Patent Grant
9549970
This application contains a Sequence Listing submitted electronically as a text file by EFS-Web. The text file, named “3923-37-PCT_ST25”, has a size in bytes of 8 KB, and was recorded on 15 Aug. 2012. The information contained in the text file is incorporated herein by reference in its entirety pursuant to 37 CFR §1.52(e)(5).
The invention relates to methods of growing yeast cultures at a neutral pH to improve yields and certain characteristics of yeast cultures. The method also relates to compositions produced by these methods.
Vaccines are one of the most cost-effective measures available to the health care industry. There remains, however, an urgent need to develop safe and effective vaccines and adjuvants for a variety of diseases, including those due to infection by pathogenic agents, cancers, genetic defects and other disorders of the immune system. Publications on vaccine, for example, Rabinovich et al., Science 265, 1401-1404 (1994), state that there is still a need for safe and heat-stable vaccines that can be administered orally and that need to be administered only a few times, preferably early in life. Also preferred are combination vaccines that can protect individuals from more than one disease, as well as vaccines that do not require an adjuvant and that can elicit mucosal immunity. To date very few, if any, vaccines meet all of these criteria.
Subunit vaccines, the development of which was made possible by recombinant DNA technology, have been disappointing to date as they exhibit only limited immunogenicity. One example is the recent clinical testing of several HIV (human immunodeficiency virus) subunit vaccines which has been stopped due not only to limited efficacy of the vaccines but also because in some cases immunized individuals showed accelerated disease progression when they were subsequently exposed to HIV; see, for example, Cohen, Science 264:1839 (1994); and Cohen, Science 264: 660 (1994). One disadvantage of subunit vaccines, as well as of killed virus and recombinant live virus vaccines, is that while they appear to stimulate a strong humoral immune response, they fail to elicit protective cellular immunity. A major conclusion at the 1994 International AIDS Conference was that there remains a need for a cytotoxic T cell-mediated response to prevent, or reduce, HIV infectivity, which to date is lacking in vaccines in the clinic. In addition, HIV vaccines tested to date have failed to elicit immunity at the mucosal surfaces where primary HIV infection occurs.
Furthermore, the only adjuvants approved for use in the United States are the aluminum salts aluminum hydroxide and aluminum phosphate, neither of which stimulates cell-mediated immunity. In addition, aluminum salt formulations cannot be frozen or lyophilized, and such adjuvants are not effective with all antigens.
Yeast cells have been used in the production of subunit protein vaccines, including some of those tested in the aforementioned HIV vaccine trials. Yeast has also been fed to animals prior to immunization to try to prime the immune response in a non-specific manner (i.e., to stimulate phagocytosis as well as the production of complement and interferon). The results have been ambiguous, and such protocols have not generated protective cellular immunity; see, for example, Fattal-German et al., Dev. Biol. Stand. 77: 115-120 (1992) and Bizzini et al., FEMS Microbiol. Immunol. 2: 155-167 (1990).
In addition to vaccines, many gene and drug therapies require efficient and specific delivery vehicles to ensure the greatest possible benefit. Lack of an adequate delivery vehicle is a major roadblock to the application of gene therapy and significantly limits the therapeutic potential of many drugs. For example, recent reports have indicated that adenovirus vectors, which are currently being tested in the clinic for gene therapy applications, are stimulating undesirable immune and inflammatory responses and do not appear to be integrating in a desired manner; see, for example, Engelhardt et al., Human Gene Therapy 5: 1217-1229 (1994) and references cited therein.
Another major hurdle for yeast vaccine technology is the manufacturing process. Yeast cells have been cultured in the laboratories for many years and standard culture conditions have been established. See, for example, Methods of Enzymology, Vol. 194, Guthrie et al., eds., Cold Spring Harbor Laboratory Press (1990). Standard operating protocols generally involve culturing yeast in media that is acidic as measured by pH levels. However, culturing yeast in acidic media may result in the yeast exhibiting different biological properties that are not optimal for using yeast as antigen-bearing vehicles for purposes of immunomodulation or making vaccines. Thus, there is a need for methods for growing yeast such that the yeast exhibit properties that make them better suited for being antigen-bearing vehicles. The invention disclosed herein in based, in part, on the discovery that while yeast can grow in acidic media, the biological properties that the yeast exhibit when grown in acidic media is not as desirable as when yeast are grown in media that is at neutral pH levels.
The disclosure of all patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety for all purposes.
The invention provides a method for growing yeast by culturing the yeast in medium wherein the media is maintained at a pH level of between 5.5 and 8 for at least 50% of time that the yeast are in culture. The invention also provides for a method for growing yeast by culturing the yeast in medium wherein the media is maintained at a pH level of between 5.5 and 8 and wherein the density of the yeast is at least 0.5 yeast units/mL.
In other aspect, the invention provides for growing yeast by culturing the yeast in medium with a pH level of at least 5.5. The invention also provides a method for growing yeast by culturing the yeast in medium wherein the media is maintained at a pH level of between 5.5 and 8. In an aspect of the invention, the yeast is Saccharomyces cerevisiae. In aspects of the invention the medium is buffered with succinate or succinic acid or the medium may additionally contain soytone. In other aspects of the invention, the yeast elicits an immune response. In other aspects of the invention, the yeast expresses an antigen, in some cases the antigen is a heterologous antigen. In some cases, the heterologous antigen is expressed on the surface of the yeast.
The invention provides for a composition comprising yeast cultured by any the methods and related aspects above.
