation of cDNA molecules in cloning methods known in the art such as, for example, RTPCR. Populations of cDNA molecules produced in this way can be inserted into a suitable expression unit as will be described later. Alternatively, for antigens whose protein sequence is known, an artificial cDNA sequence can be generated using methods known in the art, the sequence incorporating codons, which are used in high frequency by the filamentous fungal host strain to produce their own proteins endogenous In addition, site-directed or random mutagenesis can be performed on an isolated or artificial cDNA molecule that codes for an antigen from a multivalent vaccine to produce variants that do not occur in the nature of the particular subunit. Methods such as PCR priming, random or site-directed mismatch, linkage mutagenesis or chemical and physical mutagenesis can be readily used to generate a population of DNA molecules that code for rationally designated or randomly generated variants of an antigen. For example, randomly generated or rationally designated PCR primers can be used to generate random or targeted heterogeneity in an antigenic coding sequence. In the sense in which it is used in the present, a variant is that which is designated rationally when a selection criterion is used, such as, for example, protein folding or when selecting a particular white residue or region, for generation of the variant or to select the DNA molecules that code for variants. A variant is one that is randomly generated when a selection criterion is not used when generating or selecting DNA molecules for variant coding. The preferred target site for generating heterogeneity in a multivalent vaccine subunit is the immunogenic epitope and the surrounding amino acid sequences. In the case of influenza antigen genes, this type of creation or variation could focus on the naturally occurring variable regions of each antigen. As an example, each amino acid in the variable region could be changed to produce a library of known variation.

Construction of expression units encoding multivalent vaccine antigens Expression units containing a nucleotide molecule encoding an antigen from a multivalent vaccine are constructed using well-known techniques. In general, an expression unit is generated by placing the sequences encoding subunits in a functional linkage with control sequences that direct the expression of the sequences encoding subunits in the final filamentous fungal host. A variety of control elements are currently known in the art to direct the expression of a sequence encoding functionally linked proteins in either constitutive or inducible form. The choice of a control sequence will be based on the fungal strain used, the conditions employed to cultivate the fungus, the level of protein expression desired, and the nature of the expression required (eg, inducible against constitutive). One skilled in the art can readily use control sequences known in the art to generate the expression units used in the present heterocarion panel. In addition to the sequences that direct the transcription and translation of the protein coding sequence, the expression units of the present invention can also control the signal sequences, the elements for expression control that direct the export of an antigen out of the cell . A review of the secretory signals that are known in filamentous fungi is provided by Dalbey R.E., et al., TIBS 17: 474-478 (1992). One skilled in the art can easily generate expression units that contain secretory signals. Another form of expression unit of the present invention may contain a fusion protein that directs the antigen to the cell membrane via fusion to a host cell or heterologous membrane anchor sequence or to the cell surface by fusion to a host cell or a heterologous cell surface molecule. In one application, the recombination units are generated in place of the expression units. In this use, the subunit coding sequence, or the fragment of an antigenic coding sequence, is flanked by the DNA regions containing the sequences that are homologous to an integration site in the fungal host strain. The homologous sequences are then used to stimulate and direct homologous recombination between the recombination units and the host chromosome. When recombination units are used, the host strain is preferably first transformed with an expression unit containing an element for expression control followed by the sequences that are used for a directed recombination. For example, an influenza antigen can be introduced into a host fungus and then the homologous recombination units can be used to introduce heterogeneity into a target region of the host chromosome. Intermediary hosts are sometimes used to produce intermediary vectors capable of transforming the final fungal cells. The intermediate bacterial transformants can then be developed to obtain the desired amounts of DNA, which can be used to transform a desired filamentous fungal host. Examples of commonly available bacterial vectors that can serve as intermediary vectors include, for example, pBR322, pUC8 and pUC9. Additional useful intermediate vectors include pHY201, pKBY2, pTZ18R, pXl82 and pCVN2.9, pN807, pN846. In another embodiment, the antigen or antigens of interest are expressed as a fusion protein with a fungal hydrophobin, such as, for example, EAS. The fungal hydrophobins are typically expressed and secreted in large quantities. The surfaces of the fungi are then coated with the hydrophobins. It is also known that these proteins are added in solution. This aggregate characteristic allows the preparation of added recombinant proteins, which form antigenic particles. These proteins can be purified and used as antigens. When multiple antigens are expressed in the same culture, multivalent antigenic particles are produced. It will be understood that this description and disclosure of the invention is intended to cover all the modalities that remain within the spirit and scope of the invention. For example, it is within the skill of the art to insert, delete or substitute amino acids within the amino acid sequence of an open reading frame without substantially affecting the antigenicity of the molecule, and these multivalent antigens as may be generated with deletions, additions or Substitutions to the naturally occurring subunit are included in the invention.

