Link:  Pharm/Biotech Resources

Title:  Predictive assay for immune response

United States Patent:  6,962,790

Issued:  November 8, 2005

Inventors:  Ennis; Francis A. (Shrewsbury, MA)

Assignee:  University of Massachusetts Medical Center (Worcester, MA)

Appl. No.:  159172

Filed:  September 23, 1998

The present invention relates to an in vitro method for determining the ability of a vaccine composition which comprises one or more antigens or a nucleic acid molecule which encodes one or more antigens to stimulate a T cell response. In one embodiment, the method comprises the steps of: (1) contacting antigen presenting cells in culture with a vaccine composition selected from among the group of vaccine compositions, thereby, if one or more of the antigens or nucleic acid molecules can be taken up and processed by the antigen presenting cells, producing one or more processed antigens; (2) contacting the antigen presenting cells with T cells under conditions sufficient for the T cells to respond to one or more of the processed antigens; (3) determining whether the T cells respond to one or more of the processed antigens; whereby if the T cells respond to one or more of the processed antigens, then the vaccine composition stimulates a T cell response; and (4) repeating steps (1), (2) and (3) with each vaccine composition in the group, thereby identifying vaccine compositions which stimulate a T cell response; and, if one or more of the vaccine compositions stimulates a T cell response, (5) selecting at least one vaccine composition which stimulates a T cell response for assessment in one or more animals and/or human subjects.

SUMMARY OF THE INVENTION

The present invention relates to a method for assessing the ability of a candidate vaccine composition to stimulate a T cell response. In one embodiment, the invention provides a method for selecting one or more vaccine compositions from among a group of vaccine compositions for in vivo assessment, for example, in one or more animal or human subjects. Each of the vaccine compositions comprises one or more antigens or one or more nucleic acid molecules encoding one or more antigens. The method comprises the steps of: (1) contacting antigen presenting cells in culture with a vaccine composition selected from among the group of vaccine compositions, thereby, if one or more of the antigens or nucleic acid molecules can be taken up and processed by the antigen presenting cells, producing one or more processed antigens; (2) contacting the antigen presenting cells with T cells under conditions sufficient for the T cells to respond to one or more of the processed antigens; (3) determining whether the T cells respond to one or more of the processed antigens; whereby if the T cells respond to one or more of the processed antigens, then the vaccine composition stimulates a T cell response; and (4) repeating steps (1), (2) and (3) with each additional vaccine composition in the group, thereby identifying the vaccine compositions within the group which stimulate a T cell response; and, if one or more of these vaccine compositions stimulates a T cell response, (5) selecting at least one vaccine composition which stimulates a T cell response for assessment in one or more animals and/or in one or more human subjects.

In another embodiment, the invention relates to a method of selecting a vaccine composition from a group consisting of two or more vaccine compositions for assessment in one or more animals or in one or more human subjects. Each of the vaccine compositions comprises one or more antigens or one or more nucleic acid molecules encoding one or more antigens. The method comprises the steps of: (1) contacting antigen presenting cells in culture with a vaccine composition selected from among said group of vaccine compositions, thereby, if one or more of the antigens or nucleic acid molecules are taken up and processed by the antigen presenting cells, producing one or more processed antigens; (2) contacting the antigen presenting cells with T cells under conditions sufficient to produce a T cell response to one or more of the processed antigens, thereby producing a vaccine composition-stimulated T cell response; (3) measuring the vaccine composition-stimulated T cell response; (4) repeating steps (1), (2) and (3) with each of the remaining vaccine compositions in the group, thereby identifying the vaccine composition or compositions which stimulate the greatest T cell response; (5) selecting the vaccine composition or compositions which stimulate the greatest T cell response for assessment in one or more animals and/or in one or more human subjects.

In a further embodiment, the invention relates to a method for assessing the ability of a vaccine composition comprising one or more antigens or one or more nucleic acid molecules encoding one or more antigens to stimulate a protective T cell response. The method comprises the steps of: (1) contacting human antigen presenting cells in culture with the vaccine composition, thereby, if one or more of the antigens or nucleic acid molecules can be taken up and processed by the antigen presenting cells, producing one or more processed antigens; (2) contacting the antigen presenting cells with human T cells under conditions sufficient to produce a T cell response to one or more of the processed antigens, thereby producing a T cell response; (3) measuring the T cell response; and, if the T cell response is greater than a pre-selected value, (4) assessing the ability of the vaccine composition to stimulate a protective T cell response in one or more animals or in one or more human subjects.

In another embodiment, the method of the invention comprises the steps of: (1) contacting human antigen presenting cells in culture with the vaccine composition, whereby, if one or more of the antigens are taken up and processed by the antigen presenting cells, said antigen or antigens are processed by the antigen presenting cells, thereby producing one or more processed antigens; (2) contacting the antigen presenting cells of step (1) with human T cell clones which are specific for an epitope within one or more of the antigens for a period of time sufficient for the human T cell clones to respond to one or more of the processed antigens; and (3) determining whether the human T cell clones respond to the processed antigen or antigens. If the T cell clones respond to the processed antigen or antigens, the method can, optionally, further include the step of assessing the vaccine composition in one or more animals or human subjects.

Preferably, the vaccine composition includes at least one antigen which comprises a T cell epitope, and the T cells are T cell clones which are specific for a T cell epitope in at least one of the antigens. In one embodiment, the T cells are CD8⁺ T cells and the vaccine composition includes at least one antigen comprising a CD8 epitope. In this embodiment, the T cell response to the processed antigen can be, for example, T cell proliferation, cytolysis of the antigen presenting cells or the production of one or more cytokines.

In another embodiment, the T cells are CD4⁺ T cells and the vaccine composition includes at least one antigen which comprises a CD4 epitope. In this embodiment, the T cell response to the processed antigen which is determined can be, for example, T cell proliferation, stimulation of antibody production by B cells or production of one or more cytokines.

The present invention offers several advantages over prior art methods of evaluating candidate vaccine compositions. For example, the method of the invention can be completed in a relatively short time period. The present method can also be used as a first screen to determine which candidate compositions should be evaluated in much more expensive and time consuming in vivo tests. Thus, the method of the invention enables the efficient and cost effective evaluation of large numbers of potential vaccine compositions, increasing the possibility that effective vaccine compositions will be discovered.

DETAILED DESCRIPTION OF THE INVENTION

Successful vaccines deliver to a host one or more antigens derived from a pathogen, thereby stimulating an immune response which protects against subsequent challenge with the pathogen. Such vaccines can take a variety of forms, including attenuated or killed pathogens, for example, viruses or bacteria; one or more proteins or peptides derived from a pathogen or synthetic or recombinant versions of such proteins or peptides; or one or more nucleic acid molecules encoding one or more proteins or peptides from the pathogen, such as a naked DNA vaccine or a nucleic acid molecule administered in a suitable vector, such as a recombinant virus or bacterium or an immunostimulating complex. Vaccines against cell proliferative diseases, such as cancers, typically utilize proteins or fragments thereof, or nucleic acid molecules encoding proteins or fragments thereof, which are unique to diseased cells or generally more abundant in diseased cells compared to healthy cells.

Cell-mediated immunity is dependent upon lymphocytes known as B cells and T cells. B cells produce antibodies targeted against extracellular antigens. T cells recognize antigen fragments (peptides) which are displayed at the surface of a host cell. Such antigen fragments result from uptake of the antigen by a host cell, or synthesis of the antigen within the host cell, followed by cleavage of the antigen within the cell. Although it is probable that most successful vaccines elicit both T cell and B cell responses, current methods for evaluating test vaccines generally focus on antibody production by B cells, and do not assess the ability of the test vaccine to elicit a T cell response.

Foreign proteins which are synthesized within the host cell or are taken up by the host cell via specific receptors are fragmented within the cytosol of the cell. One or more of the resulting peptides can become associated with class I major histocompatibility molecules (MHC I), and the resulting complexes are then presented at the surface of the cell. These MHC I/peptide complexes are recognized by specific T cell receptors in certain CD8⁺ T cells, and the peptides so presented are referred to as CD8 epitopes.

A foreign protein can be taken up by a host cell nonspecifically via endocytosis and then fragmented into peptides in a cellular lysosomal or endosomal compartment. One or more of these peptides can then become associated with a class II major histocompatibility molecule (MHC II) to form a complex which is then presented at the surface of the host cell. These MHC II/peptide complexes are recognized by CD4⁺ T cells expressing a specific receptor which recognizes the MHC II/peptide complex. These peptides are referred to as CD4 epitopes.

Peripheral T cells in the blood and organs of the immune system (e.g. spleen and lymph nodes) exist in a quiescent or resting state. Upon interaction of T cells with an MHC/epitope complex, the T cells proliferate and differentiate into activated cells having a variety of functions. CD8⁺ T cells typically become cytotoxic upon activation and destroy antigen-presenting cells via direct contact. Activated CD4⁺ T cells provide a helper function to B cells, enabling B cells to differentiate into antibody-producing cells. Activated CD8⁺ T cells and CD4⁺ T cells release a variety of cytokines (lymphokines or interleukins), which can, for example, control differentiation of many classes of lympholytic precursor cells.

In one embodiment, the invention provides a method for selecting one or more vaccine compositions from among a group of two or more vaccine compositions for in vivo assessment in one or more animals and/or human subjects. Each of the vaccine compositions comprises one or more antigens or one or more nucleic acid molecules encoding one or more antigens. The method comprises the steps of: (1) contacting antigen presenting cells in culture with a vaccine composition selected from among said group of vaccine compositions, thereby, if one or more of the antigens or nucleic acid molecules are taken up and processed by the antigen presenting cells, producing one or more processed antigens; (2) contacting the antigen presenting cells with T cells under conditions sufficient for the T cells to respond to one or more of the processed antigens; (3) determining whether the T cells respond to one or more of the processed antigens; whereby if the T cells respond to one or more of the processed antigens, then the vaccine composition stimulates a T cell response; and (4) repeating steps (1), (2) and (3) with each vaccine composition in the group, thereby identifying vaccine compositions which stimulate a T cell response; and, if one or more of the vaccine compositions stimulates a T cell response, (5) selecting at least one vaccine composition which stimulates a T cell response for assessment in vivo.

In another embodiment, the invention relates to a method of selecting at least one vaccine composition from a group consisting of two or more vaccine compositions for assessment in one or more animals and/or human subjects. Each of the vaccine compositions comprises one or more antigens or one or more nucleic acid molecules encoding one or more antigens. The method comprises the steps of: (1) contacting antigen presenting cells in culture with a vaccine composition selected from among said group of vaccine compositions, thereby, if one or more of the antigens or nucleic acid molecules can be taken up and processed by the antigen presenting cells, producing one or more processed antigens; (2) contacting the antigen presenting cells with T cells under conditions sufficient to produce a T cell response to one or more of the processed antigens, thereby producing a vaccine composition-stimulated T cell response; (3) measuring the vaccine composition-stimulated T cell response; (4) repeating steps (1), (2) and (3) with each of the remaining vaccine compositions in the group, thereby identifying one or more vaccine compositions which stimulate the greatest T cell response; and (5) selecting the vaccine composition or compositions which stimulate the greatest T cell response for assessment in an animal or in a human. In another embodiment, one or more of the vaccine compositions producing a stimulated T cell response greater than a pre-selected value are selected for in vivo assessment. Alternatively, one or more vaccine compositions having relatively high activity compared to the remaining vaccine compositions are selected for in vivo assessment.

In a further embodiment, the invention relates to a method for assessing the ability of a vaccine composition comprising one or more antigens or one or more nucleic acid molecules encoding one or more antigens to stimulate a protective T cell response. The method comprises the steps of: (1) contacting human antigen presenting cells in culture with the vaccine composition, thereby, if one or more of the antigens or nucleic acid molecules can be taken up and processed by the antigen presenting cells, producing one or more processed antigens; (2) contacting the antigen presenting cells with human T cells under conditions sufficient to produce a T cell response to one or more of the processed antigens, thereby producing a T cell response; (3) measuring the T cell response; and, if the T cell response is greater than a pre-selected value, (4) assessing the ability of the vaccine composition to stimulate a protective T cell response in one or more animals, human subjects or a combination thereof. The pre-selected value of the T cell response is, typically, chosen to represent a vaccine composition which is particularly active in stimulating a T cell response.

In another embodiment, the method of the invention comprises the steps of: (1) contacting human antigen presenting cells in culture with the vaccine composition, whereby, if one or more of the antigens are taken up and processed by the antigen presenting cells, said antigen or antigens are processed by the antigen presenting cells, thereby producing one or more processed antigens; (2) contacting the antigen presenting cells of step (1) with human T cell clones which are specific for an epitope within one or more of the antigens for a period of time sufficient for the human T cell clones to respond to one or more of the processed antigens; and (3) determining whether the human T cell clones respond to the processed antigen or antigens. If the T cell clones respond to the processed antigen or antigens, the method can, optionally, further include the step of assessing the vaccine composition in an animal or in a human.

A "processed antigen", as the term is used herein, refers to one or more epitopes derived from an antigen which are presented at the surface of an antigen presenting cell in combination with MHC I or MHC II.

The present method assesses the ability of a candidate vaccine composition to provide in vitro an antigen to antigen presenting cells in a manner which leads to processing and presentation of one or more T cell epitopes at the surface of the antigen presenting cells in combination with MHC I or MHC II. This in vitro determination provides an efficient screen for selecting compositions for more time-consuming in vivo testing in animals or in humans. This in vivo testing can be performed using methods which are well known in the art. For example, the vaccine composition can be administered to an animal or a human, and the ability of the induced immune response, if any, to protect against subsequent challenge from the pathogen from which the antigen or antigens are derived can be determined. Alternatively, or in eonjunction with such a determination, the ability of the vaccine composition to induce in vivo the proliferation of T cells and/or antibodies which recognize one or more of the antigens can also be determined. Animals which can be used for in vivo testing include laboratory animals, domesticated animals and wild animals. Suitable examples include rodents, such as mice, hamsters, rats, guinea pigs and rabbits; primates, such as monkeys and apes; and domestic animals, such as dogs, cats, horses, chickens, cows and pigs.

The antigen presenting cells are contacted with the vaccine composition in cell culture in a suitable culture medium, as is known in the art, and under suitable conditions, such as physiological pH, and at a temperature from about room temperature to about physiological temperature, for a sufficient period of time for uptake and processing of the antigen by the antigen presenting cells. If the vaccine comprises a nucleic acid molecule, the antigen presenting cells are contacted with the vaccine composition for a sufficient amount of time for the antigen presenting cells to take up and express the nucleic acid molecule and process the resulting antigen. Generally, the antigen presenting cells are contacted with the vaccine composition for a period of several hours, for example, from about 2 to about 12 hours. Following contact with the vaccine composition, the antigen presenting cells are contacted with the T cells for a sufficient period of time for activation of the T cells and generation of a T cell effector response. Generally, this process requires several hours, for example, from about 2 to about 12 hours. Preferably, the APCs are contacted with the vaccine composition for a sufficient period time for antigen or nucleic acid molecule uptake, and then washed and placed in fresh media prior to addition of the T cells. Alternatively, the antigen presenting cells can be contacted with the vaccine composition and the T cells simultaneously or within a relatively short time interval. In this embodiment, the antigen presenting cells are contacted with the vaccine composition and the T cells for a sufficient amount of time for antigen processing and generation of a T cell response. Typically, such a process requires from about 4 to about 24 hours.

The vaccine composition, preferably, comprises at least one antigen, or a nucleic acid encoding at least one antigen, which is a protein or a peptide which comprises one or more T cell epitopes, such as one or more CD8⁺ T cell epitopes, one or more CD4⁺ T cell epitopes or a combination thereof. Preferably, the T cells are specific for a particular epitope present within the antigen. More preferably, the T cells are T cell clones derived from a single precursor T cell. In a particularly preferred embodiment, the T cells are human T cell clones.

In one embodiment, the epitope is a CD4⁺ T cell epitope and the T cells are CD4⁺ T cells. As discussed above, the effector functions of CD4⁺ T cells include releasing cytokines and stimulating B cells to become antibody-producing cells. Thus, in this embodiment, the extent of the T cell response to the antigen presenting cells can be determined by measuring T cell proliferation, the production of one or more cytokines or the stimulation of antibody production by B cells. Greater levels of T cell proliferation, antibody production or cytokine production would be expected to correlate with greater immunogenicity and potential efficacy of the vaccine composition.

In another embodiment, the epitope is a CD8 epitope and the T cells are CD8⁺ T cells. As discussed above, the effector functions of CD8⁺ T cells include lysis of antigen presenting cells and release of cytokines. Therefore, the extent of CD8⁺ T cell response to the antigen presenting cells can be determined using an assay for cell lysis or by measuring the production of one or more cytokines. The CD8⁺ T cell response can also be measured by measuring the extent of release of one or more cytokines. In general, it is expected that greater cell lysis activity or cytokine release will correlate with greater immunogenicity.

The antigen presenting cells can be selected from among any suitable cells which are potentially capable of taking up the antigen, such as a natural, purified or recombinant protein, or a nucleic acid molecule encoding the antigen, and presenting a peptide epitope derived from the antigen at the cell surface in combination with MHC I or MHC II. For example, when the epitope is a CD4 epitope, cells expressing MHC II molecules can be used. Such cells include macrophages, dendritic cells and B cells. When the epitope is a CD8⁺ T cell epitope, the antigen presenting cells can be selected from among any cells which express MHC I. In preferred embodiments, the antigen-presenting cells are professional antigen-presenting cells, such as macrophages, dendritic cells and B cells. The antigen presenting cells can be, for example, recombinant cells expressing heterologous MHC molecules. In a preferred embodiment, the antigen presenting cells are human cells. The antigen presenting cells present the proper MHC molecules and are, preferably, at least partially HLA matched with the T cells. More preferably, the APCs are autologous cells, that is, cells derived from the same donor as the T cells.

In one embodiment, the T cells are clones which are specific for a particular epitope, and the vaccine composition includes at least one antigen which comprises the epitope or at least one nucleic acid molecule encoding at least one antigen which comprises the epitope. In this embodiment, response of the epitope-specific T cell clones to antigen-presenting cells which have been contacted with the experimental vaccine composition indicates that the vaccine composition is able to effect the presentation of the epitope on the surface of the antigen-presenting cells in combination with an MHC I or MHC II molecule.

Epitope-specific T cell clones can be generated using methods which are generally known in the art (see, for example, Fathman, et al., in Paul, ed., Fundamental Immunology, second edition, Raven Press (1989), Chapter 30, the contents of which are hereby incorporated by reference in their entirety). The isolation of epitope-specific T cell clones is based on T cell biology. Generally, an animal, such as a mouse, is immunized with a preparation of antigens (a bacterial lysate, or a purified protein) or is infected with a virus, such as a wild type virus or a recombinant virus containing heterologous genes encoding one or more proteins from a pathogenic microorganism, such as a virus. The animal is then sacrificed and the peripheral blood mononuclear cells (PBMC: includes T cells, B cells, monocytes), spleen and lymph nodes are isolated. The isolated cells are then cultured in media containing a defined component of the original antigenic preparation, often a recombinant or purified protein, and the essential T cell growth factor interleukin-2 (IL-2). The only T cells which will proliferate are those which recognize MHC/epitope complex in which the epitope is derived from the antigenic preparation. These cells become activated and proliferate while the unactivated cells begin to die. The cultures are maintained for several weeks, with the media containing antigen and IL-2 being periodically replaced. Eventually, clusters of living and dividing cells (a T cell line) can be observed in some of the cultures.

The proliferating cells are generally not clonal at this point and are of limited use for assaying epitope specific T cell responses. The T cell line is, preferably, cloned through a process referred to as limiting dilution. In this method, PBMC are isolated from, for example, a mouse of the same strain as the original mouse used to isolate the T cell line. These cells, called antigen presenting cells, will serve as a source of MHC proteins and will present the MHC:peptide complex to the T cell line. The T cell line is diluted to a concentration of about 1 to 5 T cells/mL in a suspension of APCs that contains the antigen of interest and IL-2. This suspension is then transferred into, for example, round or "v"-bottom 96 well microtitre plates, so that each well contains, on average, no more than 1 T cell. The cultures are maintained for several weeks and a clone can grow out of one or more cultures.

The cells isolated by limiting dilution are the progeny of a single cell that expresses only one T cell receptor, and the clone is thus epitope-specific. However, in a situation in which the cloning procedure uses whole proteins or viruses, a single protein may contain many epitopes and the precise epitope will remain unknown. The epitope can be identified using a collection of overlapping synthetic peptides that span the entire amino acid sequence of the antigenic protein. These peptides can be used to stimulate proliferation or cytokine secretion in a direct stimulation assay, or they may be used as competitive inhibitors to block activation of the T cell clone by the antigenic protein.

Human T cell clones can also be isolated. Generally, these clones are isolated from individuals who have had an infection, for example, influenza, HIV or Dengue, or have been exposed to antigens in nature or by injection and have T cells that specifically respond to those antigens. These antigens are called "recall antigens" and include tetanus toxoid and Candida albicans extract. Human T cell clones are isolated from the PBMC.

The T-cell response to APCs treated with the test vaccine composition can be determined using a variety of assays which are known in the art. Several examples are taught by Fathman, et al., supra. For example, T cell proliferation can be measured using methods known in the art. In one embodiment, the epitope-specific T cells are mixed with irradiated antigen presenting cells and the test vaccine composition and cultured. The cells are cultured for a period of a few days to allow presentation of the epitope by the APCs and activation of the T cells. T cell proliferation is then assessed by monitoring the incorporation of ³H-thymidine into newly synthesized DNA. The APCs do not incorporate ³H-thymidine because they have been irradiated. Alternative methods for assessing proliferation that do not use radioisotopes are also known.

T cell response can also be determined by determining if one or more cytokines is released by the T cells. For this assay, APCs and the test vaccine composition are mixed and cultured. Either simultaneously or after a period of time sufficient for uptake and processing of an antigen within the vaccine composition by the APCs, T cells are added to the culture. After a period of time sufficient to allow activation of the T cells, growth of the culture is stopped, for example, by freezing. Freezing the culture lyses the cells and releases cytokines that have not yet been secreted into the culture medium. The presence or absence of cytokine in the culture medium can then be determined using known methods. Optionally, the amount of one or more cytokines in the culture medium can be determined. For example, cytokines in the culture supernatant and the cells can be measured using a bioassay, in which cell lines that proliferate only when stimulated with a particular cytokine (indicator cells) are cultured in media that is supplemented with an aliquot of the cytokine-containing culture media. The culture is maintained, typically, for 10-18 hours and ³H-thymidine is added. After an additional 6-10 hours, new DNA synthesis is measured by determining the amount of ³H incorporated into the cellular DNA. Any cytokine which is produced by the T cells upon activation can be measured. Examples of cytokines which can be determined include interferon-γ and interleukin-2.

In another embodiment, cytokine production is measured using an enzyme-linked immunosorbent assay (ELISA), for example, using reagents which are commercially available as kits. In this assay, an immobilized antibody is used to specifically capture a particular cytokine from the cytokine containing culture supernatant. Unbound proteins are washed away, and the amount of bound cytokine is determined by binding a second, labeled, antibody to the captured cytokine. This assay is quantitative and more specific than bioassays. Alternatively, cytokine mRNA levels can be quantitated using the polymerase chain reaction. Cytokine production can also be determined by staining producer T cells with labeled antibodies specific for the cytokine.

In another embodiment, the T cells are CD8⁺ T cells and the response is measured by determining whether the T cells lyse the APCs which have been treated with the test vaccine composition. In one embodiment, the APCs are transformed peripheral blood lymphocyte cell lines (B-LCL) which have been incubated with ⁵¹CrO₄^2-. The resulting ⁵¹Cr-labeled PBLs are thoroughly washed, incubated with the test vaccine composition and then exposed to the antigen-specific CD8⁺ T cells. After incubating for a sufficient period of time for epitope presentation by the B-LCLs and T cell activation, the extent of ⁵¹Cr release into the culture medium is determined. The amount of ⁵¹Cr released correlates with the extent of lysis of the B-LCLs.

The production of a T cell response can, generally, be determined by comparing the result achieved with the vaccine composition to a suitable control, as is known in the art. For example, in the ⁵¹Cr release assay discussed above, the amount of 51Cr released when the B-LCLs are treated with the CD8+ T cells can be compared to the amount released when the B-LCLs are treated with vehicle alone, referred to as the background release. Significantly (measurably) greater ⁵¹Cr release in the presence of the T cells is indicative of a T cell response. In the cytokine production assay, cytokine production by the T cells in the presence of APCs treated with the vaccine composition can be compared to cytokine production by the T cells in the absence of APCs, or in the presence of untreated APCs. Greater cytokine production in the presence of treated APCs is indicative of a T cell response.

The test vaccine composition comprises one or more antigens or one or more nucleic acid molecules which encode one or more antigens. The vaccine composition can be any of the types of vaccine compositions which are known in the art. For example, the vaccine composition can comprise an attenuated pathogen, such as a weakened bacterial strain or virus, or a killed pathogen, such as a killed bacterial strain or a killed virus. The vaccine composition can also comprise a portion of a pathogen, for example, a viral coat or bacterial membrane. In another embodiment, the vaccine composition comprises one or more proteins derived from a pathogen, for example, a protein which has been purified or partially purified from the pathogen, or a recombinant protein produced by a recombinant organism which expresses a gene derived from the pathogen which encodes the protein. Examples of suitable host organisms for the production of recombinant peptides and proteins are known in the art and include E. coli. The vaccine composition can also include one or more fragments of a protein or proteins derived from pathogen. Such protein fragments include peptides which are synthesized or recombinantly produced.

In another embodiment, the test vaccine composition includes one or more proteins, or fragments thereof, which are produced by a particular type of tumor cell. Preferably, the protein is unique to the tumor cell, i.e., not present in or on healthy cells, or is expressed in greater quantity by the tumor cell than by healthy cells. The protein can be, for example, a protein found on the surface of the tumor cell. The protein(s) can be derived from the tumor cells, for example, isolated and purified or partially purified from cultured tumor cells. The tumor cell protein(s), or a fragment or fragments thereof, can also be produced recombinantly.

In another embodiment, the vaccine composition comprises a nucleic acid molecule which encodes a protein or a fragment thereof, derived from a pathogen or a tumor cell as discussed above. For example, the vaccine composition can comprise so-called "naked DNA". The nucleic acid molecule can also be contained within a suitable vector, such as a recombinant virus, such as vaccinia virus, adenovirus, orf virus, fowlpox virus, herpes virus, varicella virus, papilloma virus, SV40, retroviruses, baculovirus and poliomyelitis virus. The vector can also be a bacterium, such as salmonella, BCG or E. coli. The nucleic acid can also be present in a liposome or another suitable vector, such as are known in the art.

As discussed above, the present invention enables the rapid assessment and comparison of a large number of potential vaccine compositions. For any given disease or pathogen, for example, a variety of antigens can be assessed. For example, a set of vaccine compositions which each include different antigens or portions of antigens from a particular pathogen can be compared. Further, for a given antigen, set of antigens, or nucleic acid molecule encoding such antigen(s), a variety of formulations can be assessed. For example, a set of vaccine compositions including the same antigen or antigens, but different vectors, adjuvants, concentrations, vehicles or excipients can be compared to determine the conditions necessary for optimal efficacy.
 

Claim 1 of 21 Claims

1. A method for selecting one or more vaccine compositions from among a group consisting of two or more distinct vaccine compositions for assessment in one or more human subjects, said vaccine compositions each comprising one or more nucleic acid molecules encoding one or more antigens which comprise the same CD8⁺ T cell epitope, said method comprising the steps of:

(a) contacting human antigen presenting cells in culture with a vaccine composition selected from among said group of vaccine compositions, thereby, if one or more of the nucleic acid molecules encoding one or more antigens which comprise said T cell epitope are taken up and processed by said antigen presenting cells, producing one or more processed antigens;

(b) contacting said antigen presenting cells of step (a) with monoclonal human T cells, wherein the monoclonal human T cells, are CD8⁺ T cells, under conditions sufficient for said T cells to respond to one or more of the processed antigens;

(c) determining the level of said T cells' response to one or more of the processed antigens;

(d) repeating steps (a), (b) and (c) with each additional vaccine composition in the group; and

(e) selecting at least one vaccine composition that exceeds a predetermined level of said T cells' response for assessment in one or more human subjects.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.