WO2011153097A1 - Methods and kits for predicting a vaccine response - Google Patents

Abstract

Described herein are kits, assays and methods for predicting a subject's immune response to a vaccine. In a typical method of predicting a subject's immune response, the level of TNF-α in unstimulated B cells is measured, and high levels of TNF-α compared to a control level of TNF-α (and optionally, high levels of one or more other markers such as CD19 and CD20) are correlated with a poor immune response to a vaccine.

Description

METHODS AND KITS FOR PREDICTING A VACCINE RESPONSE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of Provisional Application Serial No. 61/351,067 filed June 3, 2010, which is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The invention relates generally to the fields of molecular genetics, molecular biology, and medicine.

BACKGROUND

[0003] Presently, there are few methods or assays existing to predict how a subject will respond to a vaccine. Using such a method or assay, a subject determined or predicted to be a poor responder could be given higher doses of a vaccine or an additional adjuvant, for example. Methods and assays for predicting a beneficial, preferably optimal, antibody response would be useful for any vaccine therapy.

SUMMARY

[0004] Described herein are kits, assays and methods for predicting a subject's immune response to a vaccine. In order to predict a subject's optimal immune response, the level of TNF-α in unstimulated B cells (which express CD 19 and CD20 markers) is measured, and a higher level of TNF-α compared to a normal or control level of TNF-α is correlated with a poor immune response to a vaccine or exogenous antigenic challenges. The kits, assays and methods described herein may be particularly useful for predicting an immune response to a vaccine in an aged subject. Aging is characterized by a low-grade chronic pro-inflammatory status referred to as inflammaging. In the experiments described below, whether or not B cells can contribute to inflammaging by secreting pro-inflammatory cytokines, such as TNF-α, and whether or not the pro-inflammatory microenvironment seen in old mice can impair B cell function in responding to stimuli such as LPS, was examined. Although not wanting to be bound by one particular theory, one hypothesis is that in aging there is a feedback mechanism of inflammatory cytokines on B cells which lowers expression of activation-induced cytidine deaminase (AID), crucial for class switch recombination (CSR). The results described herein show that unstimulated (ex vivo) B cells from old (18-25 month old) mice make significantly more TNF-α mRNA and protein than B cells from young (3-5 month old) mice, but after stimulation the old make less than young, indicating that the old B cells show an intrinsic defect in immune response. Moreover, preincubation of B cells with TNF-α before stimulation with LPS decreases both young and old B cell responses (AID, CSR), the inhibiting effect of pre-incubation being more pronounced with longer TNF-α incubation times (e.g. 12h versus lh). The results described herein also show that pre-incubation of B cells with TNF-α, before LPS stimulation, induces tristetraprolin, a physiological regulator of mRNA stability of the transcription factor E47, which is crucial for CSR and is down-regulated in old B cells. In addition, anti-TNF-α antibody increases LPS response in young and more significantly in old cultured B cells. These results altogether clearly reveal new molecular mechanisms to generate reduced antibody responses in aging, and described herein is a molecular mechanism for TNF-α-mediated down-regulation of B cell responses. The experimental results further show that in mice, inhibiting TNF-α improves a B cell response, and in human cells, higher TNF-α levels in unstimulated B cells correlates with a poor B cell response. Based on these results, TNF-α in unstimulated B cells is proposed to be a biomarker for poor B cell responses in humans.

[0005] Accordingly, described herein is a method of predicting a subject's immune response to a vaccine. The method includes: obtaining a biological sample from the subject (e.g., a human); analyzing the sample for B cells expressing TNF-α and at least one of: CD 19 and CD20; detecting a population of B cells in the sample that express TNF-α and at least one of: CD 19 and CD20; measuring TNF-α levels in the population of B cells; and correlating a level of TNF-α in the population of B cells that is higher than a control level of TNF-α with a poor immune response to the vaccine, and a level of TNF-α in the population of B cells that is equal to or lower than a control level of TNF-α with a favorable immune response to the vaccine. In the method, the subject can be aged (e.g., an aged human). In the method, the biological sample can be obtained from the subject prior to vaccination of the subject with the vaccine. In some embodiments, the subject has been immunized with a vaccine, has cancer, auto-immune disease or a viral or bacterial infection. The biological sample can be, for example, blood, sera, plasma, and saliva. The population of cells can be a population of unstimulated B cells. The step of measuring TNF-α levels in the sample can include contacting the sample with at least a first antibody directed against TNF-α. The step of measuring TNF-α levels in the population of cells can include measuring levels of TNF-α in cells expressing CD 19 and CD20. The method can further include measuring a level of at least one of: CD 19 and CD20 in the biological sample.

[0006] Also described herein is a kit for predicting a subject's immune response to a vaccine. The kit includes at least one reagent for measuring TNF-α levels in a biological sample; at least one reagent for detecting at least one B cell marker; and instructions for use. The at least one reagent for measuring TNF-α levels in a biological sample can be, for example, an antibody directed against TNF-α. The at least one reagent for detecting at least one B cell marker can be, for example, an antibody directed against CD 19, and/or an antibody directed against CD20.

[0007] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0008] As used herein, "protein" and "polypeptide" are used synonymously to mean any peptide- linked chain of amino acids, regardless of length or post-translational modification, e.g., glycosylation or phosphorylation.

[0009] By the phrase "overexpression" is meant increased levels of mRNA and/or protein expression as compared to normal tissue.

[00010] By the term "gene" is meant a nucleic acid molecule that codes for a particular mRNA and protein.

[00011] As used herein, a "nucleic acid" or a "nucleic acid molecule" means a chain of two or more nucleotides such as RNA (ribonucleic acid) and DNA (deoxyribonucl eic acid).

[00012] An aged subject (e.g., human) is one who displays physiological signs of aging.

[00013] The terms "patient," "subject" and "individual" are used interchangeably herein, and mean a mammalian (e.g., human) subject to be treated and/or to obtain a biological sample from.

[00014] As used herein, "bind," "binds," or "interacts with" means that one molecule recognizes and adheres to a particular second molecule in a sample or organism, but does not substantially recognize or adhere to other structurally unrelated molecules in the sample. Generally, a first molecule that "specifically binds" a second molecule has a binding affinity greater than about 10⁸ to 10¹² moles/liter for that second molecule and involves precise "hand-in- a-glove" docking interactions that can be covalent and noncovalent (hydrogen bonding, hydrophobic, ionic, and van der waals).

[00015] The term "labeled," with regard to a nucleic acid, protein, probe or antibody, is intended to encompass direct labeling of the nucleic acid, protein, probe or antibody by coupling (i.e., physically or chemically linking) a detectable substance (detectable agent) to the nucleic acid, protein, probe or antibody.

[00016] When referring to a nucleic acid molecule or polypeptide, the term "native" refers to a naturally-occurring (e.g., a WT) nucleic acid or polypeptide.

[00017] The term "sample" is used herein in its broadest sense. A sample including polynucleotides, peptides, antibodies and the like may include a bodily fluid, a soluble fraction of a cell preparation or media in which cells were grown, genomic DNA, R A or cDNA, a cell, a tissue, skin, hair and the like. Examples of samples include saliva, serum, tissue, skin, CSF, blood, plasma, brain (autopsy or biopsy), and epithelial cells from skin, mouth, muscle or other bodily tissue.

[00018] As used herein, the term "treatment" is defined as the application or administration of a therapeutic agent to a patient or subject, or application or administration of the therapeutic agent to an isolated tissue or cell line from a patient or subject, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease, or the predisposition toward disease.

[00019] As used herein, the term "safe and effective amount" refers to the quantity of a component which is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this invention. By "therapeutically effective amount" is meant an amount of a composition as described herein effective to yield the desired therapeutic response. The specific safe and effective amount or therapeutically effective amount will vary with such factors as the particular condition being treated, the physical condition of the patient, the type of mammal or animal being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed and the structure of the compounds or its derivatives. [00020] Although kits, assays and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable kits, assays and methods are described below. All publications, patent applications, and patents mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. The particular embodiments discussed below are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

[00021] FIG. 1 shows increased intrinsic TNF-α levels in old B cells correlate with lower LPS response. A. Purified splenic B cells (10⁶ cells/ml) were stimulated with LPS (1 μg/ml) for 1, 3, 6, 12, 24, 48 or 96 h, or they were left unstimulated. The mRNA was extracted from unstimulated B cells (tO) cells and after 1 -96 h in culture and qPCR performed. Vertical columns represent the ACt values of TNF-α mRNA expression normalized to GAPDH±SE. Values are compared to the unstimulated young B cell value, taken as 1. Fifteen pairs of young (white) and old (black) mice were analyzed for all time-points with the exception of 12 h of stimulation, where only 4 pairs of young and old mice were compared. The differences between young and old, as determined by the two-tailed Student's t test, are indicated in the figure (* p<0.05; ** p<0,01). The difference between young 0 and young 6 h of stimulation as well as old 0 and old 6 h of stimulation is significant at p<0.01. B. Total protein extracts from 20x10⁶ purified unstimulated or 24 h LPS-stimulated splenic B cells from young and old mice were prepared and loaded in WB. A representative WB is shown out of 4 performed. Densitometric analyses (arbitrary units) of TNF-α protein expression, normalized to β-actmiSE from the 4 pairs of young and old mice were performed. Values were: unstimulated young, 10 (arbitrarily assigned); unstimulated old, 35±4; stimulated young, 45±3; stimulated old, 19±3. The difference between young and old mice (both unstimulated and LPS-stimulated) is significant at p<0.05, as determined by the two-tailed Student's t test.

[00022] FIG. 2 shows TNF-α down-regulates LPS-induced B cell responses. Purified splenic B cells (10⁶cells/ml) were stimulated with LPS (1 μg/ml ) or with TNF-α (100 ng/ml), for 1 or 7 days. Alternatively, B cells were pre-incubated with TNF-α before the stimulation with LPS for 1,3 or 12 h. Then, the mRNA was extracted and qPCR performed to evaluate expression of E47 mRNA at day 1 or AID mRNA at day 7. Supernatants were also collected at day 7 to evaluate IgG or IgA secretion by ELISA. Vertical columns represent the ACt values of E47 or AID mRNA expression normalized to GAPDH±SE, as well as IgG or IgA secretion in culture supernatants, from 10 pairs of young (white) and old (black) mice. Values are compared to the LPS-stimulated young B cell value, taken as 100. Bars outside the bold box: the difference between young and old B cells is significant at p<0.01 for LPS-induced E47, AID, IgG and IgA, as well as for TNF-α- induced AID, as determined by the two-tailed Student's t test. For TNF-α- induced AID, the difference between young and old is partially due to a 2-fold lower NF-κΒ, For TNF-α-induced IgA, the difference between young and old B cells is significant at p<0.05. Bars inside the bold box: the difference between young and old B cells is significant at p<0.01 when B cells are stimulated with LPS and TNF-α together and at p<0.05 for all the other stimuli; the difference between young B cells stimulated with TNF-α and LPS together and young B cells pre-incubated with TNF-α 3h and then stimulated with LPS is significant at p<0.01; the difference between old B cells stimulated with TNF-α and LPS together and old B cells pre- incubated with TNF-α 3h and then stimulated with LPS is significant at p<0.05 (not significant for AID).

[00023] FIG. 3 shows AID in stimulated B cells is negatively correlated with their unstimulated levels of TNF-α. TNF-α and AID mRNA were obtained as described above. Nineteen mice were evaluated. Pearson's correlation^:::-0.776, p<0.0001 (two-tailed). Old mice: squares; young mice, X. The four old mice in the "young" category (for high AID and low TNF- a) were also "young-like" for other characteristics, i.e. pre-B cell phenotype in the bone marrow.

[00024] FIG. 4 shows anti-TNF-α antibody increases LPS response in young and more significantly in old cultured B cells. Purified splenic B cells (10⁶cells/ml) were stimulated with LPS (1 μg/ml) for 7 days. Anti-TNF antibody (5-100 ng/ml) was added to B cell cultures at the beginning of culture (A). Alternatively, it was added 3 times (beginning of culture, day 2 and day 4) (B). Then, the mRNA was extracted and qPCR performed to evaluate expression of AID mRNA. Vertical columns represent the ACt values of AID mRNA expression normalized to GAPDH±SE, Values are compared to the young B cell value, taken as 100. Four pairs of young (white) and old (black) mice were analyzed. The differences between young and old, as determined by the two-tailed Student's t test, are indicated in the figure (* p<0.05; ** p<0.01; ns, not significant). [00025] FIG. 5 shows TNF-α stimulation induces TTP in splenic B cells. Purified splenic B cells (10⁶ cells/ml) were stimulated with LPS (1 μg/mI), alone or together with TNF-α, for 3 h. Alternatively, B cells were pre-incubated with TNF-α before the stimulation with LPS for 1 or 2 h. Then, the mRNA was extracted and qPCR performed to evaluate expression of TTP. Vertical columns represent the ACt values of TTP mRNA expression normalized to GAPDHiSE. Values are compared to the 3 h LPS-stimulated young B cell value, taken as 100. Five pairs of young (white) and old (black) mice were analyzed. TNF-α alone induced 10% lower levels of TTP mRNA in both young and old B cells, as compared to LPS alone. As controls for the combined TNF/LPS cultures, no significant differences were observed among LPS 1 , 2 or 3 h in both young and old B cells (91% and 101% versus 100% in the young and 255% and 347% versus 310% in the old). The differences between young and old, as determined by the two-tailed Student's t test, are indicated in the figure (* p<0.05; ** p<0.01).

[00026] FIG. 6 is a model for differential activation of CSR in B cells from young versus old individuals. Unstimulated (ex vivo) B cells from old individuals (2) make significantly more TNF-α mRNA than B cells from younger ones (1). In the presence of endogenous TNF-α in the old (2) and exogenous TNF-α added to the young (3), TNF-a,/CpG (which is the equivalent of LPS in mice) induces TTP which down-regulates the stability of E47. Arrows indicate relative values between young (1) and old (2) or young (1) and TNF-α added to young (3) and may reflect "threshold" values which need to be reached to affect further downstream effector functions.

[00027] FIG. 7 shows anti-TNF-α antibody in vivo increases LPS-induced AID mRNA expression in B cells from old but not from young mice.

[00028] FIG. 8 shows AI D in stimulated B cells is negatively correlated with levels of TNF-α in unstimulated B cells. Peripheral blood was taken from 25 young individuals (20-64 years) and 17 elderly individuals (>65 years), before (tO) and 28 days (t28) after vaccination with the seasonal flu vaccine (seasons 2009-2010 and 2010-2011). To evaluate TNF-α mRNA expression by qPCR, mRNA was isolated from B cells at tO, To evaluate AID mRNA expression by qPCR, mRNA was obtained from B cell cultures stimulated with the influenza vaccine at tO and t28. Results are expressed as fold-increase after vaccination (AID qPCR at t28/t0). A 2 fold-increase was arbitrarily defined as a positive response, based on the response seen in young subjects. Previous results have shown that this AID response positively correlates with a good anti- influenza vaccine response. Young individuals: X, Elderly individuals: ●, The p value is indicated below the horizontal axis and was evaluated by the Student's t test (two tailed).

DETAILED DESCRIPTION

[00029] Described herein are assays, methods and kits for predicting how a subject will respond to a vaccine. In a method or assay for predicting how a subject will respond to a vaccine, a blood sample is obtained from a subject prior to vaccination, TNFa levels in unstimulated B cells in the sample are measured with a control antibody of the same isotype but not specific for TNF-α, which measures the background level. Unstimulated B cells from younger individuals show no or minimal level of detection, whereas older individuals give at least 4 to 5 -fold more positive cells with fluorescence intracellular staining. Preliminary data indicate that 30% B cells from an elderly individual are positive for intracellular TNF and in mice 20% B cells from old and 4% from young are positive. Higher levels of TNF are correlated with a poor immune response, thus indicating that the subject will not respond favorably to the vaccine. The experimental results described herein reveal a novel biomarker for B cell function, i.e., TNF alpha in unstimulated B cells from the blood. B cell function is measured by AID which has previously been associated with beneficial B cell function (class switch from IgM to IgG), In general, a method or assay of predicting how a subject will respond to a vaccine includes staining B cells in the blood (i.e., blood obtained from a human subject) with either surface CD 19 or CD20 (markers for all human B cells) and intracellular TNFa. These levels of intracellular TNF-α will negatively correlate with AID (B cell response to antigen or vaccine) and with beneficial antibody titers to a vaccine. In this embodiment, antibodies to CD 19 or CD20 would each have a fluorochrome different from the one attached to TNF-α. In some methods of predicting how a subject will respond to a vaccine, TNF-α niRNA is measured by qPCR, for example. A typical kit for predicting how a subject will respond to a vaccine would include at least a first antibody directed against TNFa (e.g., PE (red)-labeled anti-TNFa). A kit can further include a second antibody to a B cell marker such as CD 19 or CD20 (e.g. FITC (green)-labeled anti B cell antibody directed against CD20 or CD 19). In such an embodiment, B cells having high levels of TNF would be yellow, and this would indicate both red and green present in the same cell. Use of TNFa as a biomarker would be applicable to discern which people, especially but not only the elderly, would be poor responders to a vaccine and could thereby be given higher doses of vaccine or an additional adjuvant, which at this time is not recommended for the entire population. Described herein are data pertaining to the seasonal vaccine response, showing a negative correlation between the initial levels of TNF in B cells and a good response. These also apply to other vaccine responses, e.g. the pandemic H1N1. Also the in vitro response of B cells to a polyclonal stimulus such as the TLR9 agonist CpG is negatively correlated with the initial levels of TNF in unstimulated B cells. Therefore, described herein is a method of predicting a beneficial/optimal antibody response which would be useful for any vaccine therapy. The assays, methods and kits described herein may find particular use, for example, in clinical settings to assist physicians who are immunizing people with vaccines. In a typical embodiment, the assays, methods and kits described herein are used with elderly subjects and other populations who are at risk of having a lowered immune response (e.g., immune deficiencies).

Biological Methods

[00030] Methods involving conventional molecular biology techniques are described herein. Such techniques are generally known in the art and are described in detail in methodology treatises such as Molecular Cloning: A Laboratory Manual, 3rd ed,, vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001 ; and Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 992 (with periodic updates). Immunology techniques are generally known in the art and are described in detail in methodology treatises such as Advances in Immunology, volume 93, ed. Frederick W. Alt, Academic Press, Burlington, MA, 2007; Making and Using Antibodies: A Practical Handbook, eds. Gary C. Howard and Matthew R. Kaser, CRC Press, Boca Raton, Fl, 2006; Medical Immunology, 6^th ed., edited by Gabriel Virella, Informa Healthcare Press, London, England, 2007; and Harlow and Lane ANTIBODIES: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988. Methods of Detecting a Subject's Immune Response to a Vaccine

[00031] Described herein are assay and methods of predicting a subject's (e.g., human) immune response to a vaccine using one or more biomarkers to identify levels of TNF-α in B cells. Typically, the subject is one who will receive a vaccine, or for whom vaccine administration is being considered. A method of predicting a subject's immune response to a vaccine can include: obtaining a biological sample from the subject; analyzing the sample for B cells expressing TNF-α and at least one of CD 19 and CD20; detecting a population of B cells in the sample that express TNF-α and at least one of CD 19 and CD20; measuring TNF-α levels in the population of B cells; and correlating a level of TNF-α in the population of B cells that is higher than a control level of TNF-α with a poor immune response to the vaccine, and a level of TNF-α in the population of B cells that is equal to or lower than a control level of TNF-α with a favorable immune response to the vaccine.

[00032] Any suitable biological sample can be used in the methods. Examples of biological samples include blood, saliva, serum, plasma. The steps of the method can be performed using any suitable protocol or assay. Examples of suitable assays include enzyme-linked immunosorbent assays (ELISAs), Western blots, flow cytometry assays, immunofluorescence assays, qPCR, microarray analysis, etc.

[00033] Any suitable reagent for detecting expression in B cells (e.g., CD19 or CD20-positive cells) of TNF-α and quantitating levels of TNF-α and/or proteins regulating or regulated by TNF- α (e.g. TTP) can be used. In a typical embodiment, an antibody (e.g., monoclonal, polyclonal, Fab fragment, etc.) specific for the protein whose presence and expression level is being analyzed is used. In some embodiments, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the kits, assays and methods described herein. Antibodies specific for TNF-α (or other proteins described herein) may be provided in a kit that incorporates at least one of these procedures to detect TNFα expression in unstimulated B cells. The kit may contain other components, packaging, instructions, or other material to aid the detection of the protein and use of the kit. [00034] Whether or not TNFa is overexpressed in B cells (e.g., CD 19 or CD20-positive) can be determined by comparing the level of TNFa expression in the sample to a baseline level (also known as a control level) and to the young responder sample for the expression of TNFa. Unstimulated B cells from younger or the occasional older individual with a good immune response will show no or minimal level of TNF-α detection, whereas older or the occasional poorly responding young individual will give at least 4 to 5-fold more positive cells with fluorescence intracellular staining.

Kits

[00035] Described herein are kits for predicting a subject's (e.g., human) immune response to a vaccine. A typical kit for predicting a subject's (e.g., human) immune response to a vaccine includes at least a first reagent for detecting the presence of and quantitating the level of TNFa protein in a biological sample (e.g., blood, plasma, serum) from the subject, and instructions for use. Typically, a kit further includes a second reagent for detecting the presence of an additional B cell marker, e.g., CD 19, CD20. In one embodiment, a kit includes a monoclonal or polyclonal antibody to TNFa, a detectable label, and instructions for use. The at least one reagent can be, for example, a TNFa -specific antibody. The at least one second reagent can be, for example, a CD19-specific antibody and/or CD20-specific antibody. A kit may include a well plate to carry the mixture of the different reagents, as well as one or more washing buffers. Optionally, kits may also contain one or more of the following: containers which include positive controls, containers which include negative controls, photographs or images of representative examples of positive results and photographs or images of representative examples of negative results.

EXAMPLES

[00036] The present invention is further illustrated by the following specific examples. The examples are provided for illustration only and should not be construed as limiting the scope of the invention in any way.

Example 1 - A molecular mechanism for TNF-α-mediated down-regulation of B cell responses [00037] Aging is characterized by a low-grade chronic pro-inflammatory status referred to as inflammaging. in the experiments described herein, whether B cells can contribute to inflammaging by secreting pro-inflammatory cytokines, such as TNF-α, and whether adding a pro-inflammatory cytokine can impair B cell function in responding to stimuli such as LPS, was investigated. A hypothesis is that in aging there is a feedback mechanism of inflammatory cytokines that act in an autocrine way on B cells, lowering the expression of AID, crucial for class switch recombination (CSR), The results described herein show that unstimulated (ex vivo) B cells from old mice make significantly more TNF-α mRNA and protein than B cells from young mice, but after stimulation the old make less than young. Moreover, pre-incubation of B cells with additional TNF-α before stimulation with LPS decreases both young and old B cell responses. B cell function was able to be restored by adding anti-TNF-α antibody before LPS in young and more significantly in old cultured B cells. To address a molecular mechanism, it was found that pre-incubation of B cells with TNF-α, before LPS stimulation, induces tristetraprolin, a physiological regulator of mRNA stability of the transcription factor E47, crucial for CSR. These results reveal new molecular mechanisms which contribute to reduced antibody responses in aging.

[00038] Inflammation is part of the protective, biological/immunological response to infections which is crucial for survival. At the same time, however, many pathologic conditions such as autoimmune diseases are sustained by the continuous activation of the inflammatory process. Inflammation plays an important role in the pathogenesis of many diseases typical of old age. Enhanced 1L-6 and TNF-α plasma levels have been associated with functional disability and mortality of the elderly. Aging is characterized by a disregulation of inflammatory and antiinflammatory networks, which results in a low grade chronic pro-inflammatory status called inflammaging (Franceschi et al., Experimental gerontology 35:879-896, 2000). The age-related increase in circulating inflammatory mediators such as cytokines and acute phase proteins are markers of the low-grade inflammation observed with aging. Age-related alterations in responses to immune stimulation, for example chronic T cell stimulation with viruses such as CMV, also contribute to low-grade inflammation by increasing the level of pro-inflammatory mediators such as TNF-α (Sansoni et al., Experimental gerontology 43:61-65, 2008). Although production of these pro-inflammatory cytokines is thought to be in part a macrophage-medi ated event, it is clear that other cell types, including stroma (i.e. epithelium and endothelium and fat), as well as T cells, produce these mediators in vivo. The possible contribution of B cells to this process has not yet been pursued and is the subject of the experiments described herein. B cells, through the secretion of cytokines such as TNF-α, have been shown to contribute to immunity against infectious agents, such as Toxoplasma gondii, Heligomosomoides polygyrus or Pneumocystis carinii by promoting expansion and differentiation of primary and memory Thl or Th2 cells. Moreover, the possible contribution of B cells and/or antigen presenting cells to the inflammatory process supports their pathogenic role in a wide range of autoimmune diseases. Previous results showed that the molecular basis for E47, and hence AID (acti vation-ind uced cytidine deaminase), and class switch recombination (CSR) of immunoglobulin (Ig) being lower in aged individuals, mice (Frasca et al., J Immunol 172:2155-2162, 2004) and humans (Frasca et a!., J Immunol 180:2741-2746, 2008), is due to decreased E47 mRNA stability (Frasca et al., J Immunol 175:6633-6644, 2005). This reduced E47 mRNA stability with age is mediated, at least in part, by binding of tristetraprolin (TTP) to the 3'UTR (18). As TTP also regulates inflammatory cytokine (TNF-α, IL-6) mRNAs similarly, it is hypothesized herein that in aging, there may be a feedback mechanism of inflammatory cytokines to attempt to down-regulate further expression of these, and that in B cells this process inadvertently also downregulates E47 and an optimal B cell immune response, including Ig CSR and AID. In the experiments described herein, two questions were addressed: 1) whether B cells can contribute to the increased inflammatory response in aging (inflammaging) by secreting more proinflammatory cytokines i.e. TNF-α than B cells from young mice and 2) whether the proinflammatory microenvironment seen in old mice, and specifically TNF-α, can reduce the ability of B cells to respond to stimuli such as LPS. The results described herein reveal new molecular mechanisms which may contribute to reduced antibody responses in aging.

Materials and Methods

[00039] Mice, definition of phenotype: Male and female young (2-4 mo of age) and old (24- 27 mo of age) BALB/c mice were purchased from the National Institutes of Aging and maintained in an AAALAC-certified facility. Mice were acclimated for at least 7 days before sacrifice. Mice with evidence of disease were not used in these studies. Most of the experiments were done with females. A few experiments were done with males. No significant differences between females and males were seen. All studies adhered to the principles of laboratory animal care guidelines and were IACUC approved. Bone marrow cells were counted and used for flow cytometry to evaluate the percentages of pro-B/pre-B cells, as previously described (Sherwood et a!., J Immunol 161 :4472-4475, 998). A moderately depleted phenotype corresponded to 25- 80% loss in pre-B cells. A severely depleted old mouse corresponded to 80% or more loss in pre- B cells and 50% loss in pro-B cells, as compared to young (Van der Put et al., Experimental gerontology 38: 1 137-1147, 2003). Except for Fig. 3, the old mice used in the experiments herein had the moderately or severely depleted phenotypes (which represent 80-90% of mice at 24-27 months of age) (Van der Put et al., J Immunol 173:818-827, 2004).

[00040] Splenic B cell enrichment: B cells were isolated from the spleens of young and old mice. Briefly, cells were washed twice with medium (RPMI 1640; Invitrogen Life Technologies) and incubated for 20 min at 4°C with anti-CD 19 Microbeads (Miltenyi Biotec), according to the MiniMacs protocol (Miltenyi Biotec) (20 μl Microbeads + 80 μl PBS, every 10⁷ cells). Cells were then purified using magnetic columns. At the end of the purification procedure, cells were 80-85% CD19-positive by cytofluorimetric analysis. After the isolation procedure was ended, cells were maintained in PBS for 3 h at 4°C to minimize potential effects of antiCD19 antibodies on B cell activation. In the experiments where macrophages were removed, B cells were isolated from the non-adherent fraction.

[00041] Cell culture: B cells were cultured in complete medium (RPMI 1640, supplemented with 10% FCS, 10 μg/ml gentamicin, 2 x 10-5 M 2-ME, and 2 mM L-glutamine). FCS was certified to be endotoxin-free. B cells (10⁶/ml) were stimulated in 24-well culture plates for different time-points (indicated in each figure) with 1 μg/ml of LPS (SIGM A). Alternatively, B cells were stimulated in 24-well culture plates for different time points with 100 ng/ml of TNF-α (PMC3014 Biosource). In some experiments, a purified rat anti-mouse TNF-α antibody (551225 BD Pharmingen) was added to the LPS-stimulated B cell cultures at the concentration of 1-50 ng/ml. The antibody was either added once at the beginning of the culture, or it was added every day. At the end of each stimulation time, B cells were counted in trypan blue to evaluate viability which was found comparable in cultures of young and old B cells (within 10%).

[00042] Preparation of total cell lysates: Before protein extraction, splenic B cells were counted using trypan blue. Cells were harvested and centrifuged in a 5415C Eppendorf micro fuge (2,000 rpm, 5 min). Total cell lysates were obtained as follows. The pellet of cultured B cells was resuspended in M-PER (Mammalian Protein Extraction Reagent, Thermo Scientific), according to the manufacturer's instructions. The amount of protein extracted from the same number of B cells is highly reproducible (90%) from one experiment to another in both young and old mice.

[00043] Western blotting (WB): For the evaluation of specific proteins in splenic B cells, protein extracts at equal protein concentration were denatured and then electro-transferred onto nitrocellulose filters. Filters were incubated with the following primary antibodies: rabbit anti- TNF-α (1/1000 diluted, Cell Signaling 3707), or with mouse anti- -actin (1/8000 diluted, SIGMA A4700) as loading control, in PBS-Tween 20 containing 5% milk. After overnight incubation with the primary antibodies, immunoblots were incubated with the following secondary antibodies: HRP-conjugated goat polyclonal anti-rabbit (1/50,000 diluted, 1 1 1-035- 003; Jackson ImmunoResearch Laboratories) or HRP-conjugated goat anti-mouse (1/16,000 diluted, 610-1319; Rockland) for 1.5 h at 4°C. Membranes were developed by enzyme chemiluminescence and exposed to CL-XPosure Film (Pierce). Films were scanned and analyzed using the Alpha] mager Enhanced Resolution Gel Documentation & Analysis System (Alpha Innotech, San Leandro CA) and images were quantitated using the AlphaEaseFC 32-bit software.

[00044] RNA extraction and cDNA preparation: n RNA was extracted from stimulated B cells (10⁶/ml) using the μMACS mRNA isolation kit (Miltenyi Biotec), according to the manufacturer's protocol, eluted into 75 μl of preheated elution buffer, and stored at -80°C until use. Ten μl of mRNA (approximately 10 ng) were used as template for cDNA synthesis in the reverse transcriptase reaction.

[00045] Quantitative PCR (qPCR): Two μl of cDNA were added to 10 μl of Taqman Master mix (Applied Biosystems no.4369016), 1 μl of forward primer, 1 μl of reverse primer, and deionized water in a final volume of 20 μl. Reactions were conducted in MicroAmp 96-well plates (Applied Biosystems, ABI no.N8010560), and run in the ABI 7300 machine. Calculations were made with ABI software. Briefly, we determined the cycle number at which transcripts reached a significant threshold (Ct) for E47, AID, TTP and GAPDH as control. A value for the amount of the target gene, relative to GAPDH, was calculated and expressed as Ct. Primers for PCR amplification of TNF-α, E47, AID, TTP and GAPDH were the following (all from ABI): Mm01 161290 (TNF-α), MmOl 17557 (Tcfe2/E47), Mm00507774 (AID), Mm00457144 (TTP), Mn99999915 (GAPDH).

[00046] Enzyme-linked immunosorbent assay (EL1SA): TNF-α concentration in serum, plasma and culture supernatants was determined by a mouse quantitative ELISA kit (eBioscience 88- 7324-22), according to the manufacturer's instructions. IgG and IgA concentration in collected supernatants of cultured B cells was determined by mouse quantitative ELISA kits (Bethyl Labs), according to the manufacturer's instructions.

Results

[00047] Increased intrinsic TNF-a levels in old B cells correlate with lo wer LPS response. In order to test the possible contribution of B cells to inflammaging, TNF-α mRNA expression and protein release by B cells from young and old mice in vitro stimulated with LPS for different time-points or left unstimulated was investigated. It was already shown that B cells from young mice can secrete TNF-α in response to in vivo infections or to LPS injection. No TNF-α production, however, was shown after in vitro stimulation of B cells from young mice with LPS from F. tularensis or E. coli. Currently there are no data on how much TNF-α is made in B cells from old versus young mice or on whether TNF-α could be released from unstimulated B cells from young and old mice. Results in Fig. 1A show that old unstimulated B cells make 5-fold more TNF-α mRNA than young B cells. The expression of TNF-α mRNA in cultures of LPS- stimulated B cells from young mice increases with the time of stimulation, the peak being at 6 h of stimulation, and then decreases. In old B cells, conversely, TNF-α mRNA expression progressively decreases with the time of stimulation and at 6 h is half the level observed in young B cells. After 6 and 12 h of LPS stimulation, B cells from old mice make not only significantly lower (absolute) amounts of TNF-α than young B cells, but also the stimulation index is even more severely impaired. At later time-points, the expression of TNF-α mRNA further decreases in both young and old B cells, but differences are not significant. Thus, it appears that ex vivo old B cells are already stimulated and are refractory to further stimulation.

[00048] TNF-α protein expression in unstimulated old B cells is 3-4-fold higher than in young B cells (Fig. IB), but after 24 h stimulation with LPS is half the value of young B cells, as evaluated by WB (also confirmed by ELISA in 10 pairs of young and old cultures, 120±11 pg/ml in young, 40±5 pg/ml in old). It was found in a series of preliminary results that the peak of TNF-α protein release in culture supernatants is between 6 and 24 h stimulation for both young and old, as evaluated by ELIS A. The level of expression of TNF-α mR A and protein in B cells was half of that of LPS-stimulated monocyte/macrophage cultures which are known as one of the primary cells making TNF-α. Both TNF-α mRNA and protein expression kinetics for monocyte/macrophages were similar to those of B cells (e.g., at 6 h young was increased 10X but old was decreased 2X).

[00049] TNF-a down-regulates LPS-induced B cell responses: Experiments were performed to show a direct relationship between the inhibitory capability of TNF-α on B cell function in young and old B cells, as well as IgA. B cells from young and old mice were stimulated with LPS and TNF-α together. LPS is used as a canonical TLR/microbial mimic stimulus. Alternatively, cultures were pre-incubated with TNF-α before the stimulation with LPS for 1, 3 or 12 h, over a total time of culture of 24 h (E47 mRNA) or 7 days (AID mRNA, IgG, IgA). Results in Fig. 2 show that stimulation with LPS and TNF-α together (given at the same time, left bars, bold box) induced a stronger response in both young and old B cells, as compared to LPS or TNF-α alone, but the response to LPS or TNF-α alone (bars outside the bold box) as well as to both LPS+TNF-α are reduced in old B cells. Pre-incubation of B cell cultures with TNF-α before the stimulation with LPS decreases both young and old B cell responses, the inhibiting effect of pre-incubation being more pronounced with longer TNF-α incubation times. The peak for E47 mRNA expression is at 24 h of LPS stimulation; at 12 h, the amount of E47 mRNA is half of that seen at 24 h; therefore ½ of the decrease we see at TNF 12 h/LPS is due to the suboptimal LPS response. These results are consistent with the hypothesis that in old B cells CSR is down-regulated by TNF-α, in particular by B cell-derived (autocrine) TNF-α. In support of this hypothesis, Fig. 3 shows that AID is negatively correlated with the levels of TNF-α in unstimulated B cells (p=0.0001) and that most old B cells are low for AID and high for initial TNF-α. This figure also emphasizes the point that not all old mice are identical and need to be analyzed individually, or first screened for markers for bone marrow (Chorinchath et al., J Immunol 156:1525-1530, 1996) before pooling cells.

[00050] Anti-TNF-a antibody increases LPS response in young and more significantly in old cultured B cells. Because it was shown above that pre-incubation with TNF-α inhibits LPS- induced B cell responses, whether an anti-TNF-α antibody added at the beginning of culture together with LPS would reverse the negative effects of TNF-α was tested. Results in Fig. 4A show that the anti-TNF-α antibody was able to increase in a dose-dependent manner AID mRNA expression in B cell cultures from old but not from young mice. AID mRNA expression was further increased not only in old but also in young B cell cultures when the antibody was added 3 times instead of once, at the beginning of culture and at day 2 and 4 (Fig. 4B). This effect was more pronounced in old B cells as compared to young B cells. The anti-TNF-α antibody did not have any effect when added to the cultures in the absence of LPS stimulation (not shown). These results suggest that the antibody was able to counteract the effects of high TNF-α levels of old B cells from the beginning of culture, thus allowing old B cells to respond to LPS in a similar way as that of young B cells.

[00051] TNF-a pre-incubation induces more TTP in old splenic B cells: In order to pursue a potential mechanism of action for TNF-α inhibiting B cell function, whether the pre-incubation of B cells with TNF-α before the stimulation with LPS can induce TTP and therefore be responsible for the reduced response observed in both young and old B cells was investigated. The hypothesis is that there is a feedback mechanism of inflammatory cytokines, especially autocrine, such as TNF-α for B cells which reduces these cytokines to a new challenge stimulus via decreased mRNA stability. This mechanism also decreases E47, AID and CSR when B cell stimulation is induced (e.g. by TLR/Ig/costimulatory mechanisms). Results in Fig. 5 show that LPS, alone or together with TNF-α, induces TTP mRNA expression in both young and old B cells, the levels in old being higher than in young B cells, as previously shown (Frasca et al., J Immunol 179:918-927, 2007). Pre-incubation of B cells with TNF-α before the stimulation with LPS increases TTP mRNA expression in both young and old B cells. Again, the effect of TNF-α is more pronounced when TNF-α is added for increased times before addition of LPS. In two preliminary experiments we showed that the degradation of TNF-α mRNA is greater in old than in young LPS-stimulated B cells.

[00052] In conclusion, results herein clearly indicate that B cells make TNF-α and therefore contribute to the systemic modification of the cellular microenvironment typical of old age. The model shown in FIG. 6 emphasizes that the increased autocrine TNF-α released by aged B cells impairs their function. The results show that unstimulated B cells from old mice make more TNF-α mRNA and protein than B cells from young mice, but after stimulation the old make less than the young, i.e. old B cells are pre-activated/hyporeactive. If B cells are pre-incubated with TNF-α before stimulation with LPS, the responses of both young and old B cells responses are inhibited. B cells can in fact be induced by TNF-α to secrete IgA but not IgG, and this response is down-regulated in old B cells, emphasizing the importance of unique stimuli for a complete evaluation of the aged B cell response. The inhibiting effect of pre-incubation with TNF-α is more pronounced with longer TNF-α incubation times. This inhibitory effect correlates with the induction of TTP, a physiological regulator of mRNA stability of the transcription factor E47, crucial for CSR, downregulated in old B cells. Finally, anti-TNF-α antibody increases the LPS response in young and more significantly in old cultured B cells. Taken together, these results show that inflammation and B cell function are directly related in old mice and should help to better understand the mechanisms leading to reduced antibody responses in aging. These will also help to design new possibilities for novel therapeutic approaches for age-related immune diseases.

Example 2 -- TNF-α is a biomarker for poor B cell response in humans

[00053] Referring to FIGS. 1-5 these data show that old murine B cells make more TNF-α and that they respond less well because of this (TN F-a was inhibited in culture with antibody to TNF-α and a good response was restored). FIGS. 7 and 8 show, in particular, data in mouse (Fig. 7, in vivo anti- TNF-α treatment) and human cells (Fig. 8), showing higher TNF-α in unstimulated cells correlates with poor specific B cell response induced by the influenza vaccine (AID - activation -induced cytidine deaminase, a gold standard biomarker for good response). Referring to FIG. 7, anti-TNF-α antibody (100 μg/mouse/day) was injected intraperitoneally for 3 consecutive days. Purified splenic B cells were stimulated with LPS for 7 days. Then, the mRNA was extracted and qPCR performed to evaluate expression of AID mRNA. Vertical columns represent the ACt values (qPCR) of AID mRNA expression normalized to GAPDH±SE. Values are compared to the young untreated B cell value, taken as 100. Two pairs of young and old mice were analyzed. Referring to FIG. 8, peripheral blood was taken from 25 young individuals (20-64 years) and 17 elderly individuals (>65 years), before (tO) and 28 days (t28) after vaccination with the season flu vaccine (seasons 2009-2010 and 2010-201 1). To evaluate TNF-α mRNA expression by qPCR, mRNA was isolated from B cells at tO. To evaluate AID mRNA expression by qPCR, mRNA was obtained from B cell cultures stimulated with the influenza vaccine at tO and t28. Results are expressed as fold-increase after vaccination (AID qPCR at t28/t0). A 2 fold-increase was arbitrarily defined as a positive response, based on the response seen in young subjects. Applicants have previously see that this AID response positively correlates with a good anti- influenza vaccine response. Young individuals: X, Elderly individuals: ·. The p value is indicated below the horizontal axis and was evaluated by the Student's t test (two tailed).

[00054] These experiments provide proof of principle in mice that inhibiting TNF-α in vivo can improve a B cell response and that TNF-α is a biomarker for poor B cell response in humans - that the unstimulated B cells make more TNF-α.

Other Embodiments

[0100] Any improvement may be made in part or all of the kits, assays and method steps. All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended to illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. Any statement herein as to the nature or benefits of the invention or of the preferred embodiments is not intended to be limiting, and the appended claims should not be deemed to be limited by such statements. More generally, no language in the specification should be construed as indicating any non-claimed element as being essential to the practice of the invention. This invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contraindicated by context.

Claims

What is claimed is:

1. A method of predicting a subject's immune response to a vaccine, the method comprising:

obtaining a biological sample from the subject;

analyzing the sample for B cells expressing TNF-α and at least one of: CD 19 and CD20; detecting a population of B cells in the sample that express TNF-α and at least one of: CD 19 and CD20;

measuring TNF-α levels in the population of B cells; and

correlating a level of TNF-α in the population of B cells that is higher than a control level of TNF-α with a poor immune response to the vaccine, and a level of TNF-α in the population of B cells that is equal to or lower than a control level of TNF-α with a favorable immune response to the vaccine.

2. The method of claim 1, wherein the subject is a human.

3. The method of claim 1 , wherein the subject is aged.

4. The method of claim 1, wherein the method comprises obtaining the biological sample from the subject prior to vaccination of the subject with the vaccine.

5. The method of claim 1, wherein the subject has been immunized with a vaccine, has cancer, auto-immune disease or a viral or bacterial infection.

6. The method of claim 1 , wherein the biological sample is selected from the group consisting of: blood, sera, plasma, and saliva.

7. The method of claim 1 , wherein the population of cells is a population of unstimulated B cells.

8. The method of claim 1 , wherein the step of measuring TNF-α levels in the sample comprises contacting the sample with at least a first antibody directed against TNF-α.

9. The method of claim 1 , wherein measuring TNF-α levels in the population of cells comprises measuring levels of TNF-α in cells expressing CD 19 and CD20.

10. The method of claim 1 , further comprising measuring a level of at least one of: CD 19 and CD20 in the biological sample.

11. A kit for predicting a subject's immune response to a vaccine, the kit comprising:

at least one reagent for measuring TNF-a levels in a biological sample;

at least one reagent for detecting at least one B cell marker; and

instructions for use.

12. The kit of claim 11 , wherein the at least one reagent for measuring TNF-α levels in a biological sample is an antibody directed against TNF-α.

13. The kit of claim 11 , wherein the at least one reagent for detecting at least one B cell marker is an antibody directed against CD 19.

14. The kit of claim 11 , wherein the at least one reagent for detecting at least one B cell marker is an antibody directed against CD20.