Nature of the original strains Because each of the original fungal strains used in the production of heterocarions of the present invention will contain a member of a population of DNA molecules that encodes an antigen of a multivalent vaccine, a fungal parent will have a core modified to contain a member of a first population of DNA molecules that codes for a first antigen or groups of antigens of a desired multivalent vaccine and each successive fungal parent will have a modified core to contain a member of a different population of molecules of DNA that codes for a different antigen or groups of antigens of a desired multivalent vaccine. For example, to produce a heterocarion that provides a divalent influenza vaccine, a fungal parent will produce a group of antigens from type A Hl NI of influenza while the other fungal parent will produce an antigenic group from the cDNA that comes from type A H3 N2 of influenza. To produce a trivalent influenza vaccine, a fungal parent will produce an antigenic group from the cDNA that comes from the Hl NI influenza type, the second fungal parent will produce an antigenic group from the cDNA that comes from the H3 N2 type of influenza and the third father fungal will produce an antigenic group from the cDNA that comes from the BHN type of influenza. The nucleotide and protein sequences are available to the public. For example, example sequences for HA genes include H3N2 (AY738729), influenza A virus (A / Leningrad / 54/1 (H1N1)) neuraminidase gene (M38309), Influenza A virus (A / Swine / Ontario / 42729A / 01 (H3N3)) neuraminidase (NA) gene (AY619975), Influenza A virus (A / Swine / Ontario / K01477 / 01 (H3N3), neuraminidase (NA) gene (AY619966) , Influenza A virus (A / Swine / Saskatchewan / 18789/02 (H1N1)) neuraminidase gene (NA) (AY619960), Influenza A virus (A / Puerto Rico / 8/34 / Mount Sinai (H1N1)) segment 6 (NC 004523), Influenza A virus (A / mallard / Alberta / 211/98 (H1N1)) neuraminidase (NA) gene (AY633214), Influenza A virus (A / mallard / Alberta / 99/91 (H1N1) )) neuraminidase gene (NA) (AY207541), and Influenza A virus (A / duck / Miyagil / 9/77 (H1N1)) neuraminidase gene (AY207534) In addition to being modified to contain a DNA molecule encoding for the antigen or group of antigens, as described above, the nuclei of each of the parent strains must contain a genome that results in a characteristic that makes the fungus dependent on the presence of a second nucleus, and / or a third nucleus and / or additional nuclei to survive under the conditions used for form heterocarions. In this way, the core of each parent confers a characteristic that could result in the failure of the fungus in which it is contained to survive under culture conditions unless the second nucleus, and / or the third nucleus and / or additional cores are also present. For example, a parent who requires a particular nutrient may be cultivated in a medium that lacks the nutrient along with a parent who does not have this requirement. If hyphae fusion occurs, the nucleus of the second parent confers ability to survive in the absence of this nutrient. The second parent, in turn, may require a different nutrient, not required by the first. Only fungi that contain both nuclei will then be able to survive when both nutrients are lacking. The required nutrient can be any substance that the cells of the fungal strain need to develop or, when absent, seriously damage the ability of the fungus strain to grow or survive.

Examples of useful nutrient requirements and relevant mutants include: (1) amino acids such as, for example, histidine (his-1 mutants to -7), proline (aga mutants), arginine (arg-11 mutants), citrulline (mutants) arg-11), asparagine (asn mutants), choline (chol-1 and chol-2 mutants), cysteine (cys-1 mutants), glutamine (gln-1 mutants), leucine (leu -1 to -4), lysine (lys -2, -4 and -5), methionine (mutants mac and mutants met-6, -9 and -10), and threonine (mutants thr-2 and -3); (2) mixtures of aromatic amino acids, such as, for example, a mixture of p-aminobenzoic acid, tyrosine, tryptophan, and phenylalanine (required by all aro strains except aro-6, aro-7 and aro-8), a mixture of tryptophan and phenylalanine (required for the aro-6 mutants), a mixture of isoleucine and valine (required for ilv-1, -2 and -3), and a mixture of phenylalanine and tyrosine (required for the pt mutants); (3) vitamins such as, for example, pantothenic acid (pan-1 mutants) and thiamine (thi-2 and thi-4 mutants); (4) purine bases such as for example, adenine (mutants ad-2 to ad-4 and ad-8), hypoxanthine (mutants ad-2 and ad-3), inosine, and guanine or guanosine (mutants gua-1 or -2);(5) pyrimidine bases such as, for example, uracil (pyr-1 to pyr-6); (6) saturated fatty acids (cel mutants) or unsaturated fatty acids such as, for example, Ci6 or Cis fatty acids having a double bond in the cis conformation at the position either 9- or 11-, fatty acids with a double bond in the trans configuration at position 9, and fatty acids with multiple cis double bonds interrupted by methylene bridges (ufa-1 and -2); (7) physiologically important ions such as, for example, potassium (trk); (8) sugar alcohols such as, for example, inositol (mutants aqu and mutants inl) and glycerol; and (9) other organic entities such as, for example, acetate (as mutants), I-ketoglutarate, succinate, maleate, formate or formaldehyde (for mutants), p-aminobenzoic acid (mutants pab-1, -2 and -3) , and sulfonamide (sfo mutants at 35 ° C). A specific example based on a nutritional requirement is the Arg B + gene that codes for the enzyme ornithine transcarbamylase. This enzyme is present in A. niger wild type. Mutants lacking this enzyme (Arg b strains) can be prepared by usual non-specific techniques, such as, for example, treatment with ultraviolet radiation, followed by selection based on an inability to grow in a minimal medium, coupled with a ability to grow in a medium that contains arginine. The fungi that contain this genome will develop in minimal media if they include an Arg B + nucleus. Also useful for forcing heterocarion formation are genes that confer resistance to any of a variety of cytotoxic agents. For example, in an alternative embodiment, one of the parents may have a requirement for nutrients as well as a resistance to a toxic effect induced by a noxious chemical, an antibiotic or virus, or stringent environmental conditions, such as, for example, variation of predetermined temperatures to which another parent is sensitive. Specific examples of harmful chemicals that can exert a toxic effect include acriflavine (resistance conferred by acr in general, with the presence of the shg gene required for resistance by acr-4 and acr-6); 3-amino-1,2,4-triazole (resistance conferred by acr-2, atr-1, cpc, leu-1 or leu-2)); dyes such as, for example, malachite green (resistance conferred by acr-3); caffeine (resistance conferred by caf-1); purine analogues (resistance to 8-azaadenine and 2,6-diaminopurine conferred by aza-1; resistance to 8-azaadenine and 8-azaguanine conferred by aza-2; resistance to 8-azaguanine and 6-mercaptopurine conferred by aza-3 Resistance to 6-methylpurine conferred by mep (3) and mep (10), cyanide (insensitivity conferred by cni-1 in the first 24 hours of growth), tetrazolium (resistance conferred by cya-6 and cya-7), cycloheximide (resistance conferred by cyh-1, -2 and -3), chromate (resistance conferred by cys-13), 2-deoxy-D-glucose (resistance conferred by dgr), edein (resistance conferred by edr-1 and -2) ), ethionine (resistance conferred by eth-1, by nap in the presence of p-fluorophenylalanine and by oxD if the ethionine is in the D form), fluorine compounds such as, for example, 5-fluorodeoxyuridine, 5-fluorouracil and -fluorouridine (resistance to all three conferred by fdu-2; resistance to 5-fluorouracil which is conferred by uc-5 in a minimal medium ammonium re; resistance to 5-fluorodeoxyuridine and 5-fluorouridine conferred to ud-1), and fluorophenylalanine (resistance conferred by fpr-1 to -6 under certain conditions); 8-azaadenine (strength conferred by mts); methane-methane sulphonate (insensitive or marginally sensitive by upr-1); surfactants such as, for example, dequalinium chloride, cetyltrimethylammonium bromide, and benzalkonium chloride (strength conferred by south-1); and metal ions such as for example, vanadate (resistance conferred by van).

Examples of antibiotics that typically exert a toxic effect include benomyl carbamate > methyl-l- (butylcarbamoylbenzimidazol-2-yl) (resistance conferred by Bml); antimycin A (insensitivity conferred by cni-1 in the first 24 hours of growth); polyene antibiotics such as, for example, nystatin (resistance conferred by erg-1 and 3); and oligomiein (resistance conferred by oli). Also useful are genes that confer resistance to extremes in various environmental conditions such as, for example, high or low temperature, lack of oxygen (resistance conferred by an), constant light (resistance conferred by lis-1, -2 and - 3) or the absence of light, UV radiation, ionizing radiation, and high or low osmotic pressures. In a particularly preferred embodiment, the resistance to a toxic effect is a resistance to an antibiotic such as, for example, hygromycin. The strains in general useful in the invention can be grown in Vogel IX Minimum Medium (N medium) in cotton-covered test tubes, with supplements that are added depending on the phenotype of the strain, such as, for example, histidine, arginine. and / or inositol. Typical strains can be obtained, for example, from Fungal Genetics Stock Center ("FGSC") and from D.D. Perkins, Stanford University. Another strain of N. crassa believed to be useful is M246-89601-2A (obtained from Dr. Mary Case, University of Georgia, Athens). This strain is a derivative of wild-type 74A, which contains a stable qa-2 mutation (M246), an arom-9 mutation (M6-11), and an inos mutation (io601). The double qa-2 mutant, arom-9, lacks the biosynthetic and catabolic dehydrokinase activities and is unable to grow in minimal medium without a supplement of aromatic amino acids, such as, for example, phenylalanine at a concentration of approximately 80 pg per my. Useful strains of A. niger (ATCC 46951) are also available from the Fungal Genetics Stock Center, as well as strains of Fusarium, Gelasinospora, and Sordaria fimicola, or can be prepared by mutagenization with UV light to form an isolate that requires ornithine or arginine to grow in a defined minimum medium. This strain, which lacks ornithine carbamoyl transferase, has been termed arg B (350 (-) 52). The means for growth of A. niger or A. nidulans are described by Cove, Biochim Biophys Acta (1966) 113: 51-56. The standard procedures generally used for the maintenance of the strains and the preparation of conidia (Davis and de Serres, Method Enzymol (1971) 17A: 79-141). Mycelia are typically grown in liquid cultures for approximately 14 hours (25 ° C), as describe in Lambowitz et al. J Cell Biol (1979) 82: 17-31.

The host strains in general can be grown in minimum means either Vogel or Fried supplemented with the suitable nutrients, such as, for example, histidine; arginine; phe, tyr, and / or trp (each of approximately 80 pg for me); p-aminobenzoic acid (approximately 2 pg per my); and inositol (approximately 0.2 mg per ml).

Many fungal strains with the characteristics desired are available to the public. However, if not are easily available, someone with experience in technique can use the selection techniques well known in the art to prepare either the mutants desired or the nuclei under engineering that provide the desired feature. The combinations Illustrative parents are shown in the following table.

Table 1. Combinations of diacarions: Father 1 Father 2 Phenotypes requires histidine tryptophan or requires arginine lysine or requires uranyl thymidine Combinations of tricarions Father 1 Father 2 Father 3 phenotypes requires histidine tryptophan arginine and tryptophan arginine histidine (provides fusion partners with) (arginine) (histidine) (tryptophan) tricarions Father 1 Father 2 Father 3 Father 4 phenotypes requires histidine tryptophan arginine leucine and tryptophan arginine leucine histidine and arginine leucine histidine tryptophan (leucine) (histidine) (tryptophan) (arginine) As seen in the table, a variety of Characteristic combinations / complementary properties can be selected to adjust various conditions of fusion. In general, the nutrient requirement is manifested by a mutant strain, while the capacity to resist certain substances can be conferred more conveniently by modifying the core with a expression system for the resistance gene.

Alternatively, the nutritional requirement can be affect using recombinant techniques such as by example, homologous recombination with a transformant vector and resistance can be conferred by low mutation conditions where toxic conditions are present.

In one embodiment of the invention, the cells hosts are converted to spheroplasts for transformation. When spheroplasts are used, a preferred method to prepare them is by digestion Enzymatic cell walls, for example, when using a mixture of chitinase / glutamase. The selection of an enzyme suitable for enzymatic digestion is within the skill of the art. The enzymes use those capable of digesting complex polysaccharides, and are among those known to be effective in preparing the fungal spheroplast of a wide variety of fungal species. Specific examples of suitable enzymes include Novozyme 234 (an impure mixture of enzymes) and beta-glucurouidase. Other suitable methods can be used to form spheroplasts. If suitable methods for cell wall penetration are identified through the use of vectors, however, whole cells of the fungal host can be used together with or in place of spheroplasts. To modify the nucleus of a fungal host strain to contain an expression unit for a DNA encoding a particular subunit of multivalent vaccine, the practice of the invention employs, unless otherwise indicated, molecular biology, microbiology, and recombinant DNA techniques that are within the experience of the technique. These techniques are fully explained in the literature. See, for example, Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); D.N. Gover et al. DNA Cloning: A Practical Approach (1985) Volumes I and II; Oligonucleotide Synthesis (M.J. Gait ed., 1984); Nuclei Acid Hybridization (Hames et al., 1985); Transcription and Translation (Hames et al., 1984); Animal Cell Culture (R. Freshhey ed. 1986); Immobilized Cells and Enzymes (IRL Press 1986); B. Perbat, A. Practical Guide to Molecular Cloning (1984).

General procedure for the transformation of N. crassa Once the population of DNA molecules that code for the multivalent antigens are placed in the expression units, the DNA molecules are used to transform host strains of a filamentous fungus, such as is described by Smart, "Heterologous dimeric proteins produced in heterokaryons". Strains of Neurospora crassa are available to the public from the Fungal Genetics Stock Center, although strains prepared independently can also be used. The mutants can be isolated again, as illustrated by Stadler et al. Genetics (1966) 54: 677-685 and Haas et al. Genetics (1952) 37: 217-26. Useful strains can also be obtained from D.D. Perkins from Stanford University. The strains are typically grown in Vogel IX minimal medium ("N medium") in test tubes capped with cotton, with suitable supplements that are added depending on the phenotype of the strain.

Spheroplasts are used as subjects for transformation. To form conidial spheroplasts, the fungus is inoculated in 25 ml of solid N medium, with appropriate supplements in four to five Erlenmeyer flasks of 125 ml, which have been covered with cotton. The cultures are grown at room temperature for 5-7 days. The conidia are collected by adding 10 ml of medium N to each flask, replacing the cotton tampon, and plugging the flask with thread. The solids are allowed to settle for a few minutes. The conidial mixture is emptied into a bag of cheesecloth subjected to an autoclave that hangs in the mouth of an Erlenmeyer flask and secured with rubber bands. The filtrate is recovered, and the concentration of conidia is determined by a hemocytometric count, with chains that count as one. A volume of 2 x 109 conidia is added to 150 ml of liquid N medium containing 1.5% sucrose and suitable supplements. The conidia are germinated in the flask capped with cotton while stirring (150-200 rpm) for 5-6 hours at room temperature to more than 75% that has germinated and the tubes for germination have 1-4 diameters of length of conidia. The cells are harvested by centrifugation at approximately 1500-2000 rpm for 10 minutes. The cell pellet is rinsed three times with water. The pellet is then resuspended in 10 ml of 1.0 M sorbitol, and the spheroplasts are prepared by enzymatic elimination of the conidial resistant cell wall with an enzyme under isotonic conditions, to avoid "bursting" of the spheroplast as form The protocol is adapted from the method of Vollmer and Yanofsky, Proc Nati Acad Sci E.U.A. (1986) 83: 4869-73. Specifically, in a sterile 250 ml Erlenmeyer flask, the conidial suspension is generally added to 50 mg of a solid enzyme distributed by Interspex under the tradename Novozyme 234. The mixture is shaken vigorously (100 rpm) at 30 ° C during approximately one hour (4 ± 10 minutes) to digest the cell wall. The process for spheroplasts formation is monitored by examining a small aliquot of the mixture microscopically under a slide. Spheroplasts can be detected because they are osmotically lysed when water is applied to one end of the slide. The process should be monitored frequently in the later stages of spheroplasts formation. The spheroplasts mixture is decanted into a sterile 15 ml conical centrifuge tube, and the spheroplasts are recovered by centrifugation at 500 rpm (10 minutes) in a rotating table-top centrifuge. The resulting pellet is rinsed twice with 10 1.0 M sorbitol and then once with the following STC solution: 91 g sorbitol; Tris. 50 mM Cl; 50 mM CaCl2; NaOH sufficient to adjust the pH to 8.0; and c.s. for 500 mi. The final spheroplasts sediment is suspended in a mixture of 16.0 ml of STC, 200 μ? of DMSO, and 4 ml of the following PTC solution: 200 g of polyethylene glycol sold under the trade name "4000" by Sigma; Tris. 50 mM Cl; 50 mM CaCl2; NaOH sufficient to adjust the pH to 8.0; and c.s. for 50 mi. The resulting suspension of spheroplast can either be used directly or stored frozen in 1.0 ml of aliquots at -80 ° C. In a tube with a screw cap of 15 ml, sterile, 2.0 μ? of 50 mM Spermidine solution, 5.0 μ? of the plasmid DNA to be transfected, such as that containing the expression system for a desired multivalent vaccine antigen together with a selectable marker such as, for example, benomyl resistance (usually at a concentration of about 1.0 mg / ml) and 5.0 μ? of a 5 mg / ml solution of heparin were mixed by the rapid passage of the tube. The spermidine solution is prepared by dissolving 12.73 ng of spermidine in 1.0 ml of TE and adjusting the pH to 8.0, and it can be stored at -20 ° C. The heparin solution is prepared by dissolving 50 mg of the sodium salt of heparin in 10 ml of STC and can be stored in frozen aliquots. The contents of the tube were briefly centrifuged (pulsed) in a tabletop centrifuge with rotating bucket and then placed in a bath with ice. Approximately 50-100 μ? of thawed spheroplasts were added to the tube. The mixture was then incubated on ice for approximately 30 minutes, although incubation periods of about 20 minutes on ice have been successful. Approximately 1 ml of PTC is added and mixed well by rapidly passing the tube. The mixture is further incubated at room temperature for about 20 minutes. A regeneration of "higher" agar is prepared when mixing: 20 ml 50x Vogel minimum medium; 825 mi of water; 182 g of sorbitol; and 28 g of agar. The upper agar was autoclaved and 100 ml of a lOx FIGS solution (containing 5 g / 1 of fructose, 2 g / 1 of inositol, 2 g / 1 of glucose, and 200 of sorbose) is added. 15 ml of the upper agar is incubated at 50 ° -55 ° C. and it is emptied into the tube containing the spheroplasts and the plasmid DNA. The content is mixed quickly by passing quickly and inverting the tube 2-3 times and then emptying evenly on a layer of "lower" agar placed on plates. The "lower" agar is prepared by mixing any required supplements, in lxN medium; by autoclaving; and adding lOx FIGS and benomyl (if benomyl resistance is used as a label) at final concentrations of lx and 0.5 g / ml respectively. A volume of 25 ml of "lower" agar is emptied into a Petri dish and allowed to harden. After the upper agar has been emptied onto the lower agar, the bubbles are removed by flaming. The plates are maintained in a vertical position until the upper agar has solidified (approximately 5 minutes). If the upper agar tends to harden prematurely, the lower agar plates can be preheated. Once the upper agar has solidified, the plates are incubated in an inverted position at 30 ° C. For the selection of N. crassa transformants, the host is cultured on the appropriate medium (the composition having only the transformed cells to use or contain an antibiotic to which only the transformed cells are resistant) and incubated at about 3 ° C. . An indication of a successful transformation can be observed approximately 24-36 hours after plaque placement. Stable transformants are usually marked after three days of growth. The incubation period to detect the transformants will vary depending on the host strain and the phenotypic marker. The selected transformants can be selected by, the expression of the desired antigen subunit by standard methods, such as, for example, a suitable ELISA, a colony transfer immunoassay, a restriction enzyme analysis, filter hybridization, subcloning by inclusive suppression, and the like. In the present invention, the recombinant techniques described above are used to produce fungal host strains expressing a desired recombinant antigen or group of antigens, each host strain having one or more characteristics that negatively affects growth under specific conditions although it can be corrected by a property conferred by one or more other cores. The resulting host strains are the mothers used to form the heterocarions of the invention. Alternatively, electroporation methods can be used to transform conidia recently collected from filamentous fungi such as, for example, Neurospora crassa (Van, D.C.

Fungal Genetics Newsletter No. 42A (Supplement) (1995)). In general, conidia are collected from crops that are 7-28 days old. The cells are washed in 1 M sorbitol solution and suspended at a final concentration of 2.5 x 109 cells / ml. Approximately 5 pg of linearized DNA was added from an aliquot of the conidial suspension and a portion of it was placed in the bottom of a cuvette for electroporation, for example a cuvette for electroporation with an aperture of 0.2 cm. An electroporator such as, for example, an InVitrogen Electroporator II, is adjusted with a voltage gradient of approximately 7.25 kb / cm and a setting of approximately 71 pF and approximately 200 ohms. After electroporation, the cells are plated in a suitable medium with or without an upper agar essentially as described above. After transformation, a stable production strain derived from each molecular variant is established by expanding the culture on a selective medium for the particular host cell and the expression unit used in each individual case.

Production of heterocarion Because all fungal host strains are selected to be conalélic with respect to all alleles of heterocarion compatibility (with the exception of the allele of quenching when the tol gene is present as explained above), when the host strains are they grow together under conditions where none of the host strains can survive alone, the fungi fuse in such a way that the heterokaryotic fungus of the invention is formed. By hyphal fusion, haploid nuclei different from host fungi coexist in a common cytoplasm. While not wishing to be bound by any theory, applicants believe that the membrane fusion resulting from the aggregation of intramembranous particles within each cell makes it possible for the cells to come into contact between the protein-free areas. The rearrangement of the lipids in the contact zones then leads to a total fusion. Because each of the parents contains a nucleus that produces different antigens from the multivalent vaccine, the resulting heterokaryon is capable of producing the complete multivalent vaccine comprising multiple antigens. The heterocarion generated in this way is stable, with the nuclei dividing approximately at the same speed.

Generation of heterocarions that produce defined multivalent vaccines The compositions and methods of the present invention employ heterocarions expressing immunogenic antigens derived from pathogenic organisms. As described above, heterocarion is generated from two or more host strains, each heterocarion produces a different multivalent vaccine. An example is a heterocarion that produces variants of the influenza antigen. To generate this strain, the conidia suspensions of each individual parent strain are mixed together on a substrate with solid media without any nutritional supplement, or in the case of host cells with a resistance gene, in media containing the cytotoxic agent. A heterocarion made from a predetermined combination of parent strains can be formed or a heterocarion library can be formed in a matrix using a microtiter plate or other convenient format. A few of the many combinations available are illustrated in the following table, Table 2, which is provided by way of example and does not mean that it is limiting in any way.

Table 2: Generation of heterokaryons for the production of multivalent vaccines The mother host cells contain genes from influenza strains AH1N1 or AH3N2 or BHN with antigenic variants a, b, c, ... d, e, f, ... g, h, i, ...

A strain for the production of individual vaccines can be generated based on the user's choice Heterocarion comprised of: strain c plus strain f plus strain h Or a panel of strains for the production of vaccines can be generated as Heterocarion one comprised of: strain plus strain d plus strain g Heterocarion two comprised of: strain plus strain plus strain h Heterocarion three comprised of: strain b plus strain f plus strain i etc., in all possible combinations, if desired, where: "A" = type A influenza; "B" = influenza type B; "H" = haemagglutinin; "N" = neuraminidase; "variants" = different combinations of antigenic classes of H and N antigens of influenza types and strains.

A typical minimum medium contains: per liter, 5.0 g of dextrose, 50.0 ml of a saline solution (below), 1.0 my trace elements (below), and 12.5 g of agar (pH 6.5 adjusted) if the medium will be in a solid form. The Saline solution contains: 120.0 g of NaN03, 10.4 g of KC1, 10.4 g of MgSO4, and 30.4 g of KH2P04. The trace element solution contains: 1.1 g of (NH4) 6 Mo7 024.4H20, 11.0 g of H3 B03, 1.6 g of CoCl2 6H2 O, 1.6 g of CuS04, 50.0 g of Na2 EDTA, 5.0 g of FeS04-7H2 O, 5.0 g of MnCl24H2 O, and 22.0 g of ZnS047H20 (pH 6.5). In this way, to maintain the filamentous heterokaryotic fungus in its heterokaryotic state, the external force is maintained. The growth of heterokaryotic fungal cells in minimal medium "forces" the strains to remain together. If the mating types are opposite, the presence of the tol gene can be used to keep the heterocarions stable (A + a). The multivalent vaccine is produced by cultivating the heterocarions of the invention under favorable conditions for the production of antigens. The antigens of the multivalent vaccine can be recovered from the culture and purified according to standard techniques adapted, of course, as necessary to preserve the structure of the antigens. Preferably, the heterocarion filamentous fungus carries an expression unit that allows the host to be cultivated to secrete the desired multivalent vaccine directly into a minimal growth medium, such that the multivalent vaccines can be purified directly from the free medium of the host. cells 0 the heterocarion filamentous fungus carries an expression unit that directs the antigens to the cell surface. The multivalent antigens produced intracellularly can be isolated from the cellular ones. Useful purification methods according to known procedures are within the skill of the art, such as, for example, exclusion by molecular size, ion exchange chromatography, HPLC, affinity chromatography, and hydrophobic interaction chromatography, and the like .

Antigens The disclosed invention is directed to the preparation of antigenic compositions, such as for example, vaccines, against pathogens, in particular against pathogens that demonstrate the ability to change their antigenic character. The eukaryotic, viral, bacterial and fungal antigens are all contemplated for use with the disclosed invention. Another use for the invention is to prepare antigenic compositions against several different antigens simultaneously. A preferred embodiment of the invention relates to the preparation of multivalent influenza vaccines. The influenza virus is constantly mutating and generating new strains. Accordingly, the list of individual genes for use in the disclosed invention could be unnecessarily limited since the invention can be applied to any strain of influenza, or any pathogenic strain for that matter. There are three types of influenza viruses, A, B and C. Type A and B viruses cause epidemics of disease almost every winter, whereas type C viruses only cause mild respiratory disease and are not considered clinically important. Type A influenza viruses are divided into subtypes based on two proteins on the surface of the virus, hemagutinin (HA) and neuraminidase (NA). The current subtypes of influenza A viruses that infect humans are A (H1N1) and A (H3N2). There is currently a great discussion regarding the avian influenza virus (H5N1), for its acronym in English) that is known to also infect humans. The influenza virus experiences a huge antigenic dispersion. Much effort is being devoted around the world to monitor the antigenic spread of the virus, so that new variant strains that emerge can be identified and then used to produce updated vaccines that more closely match the strains that probably infect people in an era of given flu. At the time of identification of an influenza strain of interest, the methods of the disclosed invention can be used to generate a multivalent antigenic material which can then be used to prepare vaccines. Another modality is aimed at the preparation of multivalent vaccines against Plasmodium, a genus of protozoa that includes four species that cause malaria in humans. Examples of the four include Plasmodium vivax and Plasmodium falciparum. A general list of pathogens against which multivalent vaccines are contemplated include the human papillomavirus human 16, 18, and 31, the immunodeficiency virus (HIV), varicella herpes virus, measles virus , Epstein Barr virus, respiratory syncytial virus, parainfluenza 3, herpes simplex virus type 1, and herpes simplex virus type 2. Suitable antigens as targets for the disclosed invention include any protein from these organisms that is capable of producing a immune response in a host.

Analysis for secreted antigens Heterocarion hosts can be stored in minimal solid media and are also grown in minimal liquid media under favorable conditions for the expression of multivalent antigens. After 2-7 days of growth, the liquid media can then be collected under sterile conditions and can be tested for the presence of each specific desired antigen by standard analytical methods including, but not limited to: ELISA, PAGE, capillary electrophoresis and spectrometry. At the time of identification of a culture that is producing the variant variant of the multivalent vaccine, the cells stored in the solid media can be expanded to fermentation cultures for a longer time by standard methods. When the growth under whatever optimal conditions is for the particular fungal host used, this expanded host culture will provide the desired product in sufficient quantities for further investigation evaluation and eventual use as a recombinant vaccine.

Analysis for antigens directed to cell surfaces Heterocarions can be constructed to express fusion proteins that display the antigens szij ^ -a.IAS R. .ta.r: í¾.,.,. T-ja.s__ -cimorf i r? ac de Tac a_s_o x a_s fnnnal O-g _I hydrophobin, such as, for example, the EAS protein of Neurospora, as well as permease proteins, such as, for example, the MTR protein of Neurospora. These proteins are useful as fusion partners directed to the cell surface with viral antigens. The fusion proteins are constructed and expressed by means well known to those skilled in the art. Individual strains expressing the antigen on the cell surface can be analyzed by standard analytical methods. These methods include, but are not limited to: direct or indirect cells marked by antibodies specific for the viral antigen, followed by detection of specific binding by various means. These means include, but are not limited to: ELISA, visual or fluorescence spectrometry, and flow cytometry. The individual strains expressing antigens can then be fused, can be retested for the simultaneous expression of the surface antigens expressed individually, and thus result in a multivalent target for the immune system, useful for recombinant vaccines, and either directly or after purification.

Analysis for non-secreted antigens If the antigens are not secreted, the cell mass in each liquid culture can be eliminated, breaking up by standard methods and the cell supernatant and the wastes analyzed for the multivalent vaccine with convenient characteristics. Once the strain producing the desired variant has been identified by standard methods, the strains stored in solid media can be used for inoculation and to produce an expanded culture. Again, when grown under optimum conditions for the particular heterocarion, this expanded host culture will provide the desired product in sufficient amounts for further evaluation and use. The following examples are offered to illustrate but not limit the invention.

Example 1 Constructs of synthetic HA genes Synthetic genes (HAO from A / New Caledonial / 20/1999 / H1N1, HA from A / Vietnam / 1194/200 / H5N1, and MI from A / Vietnam / 1194/2004 / H5Nl), were designated by the following method: For each gene, the amino acid sequence was extracted from the NCBI public database. In the HA and HAO genes, a fungal signal sequence was substituted for the native leader sequence. Using this sequence, a gene for fungal expression was translated in an inverse manner and optimized by codons using codon preferences of Neurospora crassa. The sequence was altered to a low free energy form, which was calculated to reduce the secondary structure in the nascent mRNA. The resulting gene was investigated by chains for the splicing of introns from donor and acceptor sites. After altering the sequence to remove these sites, the sequence was checked again for the transcriptional termination sites, and any random sites were removed. Then the optimized sequence was sequenced. The resulting DNA was subcloned in E. coli and then sequenced to verify the errors in the synthesis. After confirmation of the sequence, the DNA was subcloned into an expression vector (pHDKXL1 for the HA and HAO genes, pALGAM for the MI gene), and transformed into Neurospora. The HA and HAO genes were used for integration in the His-3 locus of Neurospora, and the MI gene was used for integration in the Neurospora Am gene.

EXAMPLE 2 Expression of HAO in N. crassa As analyzed in Example 1, an expression vector encoding the synthetic hemagglutinin gene 0 (HAO) with a fungal signal sequence linked to facilitate the production of proteins was generated. The transformants were selected either for prototropia with histidine (transformants pHDKXLl / HA or HAO), or for resistance to hygromycin B (pALGAM / Ml transformants), using a host strain with a mutation in histidine-3. After purification of the transformed strains by repeated scoring in the selective medium, the transformants were selected for the expression of the genes by ELISA. The secreted influenza antigens were detected in the middle from shaker flasks. Flasks containing 25 ml of minimal Vogel salts plus 0.5% yeast extract in 125 ml of Ehrlenmeyers were inoculated with approximately one million conidiaspores / ml. The samples were either grown at 26 ° C, vigorous agitation at 200 rpm, or in a static culture at the same temperature. The samples were extracted after 2, 3, 4, 5, and 6 days of growth. The ELISA was developed using antibodies against influenza proteins, acquired from BIODESIGN. Control antigens used as standards were purchased from Protein Sciences Corporation. Positive samples with ELISA were re-selected by Western blots using standard methods and reagents. Various growth conditions were tested.

Specifically, vigorous agitation and static cultures were specifically tested. Typically, the yeast was cultured for 2 to 6 days and the samples were collected for analysis. The samples were divided by SDS-PAGE and transferred to nitrocellulose for imaging. Hemagglutinin was detected using a goat polyclonal anti-HA (H1N1) antibody, followed by an anti-goat Ig-alkaline phosphate conjugate. The union was measured by colorimetric detection. A detection of the Western blot of the expression of the synthetic HAO in N. crassa is shown in Figure 7. Figure 8 shows a SDS-PAGE gel stained with Coomassie blue from two different HAO clones. Figure 9 shows a Western blot of expression of static cultures of HA05. These data demonstrate the ability of the present system to recombinantly express influenza proteins in fungi.

EXAMPLE 3 Multivalent Expression of Influenza Antigens in Fungal Heterocarps Expression vectors were prepared according to the method analyzed in Example 1. The DNA was introduced into Neurospora by electroporation and the transformants were selected according to the methods of Example 2. A multivalent mixture of influenza virus proteins from the cultures was produced because the expression vectors code for HAO, HA, and MI matrix proteins.

Claims (15)

1. NOVELTY OF THE INVENTION Having described the present invention, it is considered as a novelty and, therefore, the content of the following CLAIMS is claimed as property: 1. Filamentous fungal heterocarps characterized in that heterocarions produce a multivalent recombinant variant of an antigen, and wherein heterocarions are formed by the fusion of two or more fungal parent strains, wherein the heterokaryon requires the presence of all the parent fungal nuclei for survival, the mother fungal strains each contain an exogenously supplied nucleic acid molecule encoding for variants of antigens derived from pathogenic organisms and where the fungal mother strains are homozygous for all alleles with heterocarion compatibility.
2. 2. The heterocarions according to the claim 1, characterized in that the multivalent antigens are secreted as soluble proteins in the culture media.
3. 3. The heterocarions according to the claim 2, characterized because the multivalent antigens are aggregated into particles.
4. 4. The heterokaryons according to claim 1, characterized in that the multivalent antigens are exhibited on the surface of the fungal heterocarion.
5. 5. The heterocarions according to claim 1, characterized in that the multivalent antigens are conserved within the cytoplasm of the fungal heterocarions.
6. 6. The heterokaryons according to claim 1, characterized in that the pathogenic organism is a virus.
7. The heterokaryons according to claim 1, characterized in that the virus is one of a group composed of influenza viruses, human papillomavirus 16, human papillomavirus 18, human papillomavirus 31, varicella herpesvirus, measles virus , Epstein Barr virus, respiratory syncytial virus, parainfluenza 3, herpes simplex virus type 1, and herpes simplex virus type 2.
8. 8. The heterokaryons according to claim 1, characterized in that the virus antigens are derived from influenza type A and influenza type B.
9. 9. The heterokaryons according to the claim 4, characterized in that the antigens consist of variants of hemagglutinin and influenza neuraminidase type A and type B.
10. 10. The heterocarions according to claim 1, characterized in that each of the variants of hemagglutinin and neuraminidase type A and type B is a variant that It occurs in nature.
11. 11. The heterocarions according to claim 1, characterized in that each of the variants of hemagglutinin and neuraminidase of influenza A and influenza B is not a subunit variant that occurs in nature.
12. 12. The heterokaryons according to claim 1, characterized in that the pathogenic organism is a bacterium.
13. 13. The heterokaryons according to claim 1, characterized in that the pathogenic organism is a fungus. 1 .
14. The heterocarions according to claim 1, characterized in that the pathogenic organisms consist of a mixture of any and all combinations of viral, bacterial and fungal organisms.
15. 15. A method for producing a multivalent vaccine, the method characterized in that it comprises the step of culturing heterocarions according to claim 1, under conditions in which the exogenously supplied nucleic acid molecules are expressed to such an extent that they form a multivalent vaccine.