The invention provides for a method for producing antigen-expressing yeast by culturing yeast containing an expression system for expressing the antigen in a medium wherein the pH of the media is at least 5.5. The invention also provides for a method for producing antigen-expressing yeast by culturing yeast containing an expression system for expressing the antigen wherein the media is maintained at a pH level of between 5.5 and 8. In one aspect, the yeast is Saccharomyces cerevisiae. In other aspects, the medium is buffered with succinate or succinic acid or the medium may additionally contain soytone. In other aspects of the invention, the yeast elicits an immune response. In other aspects of the invention, the yeast expresses an antigen, in some cases the antigen is a heterologous antigen. In some cases, the heterologous antigen is expressed on the surface of the yeast. In some aspects, the heterologous antigen is more readily accessible for interaction with other cells or agents than when the yeast is grown at a pH of less than 5.5.
The invention also provides for a composition comprising yeast cultured by the method disclosed above.
The invention also provides for a method of inducing a Th1-type response in an individual by administering to the individual a composition comprising antigen-expressing yeast wherein the yeast has been cultured in a medium with a pH level of at least 5.5.
The invention also provides for a method of inducing a Th1-type response in an individual by administering to the individual a composition comprising antigen-expressing yeast wherein the yeast has been cultured in media wherein the media is maintained at a pH level of between 5.5 and 8. In one aspect, the composition comprises dendritic cells loaded with yeast which have been cultured, maintained or harvested at a neutral pH. In another aspect, the yeast is Saccharomyces cerevisiae. In other aspects, the medium is buffered with succinate or succinic acid or the medium may additionally contain soytone. In other aspects of the invention, the yeast elicits an immune response. In other aspects of the invention, the yeast expresses an antigen, in some cases the antigen is a heterologous antigen. In some cases, the heterologous antigen is expressed on the surface of the yeast. In one aspect, the Th1-type response is interferon-gamma production. In another aspect, the Th1-type response is IL-12 production.
The invention also provides for a kit for culturing yeast comprising media wherein the pH of the media is at least 5.5 and instructions for the use of the media to culture yeast. The invention also provides for a kit for culturing yeast comprising media wherein the pH of the media is maintained at a pH level of between 5.5 and 8 and instructions for the use of the media to culture yeast. In other aspects, the medium is buffered with succinate or succinic acid or the medium may additionally contain soytone. In one aspect, the kit additionally includes yeast. In some cases, the yeast is frozen or lyophilized. In some cases, the yeast has been cultured in a media of at least pH 5.5 or has been cultured in a media wherein the pH of the media is maintained at a pH level of between 5.5 and 8. In other cases, the yeast is capable of replication.
The invention disclosed herein is based on the discovery that growing yeast at a neutral pH, at least pH 5.5, or between pH 5.5 and 8, or between pH 6 and 8, results in yeast with more desirable biological characteristics. Some of these desirable characteristics, which are detailed infra, include but are not limited to, ability to grow well at increased cell density, keeping yeast cell wall pliable and sensitive to digestion with cell wall digesting enzymes, and display of antigens in a manner that makes them more accessible to other cells and/or agents.
General Techniques
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art. Such techniques are explained fully in the literature, such as, Methods of Enzymology, Vol. 194, Guthrie et al., eds., Cold Spring Harbor Laboratory Press (1990); Biology and activities of yeasts, Skinner, et al., eds., Academic Press (1980); Methods in yeast genetics: a laboratory course manual, Rose et al., Cold Spring Harbor Laboratory Press (1990); The Yeast Saccharomyces: Cell Cycle and Cell Biology, Pringle et al., eds., Cold Spring Harbor Laboratory Press (1997); The Yeast Saccharomyces: Gene Expression, Jones et al., eds., Cold Spring Harbor Laboratory Press (1993); The Yeast Saccharomyces: Genome Dynamics, Protein Synthesis, and Energetics, Broach et al., eds., Cold Spring Harbor Laboratory Press (1992); Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) and Molecular Cloning: A Laboratory Manual, third edition (Sambrook and Russell, 2001), (jointly referred to herein as “Sambrook”); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987, including supplements through 2001); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York; Harlow and Lane (1999) Using Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (jointly referred to herein as “Harlow and Lane”), Beaucage et al. eds., Current Protocols in Nucleic Acid Chemistry John Wiley & Sons, Inc., New York, 2000) and Vaccines, S. Plotkin and W. Orenstein, eds., 3rd edition (1999).
Definitions
As used herein, the general use of the term “neutral pH” refers to a pH level of at least 5.5. The neutral pH range can be between about pH 5.5 and about pH 8, preferably between about pH 6 and about 8. One of skill the art will appreciate that minor fluctuations (e.g., tenths or hundredths) can occur when measuring with a pH meter and, as such, should take this into account when determining the pH level at any given time.
As used herein, the general use of the term “antigen” refers any molecule that can be recognized by the adaptive immune system. In one aspect, an antigen is a molecule that binds specifically to an antibody. The molecule can be any portion of a protein (peptide, partial protein, full-length protein) wherein the protein is naturally occurring or synthetically derived, or part of a cellular composition (whole cell, cell lysate or disrupted cells), part of an organism (whole organism, lysate or disrupted cells) or a carbohydrate or a portion thereof. The antigen can elicit an antigen-specific humoral immune response by itself or with the use of another compound such as an adjuvant (like crushed yeast cells). In another aspect, an antigen is recognized by T lymphocytes (or T cells) in the context of major histocompatibility complexes (MHCs). In another aspect, the antigen can act as a toleragen, against the same or similar antigens that are encountered within the cells and tissues of the animal to which the antigen is administered.
In one aspect of the present invention, when referring to the stimulation of an immune response, the “antigen” can be an “immunogen” Immunogens are molecules which can elicit an adaptive immune response, e.g., induction of antibody production. The immunogen can in some cases generate memory cells that will produce antibodies which recognize the antigen upon future exposure to the antigen. As is well-known to all persons of skill in this field, immunogens can also be recognized by T lymphocytes, although the form of the immunogen recognized by T lymphocytes will be different from the form of the immunogen that the antibody recognizes.
Methods of Culturing Yeast
The invention provides for methods for culturing yeast that produces desirable characteristics, such as high expression of a desired antigen, cell wall pliability, and display of antigen.
These methods are broadly applicable to all yeast. Yeast are unicellular microorganisms that belong to one of three classes: Ascomycetes, Basidiomycetes and Fungi Imperfecti. While pathogenic yeast strains, or nonpathogenic mutants thereof can be used in accordance with the present invention, in one aspect, nonpathogenic yeast strains are used. Examples of nonpathogenic yeast strains include Saccharomyces, Candida, Cryptococcus, Hansenula, Kluyveromyces, Pichia, Rhodotorula, Schizosaccharomyces and Yarrowia. In one aspect, Saccharomyces, Candida, Hansenula, Pichia and Schizosaccharomyces are used. In yet other aspects, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Candida albicans, Candida kefyr, Candida tropicalis, Cryptococcus laurentii, Cryptococcus neoformans, Hansenula anomala, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Kluyveromyces marxianus var. lactis, Pichia pastoris, Rhodotorula rubra, Schizosaccharomyces pombe, and Yarrowia lipolytica are used. It is understood the invention is not limited to the species list above and that one of skill in the art can apply the teachings here in any type of yeast. In another aspect, Saccharomyces cerevisiae (S. cerevisiae) is used to practice the methods of the invention. S. cerevisiae is preferred due to it ease for molecular manipulation and being “Generally Recognized As Safe” or “GRAS” for use as food additives (GRAS, FDA proposed Rule 62FR18938, Apr. 17, 1997).
The pH level is important in the culturing of yeast. One of skill in the art will appreciate that the culturing process includes not only the start of the yeast culture but the maintenance of the culture as well. The yeast culture may be started at any pH level, however, since the media of a yeast culture tends to become more acidic (i.e., lowering the pH) over time, care must be taken to monitor the pH level during the culturing process.
In some aspects of the invention, the yeast is grown in a media at a pH level of at least 5.5. In other aspects, the yeast is grown at a pH level of about 5.5. In other aspects, the yeast is grown at a pH level of between 5.5 and 8. In some cases, the yeast culture is maintained at a pH level of between 5.5 and 8. In other aspects, the yeast is grown at a pH level of between 6 and 8. In some cases, the yeast culture is maintained at a pH level of between 6 and 8. In other aspects, the yeast is grown and/or maintained at a pH level of between 6.1 and 8.1. In other aspects, the yeast is grown and/or maintained at a pH level of between 6.2 and 8.2. In other aspects, the yeast is grown and/or maintained at a pH level of between 6.3 and 8.3. In other aspects, the yeast is grown and/or maintained at a pH level of between 6.4 and 8.4. In other aspects, the yeast is grown and/or maintained at a pH level of between 5.5 and 8.5. In other aspects, the yeast is grown and/or maintained at a pH level of between 6.5 and 8.5. In other aspects, the yeast is grown at a pH level of about 5.6, 5.7, 5.8 or 5.9. In another aspect, the yeast is grown at a pH level of about 6. In another aspect, the yeast is grown at a pH level of about 6.5. In other aspects, the yeast is grown at a pH level of about 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 or 7.0. In other aspects, the yeast is grown at a pH level of about 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. In other aspects, the yeast is grown at a level of above 8.
In one aspect, yeast is cultured such that the pH level of the medium does not drop below pH 5.5. In some cases, the drop below pH 5.5 is not more than 5 minutes. In other cases, the drop below pH 5.5 is not more than 10 minutes, preferably 20, 30, 40, 50 or 60 minutes. In other cases, the drop below pH 5.5 is not more than 1 hour. In another aspect, yeast is cultured such that the pH level of the medium does not drop below 5.0. In some cases, the drop below pH 5.0 is not more than 5 minutes. In other cases, the drop below pH 5.0 is not more than 10 minutes, preferably 20, 30, 40, 50 or 60 minutes. In other cases, the drop below pH 5.0 is not more than 1 hour. As such, the longer time the yeast are grown in a medium that is at least pH 5.5 or above, the better the results will be in terms of obtaining yeast with desirable characteristics described infra.
In one aspect, the use of neutral pH methods to grow yeast cells means that the yeast cells are grown in neutral pH for at least 50% of the time that the yeast are in culture. It is more preferable that the yeast are grown at neutral pH for at least 60% of the time they are in culture, more preferably at least 70% of the time they are in culture, more preferably at least 80% of the time they are in culture, and most preferably at least 90% of the time they are in culture.
In another aspect, growing yeast at neutral pH includes culturing yeast cells for at least five minutes at neutral pH, preferably at least 15 minutes at neutral pH, more preferably at least one hour at neutral pH, more preferably at least two hours, even more preferably, at least three hours or longer.
As noted earlier, as yeast grow and replicate, the cell densities become greater and the acidity level in the culture media rises. As such, it is recommended that as the yeast are cultured at a pH level of at least 5.5 and/or maintained at at least pH 5.5 as the yeast density increases. In one aspect, the yeast are grown and/or maintained between a pH of 5.5 and 8 as the yeast density is 0.5 yeast units (YU)/ml or above. In other aspects, the yeast are grown and/or maintained between a pH of 5.5 and 8 when the yeast density is at least 0.6 YU/ml or above, preferably 0.7 YU/ml or above, 0.8 YU/ml or above, 0.9 YU/ml or above, or 1 YU/ml or above.
In another aspect, the yeast are grown and/or maintained between a pH of 6 and 8 as the yeast density is 0.5 YU/ml or above. In other aspects, the yeast are grown and/or maintained between a pH of 6 and 8 when the yeast density is at least 0.6 YU/ml or above, preferably 0.7 YU/ml or above, 0.8 YU/ml or above, 0.9 YU/ml or above, or 1 YU/ml or above.
In some aspects, it is preferable at the time of harvest that the yeast culture is at a neutral pH level. In some cases, the yeast culture, at the time of harvest, will be at a pH level of between 6 and 8. In other cases, the yeast culture, at the time of harvest, will be at a pH level of between 5.5 and 8.
The culture media can be brought to a pH level of at least 5.5 by any means. In one aspect, succinic acid (and any related forms, e.g., the anion succinate) is used for buffering the culture media. As further detailed in the Examples, the use of succinate to buffer the culture media to at least pH 5.5 allows for yeast to have a doubling time of about two to two and a half hours. Succinate is available from commercially available sources (e.g., Sigma Chemicals). In other aspects, citrate may be used to bring the media to a pH of at least 5.5. One of skill in the art will be able to readily determine other buffering agents which may be used to bring the media to a pH of at least 5.5 while keeping the yeast viable. The concept of buffering agents to keep a solution at a steady pH level is well-known in the art and as such, will not be discussed in detail herein. If yeast grown according to the invention are being used for pharmaceutical formulations (e.g., vaccines), it is recommended that GMP grade material be used.
In addition, other supplements may be added to the culture media to improve the media. Other supplements which are particularly helpful to add to the culture media include soytone. Soytone is readily available from commercial sources (e.g., BD Difco). As shown in the Examples and figures, the addition of soytone to the culture media supports higher density for growth at neutral pH. Furthermore, the addition of soytone supports expression of an antigen of interest, hemagglutinin (HA) of the influenza virus.
Other additives may be added to the yeast culture for other purposes, such as inducing expression of heterologous genes. In some aspects, copper is used to induce the expression of hemagglutinin expression. However, the use of copper is not ideal at neutral pH thus, for control of inducible genes to be expressed in yeast grown at neutral pH; an additive other than copper would be recommended.
Effects of Neutral pH on Yeast Culture
The use of a neutral pH in culturing yeast promotes several biological effects that are desirable characteristics for using the yeast as vehicles for immunomodulation and/or eliciting immune responses. In one aspect, culturing the yeast in neutral pH allows for good growth of the yeast without any negative effect on the doubling time (e.g., slowing down the doubling time). The yeast can continue to grow to high densities without losing their cell wall pliability.
In another aspect, the use of a neutral pH, such as a pH of at least 5.5 or between pH 5.5 and 8, allows for the production of yeast with pliable cell walls and/or yeast that have a sensitivity to cell wall digesting enzymes (e.g., glucanase) at all harvest densities. As such, the invention provides for methods and compositions of yeast with cell wall pliability as measured by traditional assays (e.g., sensitivity to glucanase). Prior experiments had established that yeast lost its sensitivity to digestion with cell wall digesting enzymes at harvest densities of about 0.5 YU (yeast units)/ml. As such, one advantage is that comparisons done with yeast cultured in standard growth media at 0.5 YU/ml can be used for comparison with neutral pH growth at any density. This trait is desirable because yeast with flexible cell walls can exhibit unique immune responses, such as promoting the secretion of cytokines (e.g., INF-gamma) in the cells hosting the yeast. Another reason why one of skill in the art would use the neutral pH methodology is that it allows for greater accessibility to the antigens located in the cell wall. This is useful for greater immunogenicity and also for antibody detection of expressed protein, measured by standard techniques such as flow cytometry.
Yet another desirable characteristic that is observed in yeast cultured at neutral pH is the expression of antigens in a way that is beneficial for purposes of immunomodulation. In one aspect, the yeast are used as vehicles for antigen expression (see, for example, U.S. Pat. Nos. 5,830,463 and 7,083,787). The antigen may be an antigen native to yeast or alternatively, a heterologous antigen that is expressed by the yeast. In some aspects, the use of yeast for expression of antigens is helpful for development of vaccines, prophylactics, and therapeutics to combat various diseases and ailments (e.g., infectious diseases or cancer). Using neutral pH methodology, one of skill in the art can produce antigen-bearing yeast wherein the antigen is more accessible to other cells (e.g., for immune co-stimulatory functions or immune regulation) or to other agents (e.g., antibodies for detection). In addition, the use of neutral pH for some antigens, such as the influenza HA antigen, allows for release of the di-sulfide bonded HA by treatment with dithiothreitol (DTT) that is not possible when the HA-expressing yeast is cultured in media where the pH drops below pH 5. In some cases, this occurs when the pH drops below pH 5 for any length of time. In other cases, this occurs when the pH drops below pH 5 for one or a few minutes or one or more hours.
Another desirable characteristic that yeast cultured following the neutral pH methodologies exhibit is the secretion of Th1-type cytokines from cells that have been exposed to the yeast. Examples of Th1-type cytokines include, but are not limited to, interferon-gamma, IL-12, and IL-2. As further detailed in the Examples, dendritic cells that were loaded with yeast that had been grown following neutral pH protocols exhibit increased levels of interferon-gamma secretion and expression as compared to yeast grown at low (acidic) pH media. There was no reduction in the levels of IL-12 secretion when using the neutral pH culturing methods. As such, one of skill in the art can use the neutral pH methodologies disclosed herein for immunomodulation purposes, e.g., inducing a Th1-type response in an individual that is afflicted with a disease or disorder that would benefit from an enhanced Th1-type response.
