WO2008157518A1

WO2008157518A1 - Biomarkers of influenza vaccine response

Info

Publication number: WO2008157518A1
Application number: PCT/US2008/067167
Authority: WO
Inventors: Richard R. Drake; Gaurav Basu; Yuping Deng; Stefan Gravenstein
Original assignee: Drake Richard R; Gaurav Basu; Yuping Deng; Stefan Gravenstein
Priority date: 2007-06-15
Filing date: 2008-06-16
Publication date: 2008-12-24

Abstract

The present invention discloses biomarkers and biomarker combinations that have prognostic value as predictors of the immune response of an individual. In particular, the biomarkers of this invention are useful to predict and monitor the immune response of elderly individuals to influenza vaccine. The biomarkers of this invention are also useful to predict the immune response of any individual to influenza vaccine prior to the administration of the vaccine.

Description

BIOMARKERS OF INFLUENZA VACCINE RESPONSE

BACKGROUND OF THE INVENTION

Vaccination is the most cost-effective approach to prevent infections. Quantitative and qualitative analysis of vaccine immune response is a critical component for testing new vaccines. Also, early detection of the infectious agent and clinical diagnosis is crucial for treatment and crisis management. An exemplary case is influenza infections, which cause serious international public health problems annually that are particularly severe in elderly people, who account for more than 90% of influenza mortality. Moreover, the efficacy of current vaccines is only about 30-40% among the elderly population and up to 60% of the vaccinated elderly people acquire influenza infection. The reduced influenza vaccine efficacy and the increased morbidity and mortality with age are largely attributed to immune senescence. The antibody response to vaccine is known to decline with age (Webster, R. G. (2000) Immunity to influenza in the elderly. Vaccine. 18(16): 1686; Falsey, A.R., CK. Cunningham, W.H. Barker, R. W. Kouides, J.B. Yuen, M. Menegus, L.B. Weiner, CA.

Bonville, and R.F. Betts. (1995) Respiratory syncytial virus and influenza A infections in the hospitalized elderly. J Infect Dis. 172(2): 389) but the mechanism of the decline remains elusive.

For some pathogens it is the host immune response, rather than the pathogen itself, that is responsible for the morbidity and mortality from viral infections. For instance, influenza morbidity and mortality is realized primarily in older adults and caused by the immune response to influenza virus, as the virus only rarely escapes the respiratory tract and the viral pneumonia is atypically a terminal event. Specifically, elevated levels of cytokines are associated with influenza symptoms, including fever and headache (Hayden, F. G., R. Fritz, M.C. Lobo, W. Alvord, W. Strober, and S. E. Straus. 1998. Local and systemic cytokine responses during experimental human influenza A virus infection. Relation to symptom formation and host defense. Journal of Clinical Investigation. 101(3): 643.; Gentile, D., W. Doyle, T. Whiteside, P. Fireman, F. G. Hayden, and D. Skoner. 1998. Increased interleukin-6 levels in nasal lavage samples following experimental influenza A virus infection. Clinical & Diagnostic Laboratory Immunology . 5(5): 604). Although influenza virus can denude bronchial columnar epithelium, animal studies indicate the more extensive pulmonary damage is induced by reactive oxidative species (ROS) released from activated lung macrophages and may well be the most important cause of influenza mortality (Akaike, T., Y. Nogu, S. Ijiri, and K. Setoguchi. 1996. Pathogenesis of influenza virus-induced pneumonia: Involvment of both nitric oxide and oxygen radicals. Proceedings of the National Academy of Sciences of the United States of America. 93: 2448). The host immune response and viral pathogenicity are quite variable between pathogens. For example, the Spanish influenza pathogen (A/H1N1) of 1918 probably resulted in viral pathology while the 1957 (A/H2N2) and 1968 (A/H3N2) pandemics had more immune-related pathology; meanwhile no A/H1N1 strain since has caused as much morbidity or mortality as the H3N2 variants of the last 30 years. Clinically, influenza B is associated with milder illness compared to influenza A, indicating that the host immune response to these two types of virus are different. The differences in immune response will depend on the nuances of host cell pathogen recognition by macrophages/monocytes and dendritic cells at the early phase of immune response. In other words, the stimulation of the innate immune response (the 'first responder cell system') is a critically important determinant of immune-related pathogenicity.

In addition, bioengineering of the influenza virus to generate viral strains never previously seen in the human population remains a looming bioterrorism threat (Krug, R.M. (2003) The potential use of influenza virus as an agent for bioterrorism. Antiviral Res., 57, 147-150). These strains could also be engineered to be drug-resistant to current anti- influenza drugs. Introduction of such strains could have devastating consequences, essentially creating super-carriers of infection that would spread rapidly through the immune-naϊve human population.

The effectiveness of any diagnostic test depends on its specificity and selectivity, or the relative ratio of true positive, true negative, false positive and false negative diagnoses. Methods of increasing the percent of true positive and true negative diagnoses for any condition are desirable medical goals. Given the complexity of the genetic and molecular alterations that occur in each immune response, the expression patterns reflecting these complex changes, in addition to individual molecular changes themselves, may also hold vital information in predicting and diagnosing the immune response of an individual.

Proteomic research looks at the expression profile of multiple proteins within a complex test sample. Clinical proteomics can identify differentially expressed biomarkers, by comparing the proteomic profiles of differing physiological states, which can be used for diagnosis and therapeutic intervention. In addition to immunoassays, proteomic research has traditionally involved two-dimensional gel electrophoresis to detect protein expression differences in body fluid specimens between groups (Srinivas, P. R., et al, Clin Chem. 47: 1901-1911 (2001); Adam, B.L., et al, Proteomics 1 : 1264-1270 (2001)). Although two- dimensional polyacrylamide gel electrophoresis (2D-PAGE) has been the classical approach in exploring the proteome for separation and detection of differences in protein expression, it has its limitations in that it is cumbersome, labor intensive, suffers reproducibility problems, and is not easily applied in the clinical setting.

One recent technological advance in facilitating protein profiling of complex biologic mixtures is mass spectrometry (MS). Mass spectrometry-based proteomics have made it possible to detect and quantitate individual proteins and multiple proteins simultaneously while analyzing the entire proteome of test samples. The matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF)-MS method, in which protein solutions are typically premixed with a matrix and dried on a passive surface, may provide direct identification of each individual protein present in a complex biological test sample. After characterizing the protein peaks in a biological sample, the sample may be further analyzed to generate a protein profile or protein signature.

Similarly, surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) detects proteins bound to a protein chip array and facilitates the identification of a signature protein profile (Kuwata, H., et al, Biochem. Biophys. Res. Commun. 245:764-773 (1998); Merchant, M. et al., Electrophoresis 21: 1164-1177 (2000)). MALDI and SELDI technology have numerous advantages over 2D-PAGE: they are much faster, have a high throughput capability, require orders of magnitude lower amounts of the protein sample, can effectively resolve low and higher mass proteins (500-100,000 Da), and are directly applicable for assay development.

There is a need for new methods of improving the efficacy of influenza vaccinations. There is also a need for improved methods of proteomic analysis that will distinguish influenza vaccine responders from non-responders. The present invention is directed to these and other important ends.

BRIEF SUMMARY OF THE INVENTION

The invention is directed to detecting and quantifying immune responses to vaccination by proteomic profiling. The invention provides advancements to aid in assessing vaccine success and in guiding re-immunization of patients with insufficient immunity following infection. The invention provides biomarkers that have been profiled and characterized from clinical samples such as serum and nasal swabs. These biomarkers are useful for proteomic profiling for monitoring vaccine response and early detection/diagnosis of infection. The invention also provides sensitive methods and kits that may be used as an aid in the diagnosis of the immune response by detecting one or more of the biomarkers. The detection and measurement of the biomarkers of the invention, alone or in combination, in test samples, may provide information that may be correlated with a prognosis of an individual's immune response. The biomarkers may be characterized by molecular weight. The biomarkers may be resolved from other proteins in a sample by, e.g., chromatographic separation coupled with mass spectrometry, or by traditional immunoassays. In some embodiments, the method of resolution may involve Isobaric Tag for Relative and Absolute Quantitation, or iTRAQ analysis.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a differentially expressed peak observed by MALDI analysis between all the responders and all the non- responders and elderly responders at m/z 4417 (p<0.01).

FIG. 2 shows a differentially expressed peak observed by MALDI analysis between the young responders and elderly responders at m/z 2865 (p<0.01).

FIG. 3 shows a heat map of old responders longitudinal serum samples days 0,4,7,14,21.

FIG. 4 shows a heat map of old non-responders longitudinal serum samples days 0,4,7,14,21.

FIG. 5 shows a heat map of young responders longitudinal serum samples days 0,4,7,14,21.

FIG. 6 shows a heat map of young non-responders longitudinal serum samples days 0,4,7,14,21.

FIG. 7 shows single subject analysis young responder (serum) longitudinal samples. FIG. 8 shows a heat map of single subject analysis young responder (serum) longitudinal samples.

FIG. 9 shows single subject analysis young nonresponder (serum) longitudinal samples.

FIG. 10 shows a heat map of single subject analysis young nonresponder (serum) longitudinal samples.

FIG. 11 shows iTRAQ reporter ions and example ratios determined following MS/MS.

FIG. 12 shows serum/plasma iTRAQ analysis of influenza vaccine responses.

FIG. 13 shows plasma pooled samples of Trivalent Split Influenza vaccine recipients.

FIGS. 14-47 show MALDI results of responders and nonresponders.

FIGS. 48-49 show iTRAQ results from day 0 serum samples.

FIGS. 50-51 show iTRAQ results from day 28 serum samples.

FIGS. 52-53 show the results of iTRAQ analysis, listing the identities and molecular weights of the most differentially expressed proteins from the day 0 and day 28 samples.

FIGS. 54-55 show differential plasma protein expressions for four proteins from the day 0 sample.

DETAILED DESCRIPTION OF THE INVENTION

For the purposes of promoting an understanding of the principles of the invention, reference will now be made to preferred embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alteration and further modifications of the invention, and such further applications of the principles of the invention as illustrated herein, being contemplated as would normally occur to one skilled in the art to which the invention relates.

In an aspect of the invention, differentially expressed proteins among responders and non responders of elderly and young subjects have been identified.

An isotope labeling strategy that can characterize lectins captured from serum glycoprotein fractions involves the use of amine-reactive iTRAQ reagents (Applied Biosystems) (Ross PL, Huang YN, Marchese JN, Williamson B, Parker K, Hattan S, Khainovski N, Pillai S, Dey S, Daniels S, Purkayastha S, Juhasz P, Martin F, Bartlet-Jones M, He F, Jacobson A, and Pappin DJ. Multiplexed protein quantitation in saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. MoI Cell Proteomics 2004; 3: 1154- 1169; Wiese S, Reidegeld KA, Meyer HE, Warscheid B. Protein labeling by iTRAQ: A new tool for quantitative mass spectrometry in proteome research. Proteomics 2007; 7:340-350) to differentially label peptides with stable isotopes. As shown in Figures 11 and 12, iTRAQ reagents consist of three functional groups: 1) a reactive N-hydroxysuccinimide (ΝHS) ester, which coval entry links the reagent to peptides by reacting with primary amines on lysine side chains and on the amino terminal ends, 2) a mass balance group, and 3) an isotopically labeled reporter group. The latter two groups constitute an isobaric tag that can be varied with stable isotopes to generate four chemically equivalent forms of the iTRAQ reagent. Within the mass spectrometer during the second phase of peptide dissociation, the reporter groups are released. Because of the stable isotope tags incorporated, each generates an ion signal with a distinct m/z that may be used to quantify the amounts of peptide to which it was attached. Thus, four samples may be compared simultaneously and differences in the amount of a given peptide determined quantitatively. The full-length proteins may then be identified using any method known in the art, including but not limited to using SEQUEST and MASCOT database search algorithms.

The iTRAQ reagents offer several advantages over previously used sulfhydryl- reactive ICAT reagents. 1) Since amine groups are more abundant than cysteinyl sulfhydryl groups, iTRAQ enables quantitative analysis of more peptides per protein relative to ICAT, thereby improving protein coverage and the accuracy of quantification. This feature also enables quantification of proteins that lack cysteines. 2) Up to four distinctly labeled samples may be analyzed in a single MS experiment; it is expected that new kits with 8 isobaric tags will soon be available. This requires less sample and generally involves fewer chromatographic steps than with binary isotope methods (like ICAT or ¹⁸O). 3) Overall quantification should be improved since detection of a reporter ion occurs during MS/MS and does not require reconstruction of ion chromatograms.

The invention provides biomarkers that are predictive of immune response. The technique employs molecular profiling approaches that have extremely high sensitivity and specificity to detect and identify low concentrations of differentially expressed biomarkers in a biofluid or test sample, including but not limited to, serum and plasma. Using iTRAQ methods, the inventors have identified protein biomarkers that are differentially expressed between responding and non-responding vaccine recipients. The inventors have utilized standard hemagglutination assays to determine the phenotypic status of test subjects, i.e., whether the test subjects were responders or non-responders to influenza immunization.

The term "biomarker" as used herein refers to an organic biomolecule, the presence of which in a sample may be useful to determine the phenotypic status of a subject (e.g., whether an individual has or has not generated an immune response to a vaccine), or may be predictive of a physiological outcome (e.g., whether an individual is likely to generate an immune response to a vaccine (a responder) or is unlikely to do so (a non-responder)). Presently preferred biomarkers according to the invention include proteins, protein fragments and peptides. The biomarkers may be differentially present in a biological sample or fluid, such as but not limited to blood plasma or serum. The biomarkers may be isolated by any method known in the art, based on their mass, their binding characteristics, or other physicochemical characteristics. For example, a test sample comprising the biomarkers may be subject to chromatographic fractionation, as described herein, and subject to further separation by, e.g., acrylamide gel electrophoresis. Knowledge of the identity of a biomarker also allows its isolation by immunoaffinity chromatography, as well as its detection by immunodiagnostic or other methods. As used herein, the term "detecting" includes determining the presence, the absence, or a combination thereof, of one or more biomarkers.

It is also contemplated to measure the quantity of a biomarker which, for example, is differentially expressed depending on phenotypic status. The quantity of a biomarker may be represented by the peak intensity as identified by mass spectrometry, for example, or concentration of the biomarker, and may be quantified by any method known in the art. A biomarker is considered to be differentially present between different phenotypic statuses if the mean or median expression level of the biomarker in the different groups differs in a statistically significant manner. Common tests for statistical significance include, among others, t-test, ANOVA, Kruskal-Wallis, Wilcoxon, Mann-Whitney and odds ratio. A single biomarker, or a combination of biomarkers that is or are differentially expressed, provides a measure of the relative risk or probability that a subject belongs to one phenotypic status or another, assuming statistical significance. Therefore, such biomarkers are useful as biomarkers for disease (diagnostics), therapeutic effectiveness of a drug (theranostics), drug toxicity, and predicting and identifying an immune response. A biomarker according to the present invention will typically differentially be present in test samples from individuals who respond to vaccine relative to those with little or no vaccine response. However, some biomarkers, while not being differentially expressed between two or more phenotypic classes may, nevertheless, be considered as biomarkers in accordance with the invention to the extent that they aid in delineating subsets of groups in a classification tree.

In accordance with the invention, at least one biomarker may be detected. It is to be understood, and is described herein, that one or more biomarkers may be detected and subsequently analyzed, including several or all of the biomarkers identified. Further, it is to be understood that the failure to detect one or more of the biomarkers of the invention, or the detection thereof at levels or quantities that may correlate with immune response, may be useful and desirable as a means of selecting the most favorable vaccine regimen, and that the same forms a contemplated aspect of the invention.

The invention provides biomarkers that may be used to distinguish individuals with differential immune responses. The biomarkers may be characterized by mass-to-charge ratio as determined by mass spectrometry, by the shape of their spectral peak in time-of- flight mass spectrometry and by their binding characteristics to adsorbent surfaces. These characteristics provide one method to determine whether a particular detected biomolecule is a biomarker of this invention. These characteristics represent inherent characteristics of the biomarkers and not process limitations in the manner in which the biomarkers are discriminated. It will be appreciated that once a biomarker according to the invention is identified, for example by other methods known in the art of detecting and/or quantifying mass spectrometry, that biomarker may be employed. The biomarkers of the invention may be characterized by their mass-to-charge ratio as determined by mass spectrometry. The mass-to-charge ratio of each biomarker is provided as "M." Thus, for example, M2454.00 has a measured mass-to-charge ratio of 2454.00. The mass-to-charge ratios are determined from mass spectra generated on any appropriate commercially available mass spectrometer. Preferably, the instrument will have a mass accuracy of about +/- 0.3 percent. Additionally, the instrument will preferably have a mass resolution of about 400 to 1000 m/dm, where m is mass and dm is the mass spectral peak width at 0.5 peak height. The mass-to-charge ratio of the biomarkers is determined using appropriate commercially available software. Preferably, the software will assign a mass-to- charge ratio to a biomarker by clustering the mass-to-charge ratios of the same peaks from all the spectra analyzed, as determined by the mass spectrometer, taking the maximum and minimum mass-to-charge-ratio in the cluster, and dividing by two.

In one aspect, methods are provided for measuring the amount of biomarker present in a sample in a clinical setting. Standard clinical methods for measuring the amount of biomarker present in a sample include, but are not limited to, ELISA, microsphere-based immunoassay, lateral flow test strips, antibody based dot blots or Westerns. Antibodies which may be used in any of these immunoassays include, but are not limited to, monoclonal or polyclonal antibodies to any of the proteins listed in Tables 52 and 53 or as disclosed herein. Also provided by the invention are kits for assessing immune response comprising antibodies specific for at least one biomarker and reagents for conducting an immunoassay. The kits may comprise reagents for conducting immunoassays, such as ELISA or microsphere-based immunoassays. The kits may further comprise reagents for lateral flow test strips. At least a portion of the antibodies for the protein biomarkers may be embedded in a lateral flow test strip. The kits may further comprise a finger-stick device. Such devices may be modifications of commercially available devices for assessing biomarkers, such as the Quikread CRP finger-prick device (Orion Diagnostica, Finland) which measures the level of C-reactive protein.

Also provided are kits for detecting and measuring the amount of biomarker in a patient. Said kits may comprise antibodies specific for any one of a plurality of biomarkers and reagents for conducting an immunoassay. Said kits may further comprise antibodies specific for any number of a plurality of biomarkers and reagents for conducting an immunoassay. There may be more than one set of reagents present in the kit. For example, if the kit is intended for use with just ELISA, than only reagents for ELISA will be present. If the kit is intended for use with both ELISA and a microsphere-based immunoassay, then reagents for both ELISA and microsphere-based immunoassay will be present. If the kit is intended for use with ELISA, a microsphere-based immunoassay, and with lateral flow test strips, then reagents for all three immunoassays will be present, etc.

If lateral flow test strips are to be used to conduct the immunoassay, then the antibodies within the kit will be embedded in the lateral flow test strips. Also, the kits may be intended for use with a combination of immunoassays. So, for example, the antibodies for any one of the biomarkers may be present in the kit as free antibodies, but a portion of them may also be embedded in a lateral flow test strip.

In another aspect, the invention provides methods for detecting, quantitating, or detecting and quantitating potential immune response to influenza vaccine prior to the administration of the vaccine, comprising detecting and/or quantitating at least one biomarker in a biological sample from a subject and correlating said detection and/or quantitation with a determination of likelihood of immune response to influenza vaccine, said at least one biomarker comprising one or any combination of a plurality of the proteins in a group of proteins, selected from the group consisting of the proteins listed in Figure 52, Serum Amyloid A protein, and Alpha 1 -Acid Glycoprotein 1.

In another aspect, the invention provides methods for detecting, quantitating, or detecting and quantitating potential immune response to influenza vaccine following the administration of the vaccine, comprising detecting and/or quantitating at least one biomarker in a biological sample from a subject and correlating said detection and/or quantitation with a determination of immune response to said influenza vaccine, said at least one biomarker comprising one or any combination of a plurality of the proteins in a group of proteins, selected from the group consisting of the proteins listed in Figure 53 and Alpha- 1 -Acid Glycoprotein 1.

In another aspect, the invention provides methods for detecting, quantitating, or detecting and quantitating potential immune response to influenza vaccine prior to the administration of the vaccine, comprising detecting and/or quantitating at least one biomarker in a biological sample from a subject and correlating said detection and/or quantitation with a determination of likelihood of immune response to influenza vaccine, said at least one biomarker comprising one or any combination of a plurality of proteins selected from the group consisting of: Apolipoprotein A-I, Alpha 2-HS Glycoprotein, Vitronectin, Serum Amyloid A protein, Alpha 1-Acid Glycoprotein, Ankyrin repeat domain-containing protein 20Al, Laminin beta-4 chain, