THERAPEUTIC VACCINES

DESCRIPTION

BACKGROUND OF THE INVENTION

Field of the Invention The invention generally relates to methods for assessing the state of a subject's immune system after administration of an immune stimulant or inhibitor of interest in order to evaluate, monitor, or treat a disease or condition, e.g. a cancer vaccine. In particular, the invention provides methods for detecting a product or patterns of products produced by the immune cells of the subject in response to administration of the immune stimulant or inhibitor; the products or pattern of products that is detected serves as an indicator of the subject's functional immune response to treatment.

Background of the Invention A vaccine may be generally defined as a biological preparation that improves immunity to a particular disease. Vaccines typically contain at least one agent that resembles or contains portions of a disease-causing entity, or an entity that is characteristic of a disease. Upon administration to a subject, the agent stimulates the subject's immune system to recognize the agent (and hence the disease-related entity which it resembles) as foreign, destroy it, and "remember" it. Thereafter, the immune system can more easily recognize and destroy similar disease-related entities when they are encountered. Vaccines can be prophylactic (e.g. intended to prevent or ameliorate the effects of a future disease), or therapeutic (e.g. intended to treat or ameliorate the effects of incipient or established disease).

Therapeutic vaccines (e.g. against cancer) are currently of great interest, with some promising vaccine candidates having already been developed and approved for use by the United States Food and Drug Administration (FDA). Unfortunately, current methods for monitoring the efficacy of therapeutic vaccines, and hence to guide medical practitioners' use of the same, are rudimentary at best. In particular, there is a need in the art to identify biomarkers which are indicative of the functional immune response of individuals to the administration of therapeutic vaccines, and to develop in vitro diagnostic (IVD) assays capable of quickly and reliably detecting the biomarkers in order to assess the efficacy of the vaccines.

Single biomarkers that provide actionable results when measured in a univariate manner are uncommon. Thus, most current genomic, proteomic and gene expression research is directed toward discovery of multivariate patterns of multiple biomarkers that are identified experimentally and computationally.

Many clinically useful IVD assays are proteomic in nature and are based on the measurement of molecular concentrations of proteins and/or peptides. Computational analysis has been used to identify protein/peptide biomarkers and expression patterns of multiple biomarkers with clinical utility. Incorporation of such biomarkers into data reduction algorithms that facilitate actionable clinical decision making has greatly improved the utility of protein/peptide biomarkers in multiplex IVD assays.

Unfortunately, assays that are currently widely used for analyzing cellular immune responses remain rudimentary. They are often not based on protein/peptide multiplex assays, but instead, most simply measure the extent of redness and swelling at the injection site following administration of the vaccine or components of the vaccine under the skin, count the number of cells present, or use traditional methods to characterize cell types present as a percentage of the total white blood cell count, as with the classical complete blood count.

More sophisticated cellular assays that are capable of evaluating not just cell type and numbers, but functional responses of cells involved in cellular immune responses, have not yet been standardized or submitted for (FDA) clearance. Such assays are only performed in laboratories in the context of basic research, or in highly specialized clinical laboratories, and this limits their use. For example, the Elispot assay characterizes certain aspects of cellular immune reactivity by measurement of the production and secretion of specific proteins, but is highly labor intensive, has not been well standardized, and is thus confined to use in specialized clinical settings. While this type of assay has a number of advantages, it would be advantageous to have some simpler assays that might be employed more widely.

There is a need for the development of straightforward, reliable yet facile multiplex assays that evaluate functional responses of the immune system, particularly after the administration of an immune stimulant (or inhibitor) such as a therapeutic vaccine. Such multiplex assays should be amenable to use in standard medical settings.

SUMMARY OF THE INVENTION The invention provides methods and assay kits for determinations of the immune status of a subject before, and after, at least one administration of an immune modulating agent (IMA) to the subject, for example, to evaluate a patient's immune status prior to administration of an IMA to determine if they are a good candidate for the treatment, to evaluate how a patient is responding to treatment with the IMA, and for other reasons as part of, for example, a treatment for a disease or condition of interest. The methods and assays of the invention may provide a way to assess whether or not the administration of the IMA has had a desired effect on the subject, e.g. stimulating or inhibiting the immune system of the subject. The methods and assays may have wide applicability and may be used in clinical settings as a tool for evaluating and/or monitoring the effectiveness of treatments which modify the immune system.

According to an embodiment of the invention, and with reference to Figure 1 , an IMA is administered to a subject, typically a subject that has or is at risk of developing a disease or condition of interest. Thereafter, a biological sample is obtained from the subject. Immune cells in the sample are then exposed to (stimulated by) the IMA in vitro and the functional response of the immune cells to the IMA is determined. In some embodiments, a biological sample will also have been obtained from the subject prior to the first administration of an IMA and tested using the invention in order to establish a

patient-specific baseline (i.e. prior to treatment) functional response pattern that can be compared with the pattern observed after administration of an IMA.

According to another embodiment of the invention, a patient's immune status prior to administration of an IMA is determined. A biological sample is obtained from the patient and is exposed to (stimulated by) the IMA in vitro and the functional response of the immune cells to the IMA is determined. Based on the response, a clinician may be able to identify if the patient is a good candidate for the treatment.

In some embodiments, the immune cells are separated from the biological sample prior to IMA exposure. In other embodiments, the biological sample as a whole is exposed to the IMA and the immune cells are separated from the sample afterwards, prior to assessment of the products they produce. In yet other embodiments, the immune cells are not separated from the sample, e.g. an on-board cell capture/labeling and analyte detection system is employed.

The functional immune response of the immune cells may be determined by multiplex detection of a plurality of products or analytes produced by the immune cells in response to stimulation by the IMA. The group of products that is detected may be referred to herein as a "pattern", "pattern of expression", "functional response pattern", or other similar terms. The pattern of analytes that is detected is compared to a predetermined, characteristic (i.e. expected) response pattern that was previously established in control subjects. In one embodiment, if administration of the IMA to the subject was successful, then immune cells in the biological sample will produce a characteristic, predetermined detectable pattern of products in response to in vitro exposure to the IMA(s). If this occurs, then a trained medical practitioner may conclude that the treatment of the subject with IMA was successful, e.g. the immune cells of the subject have been successfully primed to produce a desired set of products by administration of the IMA. On the other hand, if administration of the IMA to the subject was not effective, then the immune cells in the biological sample will not respond to stimulation by the IMA by producing the desirable pattern of products. This latter outcome may result in a conclusion, by trained medical personnel, that IMA administration should be repeated, or repeated in a different manner (e.g. with a higher dose, with different IMA(s), etc.), that a different type of treatment should be tried to treat the disease, or that the treatment protocol should be altered in some other manner. In still further embodiments of the invention, the functional response pattern of a subject may be different after treatment with the IMA than it was prior to treatment with the IMA, and this difference could be evaluated with respect to whether the change in the functional response pattern is expected or not expected.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Schematic representation of the administration of IMA(s) to a subject and the subsequent in vitro detection of, as an exemplary embodiment, a pattern of products produced by immune cells from the subject.

DETAILED DESCRIPTION The invention provides methods and assays for interrogating the immune status of an individual before and/or after administration of at least one immune modulating agent (IMA) (a therapeutic immune stimulant or inhibitor). In one embodiment, the methods and assays detect, in vitro, whether or not prior in vivo administration of the IMA to the subject caused cells in the immune system of the subject to respond in a desired manner. If the IMA is an immune stimulant, the intended result is that the immune cells become competent to mount a successful immune response to the IMA. If the IMA is an immune inhibitor or suppressant, then the desired response may be a decrease in responsiveness of the immune cells or production of a pattern of analytes associated with suppressive response from the immune cells. Either way, if at least one product (or a pattern of products) associated with at least one immunological response to the IMA is detected and/or is detected in expected amounts when immune cells obtained from the subject are exposed to the IMA, or one or more components of the IMA in vitro, then one may conclude that the previous administration of the IMA to the subject did result in activation (or suppression) of the immune system with respect to the IMA. In this embodiment, if the product is not detected or the expected pattern of analytes is not observed, the previous administration was not successful.

In an embodiment, a pattern of biomarker molecule production (a plurality of products is detected) similar to a predetermined desired pattern is preferably detected when immune cells obtained from the subject are exposed to the IMA, or one or more components of the IMA, in vitro. By detecting the pattern of biomarker molecule production, one may conclude that the previous administration of the IMA to the subject did result in activation (or suppression) of the immune system with respect to the IMA. However, if the characteristic biomarker pattern is not detected, then it may be concluded that the IMA administration was not successful, i.e. the immune system of the subject did not mount a successful response to the IMA, or was not inhibited.

If the IMA is, for example, a mimic of a disease causing entity (e.g. an infectious agent) or of an entity associated with a disease (e.g. a cancer cell), medical or clinical personnel may use this information to conclude whether or not it is likely that the immune system of the subject will mount a successful response to, and hence eliminate, the disease causing entity or the entity associated with a disease, when encountered in the future.

Alternatively, if the IMA is an immune inhibitor, medical or clinical personnel may use this information to conclude whether or not the immune system has been sufficiently repressed.

In another embodiment of the invention, a patient's immune status prior to administration of an IMA is determined. A biological sample is obtained from the patient and is exposed to (stimulated by) the IMA in vitro and the functional response of the immune cells to the IMA is determined. Based on the response, a clinician may be able to identify if the patient is a good candidate for the treatment.

By immune modulating agent (IMA) we mean an agent or group of agents that modulate or modify at least one functional immune response of the immune system of a subject. IMAs may stimulate the immune system, or may inhibit the immune system. In the case of immune stimulation, the IMA (or an IMA preparation, which may include multiple agents) generally comprises antigens to which it is desirable for the subject to elicit an immune response. Such antigens include but are not limited to those which occur in or on (e.g. on the surface of) or are otherwise associated with cells which it is desirable to eliminate from the body, e.g. infectious agents (bacteria, disease causing protozoans, viruses, fungi, etc.), cancer cells, auto-reactive cells, etc.. In addition, other immune stimulating agents may be included in an IMA composition, examples of which include but are not limited to: nucleic acid sequences (e.g. CpG), polysaccharides, glycoproteins, lipoproteins, ligands that bind specific cellular receptors, cell surface ligands which bind to receptors on other cells but which do not function as receptors themselves, cellular receptor molecules (e.g. co-stimulatory receptors such as B7 and B7.1 ; inhibitory receptors such as CTLA-4, PD-1), mimics of natural cellular receptors, antibodies against cellular receptors, histocompatibility molecules (e.g. MHC class I and class II molecules), disease modified biomolecules (e.g. normal proteins with a mutated amino acid sequence, normal proteins with altered post-translational modifications such as phosphate or carbohydrate groups added, deleted, or attached at different positions than the normal biomolecule), etc.

For the assay, a biological sample from the patient or subject that received or will receive the IMA is used. The sample is exposed to either the IMA or one or more components of the IMA.

In one embodiment of the invention, the IMA comprises cancer cell antigens in the form of antigenic proteins or peptides from or associated with cancer cells, and

administration of the IMA is intended to immunize cancer patients against those

proteins/peptides, in the hope of stimulating an immune reaction that will kill cancer cells in the body of the patient. Tumor antigens are generally divided into two broad categories: shared tumor antigens; and unique tumor antigens. Shared antigens are expressed by many tumors, whereas unique tumor antigens result from mutations induced through physical or chemical carcinogens, and are therefore expressed only by individual tumors. Therapeutic cancer vaccines against both categories of tumor antigens are currently being developed for the treatment of breast, lung, colon, skin, kidney, prostate, and other cancers. In yet another approach, a cancer vaccine may contain whole tumor cells. The assays of this invention, which could include either shared or unique tumor antigens as the IMA (or components thereof) which is added to the biological sample from the patient that is receiving or will receive treatment with the IMA can help assess whether the cancer patient will be a good candidate for treatment with the IMA, help assess the dosing of the IMA provided to the patient during treatment, and help in assessing that the treatment is progressing in a desired or non-desired manner.

Those of skill in the art will recognize that the amount of an IMA that is administered to a subject may vary from subject to subject, from IMA to IMA, or for other reasons, and that skilled medical professionals (e.g. physicians or other medical or clinical personnel) will be able to determine the amounts or range of amounts. The suggested amount may be determined using methodology known to those of skill in the art, e.g. using data obtained in clinical trials. Those of skill in the art will also recognize the caveats associated with administration, e.g. in the case of administering antigens, the amount or conditions should be sufficient to avoid the development of tolerance to the antigen in the subject.

Other approaches to therapeutic anti-cancer vaccination are known to those of skill in the art, and the methods and assays of the invention may be used to evaluate the outcome of treatment with these agents as well. For example, the BioVex Inc, product OncoVEX GM-CSF is a version of herpes simplex virus which is genetically engineered to replicate selectively in tumor tissue and to express the immune stimulatory protein

granulocyte-macrophage colony-stimulating factor (GM-CSF). This factor enhances the anti-tumor immune response to tumor antigens released following lytic virus replication, providing an in situ, patient specific anti-tumor vaccine as a result.

In other embodiments, the IMA may be an immune inhibiting agent. In this case, the IMA is administered e.g. to persons with an autoimmune disease. Exemplary inhibiting agents that may be included in an IMA include but are not limited to anti-CTLA-4, antibodies that block binding or transit of cells into tissues (e.g. anti-integrin antibodies) or cell differentiation (e.g. IL-2, TLRs) factors, agents that block that activity of

pro-inflammatory proteins (e.g. anti-T F-a, anti-IL-lR), inhibitors of intracellular cell signaling pathways associated with immune response (e.g. mTOR, JAK/STAT), inhibitors of metabolism (e.g. MTX), etc.

The invention encompasses assays which indicate the immune status of a subject prior to and/or in response to (i.e. after) administration of an IMA. The product(s) associated with an immunological response can be identified from medical resource materials, or can be identified in the practice of this invention by testing biological samples from control subjects (apparently healthy people as well as people with the disease or suspected of having the disease but not treated with the IMA) as well as biological samples obtained following administration of the IMA to subjects with the disease or suspected of having a disease, and based on testing of biological samples of these subjects identifying at least one product which is common for all or a majority or a significant fraction of the subjects with the disease or suspected of having the disease (i.e., the product is in a sample from every subject or a majority of the subjects). A pattern of products could be identified by detecting a plurality of different products, one or more of which is in a sample from, for example, all or a majority or a significant fraction of the subjects).

An embodiment of the invention contemplates using the assay to identify people capable of responding to an IMA, and which are therefore good candidates for receiving the IMA as a treatment regimen. In practice, biological samples from a plurality of candidates would be exposed to the IMA or a component thereof, and, based on the detection or non-detection of expected product(s) associated with the immunological response, a subject can be identified as a good candidate to receive treatment with the IMA.

While the invention could, in certain embodiments, utilize only one product associated with one immunological response (i.e., one biomarker), it is envisioned that the invention would encompass multiplex assays. By "multiplex" we mean an assay that simultaneously measures properties of multiple analytes or chemical compounds (e.g.

biomolecules) of interest (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) in a single assay. Generally, as used herein and as described in detail below, analytes generally include biological products (chemical compounds, etc.) that are produced by immune cells.

In order to carry out the methods and assays of the invention, control expression patterns (both positive and negative) may be determined based on results obtained in control subjects. Exemplary control subjects may include, but are not limited to, e.g. subjects who are free of the disease or condition of interest and who have never received the IMA;

subjects who are free of the disease/condition of interest and who have previously received the IMA; subjects confirmed as having the disease/condition of interest but who have never been treated by the IMA; and/or subjects confirmed as having the disease of interest who have been treated by the IMA (e.g. subjects who have been successfully and/or

unsuccessfully treated with the IMA), etc.

In an embodiment, the exemplary multiplex analyte patterns of the invention are generally "characteristic of or "associated with" functional response patterns of individuals who respond positively to exposure to the IMA. For example, in the case of administering an IMA to stimulate immune response against a tumor (e.g., a therapeutic cancer vaccine) or suppress an autoimmune response (e.g., in MS, RA, or IBD), the exemplary multiplex analyte patterns which are desired are those associated with functional response patterns of individuals who respond positively to exposure to the IMA (e.g., activated for a therapeutic cancer vaccine, or suppressed for an autimmune disease like MS, RA, and IBD). For example, one typically would not administer an IMA to individuals with healthy unimpaired immune systems. Those of skill in the art are familiar with techniques for identifying previously unknown patterns of expression associated with particular immune responses.

Briefly, this is usually accomplished by multiplexing, using known techniques, a number of biological samples from control individuals as described above. This may include both the accumulation of data from positive controls (subjects whose immune systems respond "normally" to IMA exposure, e.g. by attacking cancer cells, or by not attacking the subject's tissues in the case of autoimmune diseases, etc., who may be referred to herein as

"responders), and negative controls (e.g. subjects who have not been exposed to the IMA; or subjects whose immune systems do not respond to IMA exposure, or whose immune systems respond in a manner that is not sufficient to successfully combat the disease or condition, etc., who may be referred to herein as "non-re sponders"). In some embodiments, the control subjects may be matched based on age, gender, genotype, ethnicity, medical and health history, etc. In other embodiments, information obtained from a variety of subjects with various traits, without accounting for such factors. In the case where age, gender, etc. are taken into account, the objective is generally to use information from such a group when analyzing data obtained in an experimental subject that also fits the group's profile.

Biomarkers that appear, for example, in a majority or a significant fraction, e.g., greater than at least about 50% of the positive control samples (or possibly in higher percentages, e.g. about 60, 65, 70, 75, 80, 85, 90, 95, or even 100% of positive control samples) but which do not appear in negative control samples, may be selected as biomarkers that can be used in the multiplex assays of the invention. Alternatively, the difference between positive (responder) and negative (non-responder) controls may not be absolute in that negative controls may exhibit one or more of the biomarkers in the pattern, but in lesser amounts, e.g. in less than at least about 50% of the negative control samples (or possibly in lower percentages, e.g. about 40, 35, 30, 25, 20, 15, 10, 5, or even 0% of negative control samples). Alternatively, or in addition, the biomarkers may occur with the same frequency (e.g. may be in 40% of both positive and negative controls samples) but the amount of biomarker is significantly greater in the positive control, e.g. the amount of a biomarker is about 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, lOx or even more (e.g. 50, 100, 500 or even lOOOx) abundant in the positive control than in the negative control. Those of skill in the art will recognize that the reverse may also occur, i.e. a responder may have much less of a biomarker than a non-responder.

The total number of biomarkers in a multiplex of the invention may vary widely, but will generally be in the range of at least about 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, e.g., 15, 20, 25, 30, 35, 40, 45, or 50 or more.

In order to detect prior priming of a subject's immune system via exposure to an IMA, a biological sample is obtained from a subject to whom the IMA has been

administered or is suspected to have been administered, and at least one type of immune cell in the sample is exposed to the IMA, or a component thereof, in an in vitro setting, i.e. the IMA or components thereof is or are used to stimulate the immune cells in the sample. Those of skill in the art are familiar with obtaining biological samples, which are frequently but not always biological fluids such as blood (e.g. peripheral blood), serum, synovial fluid, saliva, urine, spinal fluid, tissues, fluids originated from disease sites etc. However, in some embodiments various tissues may be sampled or be included in the sample, e.g. biopsy tissue samples. Once obtained, the biological sample (or immune cells isolated from the biological sample) is or are exposed to the IMA in an attempt to stimulate the cells. The exposure time and conditions such as temperature and CO2 concentration are optimized using techniques that are known to those of skill in the art. In some embodiments, co-stimulants such as phytohaemaglutinin (PHA), lipopolysaccharide (LPS), antigens, superantigens such as Staphylococcal enterotoxin A (SEA) and Staphylococcal enterotoxin B (SEB), anti-CD3, anti-CD28, etc. are also added to the reaction.

Generally, in vitro stimulants may be added directly to test samples and then incubated at the desired temperature such as 37°C for a suitable period of time (e.g. generally from about 0 to about 48 hours). The stimulation condition and time may be optimized so the appropriate signal to noise ratio is obtained. The stimulants may be prepared in solution, dried-down or lyophilized forms, and be supplied in the same collection tubes or in separate tubes. Several assay controls may be included and run side by side with the test samples. In addition, negative controls are generally included, e.g. the assay without IMA stimulation or with non-specific antigens. Positive controls might be the assay plus a mitogen such as PHA, LPS, superantigens such as SEA and SEB, anti-CD3, anti-CD28, etc.

Following stimulation of the cells in the sample with an IMA (or components thereof), the functional response of the immune cells in the sample to the IMA is monitored, for example, by detecting or measuring the production of a plurality or panel of substances known to be associated with immune activation, i.e. substances known to be associated with functional immune responses. The detected molecules may be produced specifically and exclusively in response to a particular IMA (or a specific antigen present in an IMA), or they may be produced in response to several different antigens in the IMA. In addition, detected biomarker molecules may be general indicators of cell activation, examples of which include but are not limited to intracellular ATP, PCNA, and calcium ions as well as specific markers of immune cell responses such as interferon-gamma (IFN-γ), interleukin-2 (IL-2), various micro-RNA (miRNA) or messenger RNA (mRNA) molecules. The immune cells present in a biological sample which may be interrogated using the multiplex assay of the invention include but are not limited to various populations or subpopulations such as: B cells, T cells, various subpopulations of B and/or T cells, monocytes, macrophages, dendritic cells, etc. Further, profiles or panels of markers from two different cell types from a patient may be compared, e.g. T helper cells vs T regulatory cells, or T regulatory cells vs B regulatory cells, etc. Alternatively, one or more subpopulations of immune cells such as CD4+ or CD8+ may be used in the assay, or specific profiles associated subpopulations of immune cells may be measured.

Exemplary biomarker molecules which may be detected include but are not limited to: IFN-γ, cytokine profiles or panels of markers, intracellular ATP concentration or other cell functions as the result of immune cell activation, etc.; the expression products of one or a plurality of genes e.g. one or a plurality of mRNA molecules, which may generate a unique, detectable mRNA profile; one or a plurality of miRNAs may generate a unique, detectable miRNA profile; one or a plurality of protein or peptides, which may generate a unique, detectable protein/peptide profile; one or more substances that are produced by activated immune cells in response to exposure to an activating antigen such as an IMA, e.g. ATP, IFN-γ, glucose, NADH, etc. Those of skill in the art are familiar with techniques for detecting such molecules, e.g. using ELISA, nucleic acid amplification (e.g. PCR), sequencing of peptides/proteins and/or nucleic acids, HPLC analyses, gel electrophoresis and staining, mass spectrometry, etc.

Those of skill in the art will recognize that if the IMA is an immune stimulating agent and a positive functional immune response is detected, then the amount of biomarker that is detected will generally increase, although this need not always be the case, since stimulation of some responses may attenuate expression of others. In addition, if the IMA is an immune inhibitor, then the amount of biomarker that is detected will generally decrease, although this too may not always be the case, since a decrease in the amount of one substance may cause a compensatory increase in one or more other substances. These possibilities are taken into account by the prior establishment of expression patterns in control subjects, as described above.

In some embodiments, interpretation of the results obtained using the assays and methods of the invention are facilitated by provision of a "scale" or "rating system" based on results obtained with control subjects. For example, the quantity or level (i.e. the measured or detected amount) of substances produced by stimulated cells may be compared, e.g. on a scale of 1 to 10, with the amount that is typically or on average detected in negative control subjects e.g. 2 or less, and the amount for positive control subjects, e.g. 8 or more. Other intermediate response values may be assigned numeric values of e.g. 3-7, with 3 being the mildest response and 7 being the strongest. Such values may also be used as target values for treatment, e.g. for a patient who starts with a response level of 6 or 7, a reasonable target range to attain by using IMA administration might be preferably 8 or more, if no side effects are experienced from the IMA treatment. Those of skill in the art will recognize that there are many possible permutations of such numeric reference systems and many ways of expressing them, (e.g. 1-100, using decimals e.g. 1.0 to 10.0, or percentages, etc., including "all or nothing" binary systems where a simple "yes" or "no" answer is provided to the question: Is a functional immune response detected?) and all such expression systems are intended to be encompassed by the present invention. The use of such scales may facilitate automation of the assays of the invention. The scale of results may also be expressed in the format of a ratio in which the numerator is the measured response (e.g. cytokine) and the denominator is a unit of measurement of the sample (e.g. volume, # of cells, per cell, etc.).

The final step in one embodiment of the method involves interpreting and then drawing a conclusion from the results of the assay. If a pre-determined characteristic pattern of biomarkers is detected, then it is likely that the subject's immune system is responding to the IMA in a favorable or desired manner. For example, if the desired response is immune stimulation, then the subject may mount a robust immune response to the IMA. This may bode well for the subject, as they are also then likely to mount a robust immune response to the entity represented or mimicked by the IMA. Conversely, for IMAs that are designed to inhibit one or more immune responses (e.g. anti-rheumatoid arthritis or anti-MS IMAs), the pattern may indicate a cessation or lessening of one or more immune response indicators if administration is successful. On the other hand, if the pre-determined characteristic pattern of biomarkers is not detected when an increased response is desired, or if a characteristic pattern is still detected when a decreased response is desired, then administration of the IMA has not been successful, and other treatment strategies may need to be considered.

In some embodiments of the invention, the detected results are compared results that are obtained in at least one of:

i) control subjects to whom the therapeutic immune stimulant or inhibitor has not been previously administered;

ii) control subjects who do not have the disease or condition of interest;

iii) patients with the disease or condition of interest to whom the therapeutic immune stimulant or inhibitor has not been administered;

iv) patients with the disease or condition of interest who do not respond successfully to treatment with the therapeutic immune stimulant or inhibitor;

v) patients with overt symptoms of the disease or condition of interest who successfully respond to treatment with the therapeutic immune stimulant or inhibitor;

vi) patients with the disease or condition of interest who do not have symptoms of the disease or conditions, other than an abnormal immune system status, and who successfully respond to treatment with the therapeutic immune stimulant or inhibitor;

vii) patients with the disease or condition of interest who do not respond successfully to treatment with the therapeutic immune stimulant or inhibitor;

viii) the patient whose immune system is being assessed but to whom the immune stimulant or inhibitor has not been administered;

ix) the patient whose immune system is being assessed and to whom the immune stimulant or inhibitor has been administered at least once; and

x) patients who are positive for the presence of the at least one product associated with a functional immunological response to the one or more components of the therapeutic immune stimulant or inhibitor but who have not developed other symptoms of the disease or condition of interest.

In some embodiments of the invention, the multiplex assays are used for immediate monitoring in the aftermath of IMA administration, e.g. a biological sample is obtained and assessed at from about 1 or 2 to about 30 days (e.g. about 1 , 2, 5, 10, 15, 20, 25 or 30 days) after administration of the IMA. In other embodiments, the patient's response to IMA administration is monitored thereafter, e.g. at intervals of several days, weeks or months, or even indefinitely, e.g. at twice-yearly or yearly intervals, in order to monitor and assess the state of the immune system of the patient on an ongoing basis. If and when a decrease in immune response is detected (or an increase, in the case of administration of an immune inhibitor), then the IMA may be re-administered to boost (or inhibit) the immune response.

The immune system status of an individual with a number of different diseases or conditions may be determined using the methods of the invention, including but not limited to: cancer, various infectious diseases, various immune disorders, autoimmune diseases such as inflammatory bowel disease (IBD), rheumatoid arthritis (RA), multiple sclerosis (MS), etc. The immune status of an individual with any condition or constellation of symptoms that is/are generally recognized by health care professionals as being detrimental to, or potentially detrimental to, the health and well-being and/or longevity and/or quality of life of an individual, and which may be treated by administration of an IMA, may be subject to analysis using the methods and assays described herein.

The overall workflow, including IMA administration, sample collection, stimulation and cell function measurement may be adapted to an automated platform(s). For example, a gene or protein chip which binds and measures nucleic acids or proteins, respectively, may be used to detect a partem of products, etc. The isolation/selection of blood cell

subpopulations either before or after IMA stimulation might be necessary or desirable for obtaining or to maximize specific and reproducible functional immune responses.

In an exemplary embodiment, the invention provides a method of assessing the efficacy of a vaccine such as a cancer vaccine. Those of skill in the art are familiar with various cancer vaccines, which typically are comprised of antigens derived from cancer cells, or cancer cells that have been inactivated, or portions of cancer cells, etc. While the invention has been described in terms of its preferred embodiments, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the appended claims. Accordingly, the present invention should not be limited to the embodiments as described above, but should further include all modifications and equivalents thereof within the spirit and scope of the description provided herein.