Compositions of Yeast Grown Using Neutral pH Methodology
The invention also contemplates compositions comprising yeast which are grown using the neutral pH methodologies disclosed herein. In one aspect, the composition comprises yeast that express native antigens, either on its surface or internally or both. This composition can be useful for various purposes, such as administration as an adjuvant. In another aspect, the composition comprises yeast that express heterologous antigens, either on its surface or internally or both. This composition can be useful for various purposes, such as immunomodulation in an individual in need thereof and the development of vaccines.
These compositions can also include pharmaceutically acceptable excipients and/or carriers. Pharmaceutically acceptable carriers may include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like. The formulation of compositions comprising yeast grown under neutral pH conditions with a pharmaceutically acceptable excipient is generally routine for one of skill in the art.
Kits of the Invention
The invention contemplates kits comprising media components for culturing yeast under neutral pH conditions. In one aspect, the kit includes media components containing succinate or succinic acid which can be used to bring the media to a pH of at least 5.5 and a set of instructions for its use. In another aspect, the kit further includes soytone as an additional component. In another aspect, the kit further includes yeast cells. The yeast cells can be frozen for starting a culture using the protocols disclosed herein. In another aspect, the yeast cells can have already been cultured by the methods disclosed herein prior to being frozen for packaging as part of the kit. In another aspect, the yeast cells can be lyophilized and optionally be included in the kit. In another aspect, the kit comprises yeast prepared according to the methods disclosed herein that is capable of replication.
The following examples are provided to illustrate certain aspects of the invention. They are not intended to limit the invention in any manner.
Various types of media can be used to culture yeast and be adjusted such that the pH level is neutral. Several examples of media which can be used are given below, however, it is to be understood that the invention is not limited to the use of these media components or media protocols.
One standard media recipe is ULDM media which is as follows:
Another standard media recipe is UL2 media which is as follows:
Another standard media recipe is UL3 media which is as follows:
Another standard media recipe is UL4 media which is as follows:
Another standard media recipe is UDM media which is as follows:
Another standard media recipe is U2 media which is as follows:
Another standard media recipe is U3 media which is as follows:
Another standard media recipe is U4 media which is as follows:
Standard media formulations may be supplemented with additional amino acids. The following protocols are exemplary media formulations.
The ULDMaa media formulation is as follows:
The UL2aa media formulation is as follows:
The UL3aa media formulation is as follows:
The UDMaa media formulation is as follows:
The U2aa media formulation is as follows:
The U3aa media formulation is as follows:
In another aspect, succinate-containing buffered media is used. Examples of succinate-containing yeast media are below. The UDMS media formulation, adjusted to pH 6.9 is as follows:
The U2S media formulation, adjusted to pH 6.9 is as follows:
The U3S media formulation, adjusted to pH 6.9 is as follows:
The U4S media formulation, adjusted to pH 6.9 is as follows:
The effect of media pH on cell growth and culture pH were tested, as shown in
The effect of media pH was tested to determine if it had any effect on cell wall thickness. Growth media and conditions were the same as in Example 1. Cultures were harvested at densities ranging from 0.5 to 2.0 YU/mL. In the legend for
As can be observed in
Thus, the results indicate that growing yeast buffered at about pH 5.5 or higher keeps the cell wall pliable and sensitive to digestion with cell wall digesting enzymes (e.g., making spheroplasts with lyticase/glucanase) at all harvest densities. In contrast, with the standard process commonly used in many yeast laboratories, the sensitivity was lost at harvest densities >0.5 YU/mL. For ease of comparison, 0.5 YU/mL with standard growth media is often used for comparison with neutral pH growth at any density.
A fusion protein denoted TK75-15 was engineered to express influenza HA protein on the cell wall using the Aga2 sequence, driven by the TEF2 promoter. In this construct, the protein was constructed with the HA sequence C-terminal to the Aga2 sequence. This protein, when expressed in cells that also express Aga1p (in this case, driven by the CUP1 promoter), localizes to the outer cell wall of the yeast cell, as well as to the cytosol. The fusion protein comprising the influenza HA antigen is a single polypeptide with the following sequence elements fused in frame from N- to C-terminus (the amino acid sequence of the fusion protein being represented herein by SEQ ID NO:1): 1) the full length S. cerevisiae Aga2 protein sequence (positions 1 to 87 of SEQ ID NO:1), including its natural 18 amino acid ER-targeting signal sequence (positions 1 to 18 of SEQ ID NO:1; 2) a spacer to separate the Aga2 from the HA body (positions 88 and 89); 3) influenza HA protein lacking its signal sequence (positions 90 to 600 of SEQ ID NO:1), and lacking 36 C-terminal residues of HA, thus eliminating its C-terminal membrane anchor and cytoplasmic tail; 4) a triglycine spacer to separate the body of HA protein from the histidine tag (positions 601-603 of SEQ ID NO:1); and 5) a C-terminal hexahistidine tag (positions 604-609 of SEQ ID NO:1). A nucleic acid sequence encoding the fusion protein of SEQ ID NO:1 is represented herein by SEQ ID NO:2. This fusion protein and the Tarmogen expressing it can be called 75-15.
The protein sequence used is as follows (SEQ ID NO:1):
The corresponding nucleic acid sequence is as follows (SEQ ID NO:2):
Different buffering agents were tested on 75-15 cells to determine its effect on cell growth and also the effect on the culture pH. These buffers are shown in
The contribution of other agents added to the yeast culture media was tested and the results are shown in
Additional experiments were conducted using different concentration of soytone.
The accessibility of particular antigens to interactions from other agents, such as an antibody for detection, was assessed using varying pH levels of the media.
The effect of culturing yeast at neutral pH on cytokine production was examined by using dendritic cells (DC) loaded with YVEC yeast (not expressing a heterologous antigen) grown in media where the pH was or was not maintained above pH 5.5. Mouse bone marrow-derived dendritic cells were loaded with 10 yeast per DC by incubating together in RPMI media for 48 hr at 37° C. Supernatants were then analyzed for secreted cytokines.
This patent application claims the benefit of priority under 35 U.S.C. §120 and is a continuation of U.S. patent application Ser. No. 12/525,045, filed Jan. 29, 2010, which is a national stage application under 35 U.S.C. §371 of PCT Application No. PCT/US2008/052843, filed Feb. 1, 2008, which claims the benefit of priority under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 60/899,281, filed on Feb. 2, 2007. Each of U.S. patent application Ser. No. 12/525,045, PCT Application No. PCT/US2008/052843, and U.S. Provisional Patent Application No. 60/899,281 is hereby incorporated by reference in its entirety.
| Number | Name | Date | Kind |
|---|---|---|---|
| 4775622 | Hitzeman et al. | Oct 1988 | A |
| 5234830 | Oshima et al. | Aug 1993 | A |
| 5310654 | Isberg et al. | May 1994 | A |
| 5413914 | Franzusoff | May 1995 | A |
| 5830463 | Duke et al. | Nov 1998 | A |
| 5858378 | Bostwick | Jan 1999 | A |
| 5919651 | Hitzeman et al. | Jul 1999 | A |
| 7083787 | Duke et al. | Aug 2006 | B2 |
| 7439042 | Duke et al. | Oct 2008 | B2 |
| 7465454 | Franzusoff et al. | Dec 2008 | B2 |
| 7563447 | Franzusoff et al. | Jul 2009 | B2 |
| 7595060 | Duke et al. | Sep 2009 | B2 |
| 7625569 | Duke et al. | Dec 2009 | B2 |
| 7632511 | Duke et al. | Dec 2009 | B2 |
| 7736642 | Duke et al. | Jun 2010 | B2 |
| 7745128 | Guo et al. | Jun 2010 | B2 |
| 8007816 | Duke et al. | Aug 2011 | B2 |
| 20030035810 | Caplan | Feb 2003 | A1 |
| 20070172503 | Selitrennikoff et al. | Jul 2007 | A1 |
| 20080003239 | Duke et al. | Jan 2008 | A1 |
| 20090098154 | Franzusoff et al. | Apr 2009 | A1 |
| 20090142366 | Franzusoff et al. | Jun 2009 | A1 |
| 20090142367 | Franzusoff et al. | Jun 2009 | A1 |
| 20090304741 | Duke et al. | Dec 2009 | A1 |
| 20100034840 | Apelian et al. | Feb 2010 | A1 |
| 20100104604 | Selitrennikoff et al. | Apr 2010 | A1 |
| 20100111912 | Apelian et al. | May 2010 | A1 |
| 20100150963 | Duke et al. | Jun 2010 | A1 |
| 20100189749 | Franzusoff et al. | Jul 2010 | A1 |
| 20100196411 | Duke et al. | Aug 2010 | A1 |
| 20100215678 | Guo et al. | Aug 2010 | A1 |
| 20110150909 | Franzusoff et al. | Jun 2011 | A1 |
| 20110301329 | Van Urk et al. | Dec 2011 | A1 |
| Number | Date | Country |
|---|---|---|
| 0414404 | Feb 1991 | EP |
| 2486400 | Jan 1982 | FR |
| 05-9124 | Jan 1993 | JP |
| 06-277086 | Oct 1994 | JP |
| 2002-291480 | Oct 2002 | JP |
| 2007-863 | Jan 2007 | JP |
| 10-0507665 | Aug 2005 | KR |
| WO 9835049 | Aug 1998 | WO |
| WO 2008115610 | Sep 2008 | WO |
| WO 2010033841 | Mar 2010 | WO |
| WO 2010065626 | Jun 2010 | WO |
| WO 2010121180 | Oct 2010 | WO |
| WO 2011032119 | Mar 2011 | WO |
| WO 2011115914 | Sep 2011 | WO |
| Entry |
|---|
| Notice of Allowance with English Translation for China Patent Application No. 200880010797.6, dated Mar. 25, 2014 4 pages. |
| Intention to Grant for European Patent Application No. 08714176.8, dated May 2, 2014 7 pages. |
| Notice of Allowance with English Translation for Japan Patent Application No. 2009-548478, dated May 27, 2014 2 pages. |
| Official Action with English Translation for Taiwan Patent Application No. 096103983, dated Mar. 21, 2014 8 pages. |
| Decision to Grant for European Patent Application No. 08714176.8, dated Sep. 18, 2014 2 pages. |
| Official Action for India Patent Application No. 5607/DELNP/2009, dated Dec. 17, 2014 3 pages. |
| Official Action for U.S. Appl. No. 12/525,045, mailed Jan. 15, 2015 9 pages. |
| Ausubel, F.M. et al. eds. (2001). Current Protocols in Molecular Biology, vol. 1, John Wiley & Sons, Inc., Supplement 55, pp. 1-11, (Table of Contents Only.). |
| Barnett, J.A. (Jun. 6, 2008). “A History of Research on Yeasts 12: Medical Yeasts Part 1, Candida albicans,” Yeast 25(6):385-417. |
| Beaucage, S.L. et al. eds. (2000). Current Protocols in Nucleic Acid Chemistry, John Wiley & Sons, Inc: New York, NY, Supplement 37, pp. 1-7, (Table of Contents Only.). |
| Bizzini, B. et al. (1990). “Use of Live Saccharomyces cerevisiae Cells as a Biological Response Modifier in Experimental Infections,” FEMS Microbiol. Immunol. 64(3):155-167. |
| Brake et al. “a-Factor-directed synthesis and secretion of mature foreign proteins in Saccharomyces cerevisiae.” PNAS, Aug. 1984, vol. 81, pp. 4642-4646. |
| Brenner, C. et al. (Feb. 1992). “Structural and Enzymatic Characterization of a Purified Prohormone-Processing Enzyme: Secreted, Soluble Kex2 Protease,” Proc. Natl. Acad. Sci. 89:922-926. |
| Broach, J.R. et al. eds. (1991). Genome Dynamics, Protein Synthesis, and Energetics the Molecular and Cellular Biology of the Yeast Saccharomyces, Cold Spring Harbor Laboratory Press: Plainview, NY, pp. v, (Table of Contents Only.). |
| Chu et al. “Fermentation Process Optimization of Recombinant Saccharomyces cerevisiae for the Production of Human Interferon-a2a,” Applied Biochemistry and Biotechnology Part A, 2003, vol. 111, No. 3, pp. 129-137. |
| Cohen, J. (Jun. 17, 1994). “Will Media Reports KO Upcoming Real-Life Trials?” Science 264:1660. |
| Cohen, J. (Jun. 24, 1994). “U.S. Panel Votes to Delay Real-World Vaccine Trials,” Science 264:1839. |
| Engelhardt, J.F. et al. (Oct. 1994). “Prolonged Transgene Expression in Cotton Rat Lung with Recombinant Adenoviruses Defective in E2a,” Human Gene Therapy 5(10):1217-1229. |
| Eto et al. Immunization with Recombinant Escherichia coli Expressing Retinal S-Antigen-Induced Experimental Autoimmune Uveitis (EAU) in Lewis Rats, Cellular Immunology, Mar. 1993, vol. 147, No. 1, pp. 203-214. |
| Fattal-German, M. et al. (1992). “Assessment of the Anti-Viral Effect of a Short-Term Oral Treatment of Mice with Live Saccharomyces cerevisiae Cells,” Dev. Biol. Stand. 77:115-120. |
| Franzusoff, A. et al. (Apr. 1, 2005). “Yeasts Encoding Tumour Antigens in Cancer Immunotherapy,” Expert Opinion on Biological Therapy 5(4):565-575. |
| Franzusoff, A. et al. (Feb. 17, 1995). “Biochemical and Genetic Definition of the Cellular Protease Required for HIV-1 gp160 Processing,” The Journal of Biological Chemistry 270(7):3154-3159. |
| Fujita et al. “Studies in the development of Japanese encephalitis vaccine: expression of virus envelope glycoprotein V3 (E) gene in yeast.” Bulletin of the World Health Organization, Feb. 1987, vol. 65, No. 3, pp. 303-308. |
| Guthrie, C. et al. eds. (1991). “Guide to Yeast Genetics and Molecular Biology,” vol. 194 in Methods in Enzymology, Academic Press, Inc: San Diego, CA, pp. v-ix, (Table of Contents Only). |
| Haller, A.A. et al. (Feb. 9, 2007). “Whole Recombinany Yeast-Based Immunotherapy Induces Potent T Cell Responses Targeting HCV NS3 and Core Proteins,” Vaccine 25(8):1452-1463. |
| Harlow, E. et al. (1988). Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, pp. iii-ix, (Table of Contents Only.). |
| Harlow, E. et al. (1999). Using Antibodies. A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, pp. 1-5, (Table of Contents Only.). |
| Jones, EW. et al. eds. (1992). Gene Expression the Molecular and Cellular Biology of the Yeast Saccharomyces, Cold Spring Harbor Laboratory Press: Plainview, NY, pp. v-vi, (Table of Contents Only.). |
| Kim et al. “Culture Method to Enhance the Productivity of Hepatitis B Surface Antigen (pre S3 + S Ag) With Recombinant Saccharomyces cerevisiae,” Biotechnology Techniques, Apr. 1996, vol. 10, pp. 233-238. |
| Klepfer et al. “Characterization of rabies glycoprotein expressed in yeast.” Archives of Virology, 1993, vol. 128, No. 3-4, pp. 269-286. |
| Lu, Y. et al. (Aug. 1, 2004). “Mutation-Selective Tumor Remission with Ras-Targeted, Whole Yeast-Based Immunotherapy,” Cancer Research, 64:5084-5088. |
| Matousek, J.L. et al. (Jan. 2003). “Evaluation of the Effect of pH on in vitro Growth of Malassezia pachydermatis,” Canadian Journal of Veterinary Research 67(1):56-59. |
| Moore et al. “Novel yeast-based vaccine against HIV-SF2 gp160 promotes a cytotoxic 43-62 cell response.” FASEB Journal 1996, vol. 10, No. 6, p. A1473 ZP002186594. |
| Mullis, K.B. et al. (1994). The Polymerase Chain Reaction, Birkhauser: Boston, MA, pp. xv-xvii, (Table of Contents Only.). |
| Plotkin, SA et al. (1999). Vaccines. Third Edition, W.B. Saunders Company: Philadelphia, PA, pp. xvii-xix, (Table of Contents Only.). |
| Pringle, J.R. et al. eds. (1997). Cell Cycle and Cell Biology The Molecular and Cellular Biology of the Yeast Saccharomyces, Cold Spring Harbor Laboratory Press: Plainview, NY, pp. v-vi, (Table of Contents Only.). |
| Rabinovich, N.R. et al. (Sep. 2, 1994). “Vaccine Technologies: View to the Future,” Science 265:1401-1404. |
| Romani, L. et al. (Feb. 1, 1993). “CD4+ Subset Expression in Murine Canadidiasis Th Responses Correlate Directly with Genetically Determined Susceptibility or Vaccine-Induced Resistance,” Journal of Immunology, 150(3):925-931. |
| Rose, M.D. et al. (1990). Methods in Yeast Genetics, A Laboratory Course Manual, Cold Spring Harbor Laboratory Press: Plainview, NY, pp. iii-iv, (Table of Contents Only.). |
| Sambrook, J. et al. (1989). Molecular Cloning, A Laboratory Manual. Second Edition, Cold Spring Harbor Laboratory Press: Plainview, NY, pp. xi-xxxviii, (Table of Contents Only.). |
| Sambrook, J. et al. (1989). Molecular Cloning, A Laboratory Manual. Third Edition, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, pp. v-xx, (Table of Contents Only.). |
| Schreuder et al. “Yeast expressing hepatitis B virus surface antigen determinants on its surface: implications for a possible oral vaccine.” Vaccine, Apr. 1996, vol. 14, No. 5, pp. 383-388. |
| Serrano, R. et al. (Dec. 2002). The Transcriptional Response to the Alkaline pH in Saccharomyces cerevisiae: Evidence for Calcium-Mediated Signalling, Molecular Microbioloqy 46(5): 1319-1333. |
| Sheng et al. “Mannan derivatives induce phenotypic and functional maturation of mouse dendritic cells,” Immunology, Jul. 2006, vol. 118, No. 3, pp. 372-383. |
| Sinai et al. “Enhancement of Resistance to Infectious Diseases by Oral Administration of Brewers Yeast,” Infection and Immunity, May 1974, vol. 9, No. 5, pp. 781-787. |
| Skinner, FA et al. eds. (1980). Biology and Activities of Yeasts the Society for Applied Bacteriology Symposium Series No. 9, Academic Press Inc: New York, NY, pp. ix-xii, (Table of Contents Only.). |
| Stubbs, A.C. et al. (May 2001). “Whole Recombinant Yeast Vaccine Activates Dendritic Cells and Elicits Protective Cell-Mediated Immunity,” Nature Medicine 7(5):625-629. |
| Valenti, P. et al. (1986). “Interaction Between Lactoferrin and Ovotransferrin and Candida Cells,” FEMS Microbiology Letters 33(2-3):271-275. |
| Valenzuela et al. “Antigen engineering in yeast: Synthesis and assembly of hybrid hepatitis B surface antigen-Herpes simplex 1 gD particles,” Bio/Technology, Apr. 1985, vol. 3, pp. 323-326. |
| International Search Report for International (PCT) Patent Application No. PCT/US2008/052843, mailed Sep. 22, 2008 4 pages. |
| International Preliminary Report on Patentability for International (PCT) Patent Application No. PCT/US2008/052843, issued Aug. 4, 2009 7 pages. |
| Official Action for Australia Patent Application No. 2008214029, dated May 18, 2012 2 pages. |
| Official Action with English translation for China Patent Application No. 200880010797.6, issued Nov. 24, 2011 10 pages. |
| English Translation of Official Action for China Patent Application No. 200880010797.6, dated Jul. 30, 2012 4 pages. |
| English Translation of China Patent Application No. 200880010797.6, dated Apr. 16, 2013 5 pages. |
| Examination Report mailed on Aug. 23, 2010, for European Patent Application No. 08714176.8, filed on Feb. 1, 2008, 6 pages. |
| Examination Report mailed on Jan. 25, 2010, for European Patent Application No. 08714176.8, filed on Feb. 1, 2008, 4 pages. |
| Response to Examination Report mailed Jun. 3, 2010, for European Patent Application No. 08714176.8, filed on Feb. 1, 2008, 9 pages. |
| Official Action for European Patent Application No. 08714176.8, dated Sep. 13, 2011 7 pages. |
| English translation of Official Action for Israel Patent Application No. 200176, dated Aug. 10, 2011 2 pages. |
| English Translation of Official Action for Israel Patent Application No. 200176, dated Jan. 29, 2013 2 pages. |
| English Translation of Official Action for Japan Patent Application No. 2009-548478, mailed Jan. 29, 2013 4 pages. |
| Official Action with English Translation for Taiwan Patent Application No. 096103983, dated Aug. 15, 2012 9 pages. |
| Official Action for U.S. Appl. No. 12/525,045, mailed Jan. 23, 2012 Restriction Requirement. |
| Official Action for U.S. Appl. No. 12/525,045, mailed Jun. 19, 2012 7 pages. |
| Official Action for U.S. Appl. No. 12/525,045, mailed Dec. 27, 2012 10 pages. |
| Notice of Acceptance for Australia Patent Application No. 2008214029, dated Jan. 21, 2014 2 pages. |
| Official Action for Canada Patent Application No. 2,676,783, dated Dec. 20, 2013 3 pages. |
| English Translation of Official Action for China Patent Application No. 200880010797.6, dated Nov. 19, 2013 4 pages. |
| Official Action with English Tranlation for Japan Patent Application No. 2009-548478, mailed Oct. 29, 2013 7 pages. |
| Official Action for Canada Patent Application No. 2,676,783, dated Apr. 8, 2015 3 pages. |
| Notice of Allowance for U.S. Appl. No. 12/525,045, mailed Apr. 21, 2015. |
| Official Action with English Translation for Taiwan Patent Application No. 096103983, dated May 29, 2015, 5 pages. |
| Notice of Allowance for Canada Patent Application No. 2,676,783, dated Oct. 9, 2015, 1 page. |
| Official Action with English Translation for Taiwan Patent Application No. 096103983, dated Nov. 17, 2015, 5 pages. |
| Number | Date | Country | |
|---|---|---|---|
| 20130309269 A1 | Nov 2013 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60899281 | Feb 2007 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12525045 | US | |
| Child | 13798725 | US |
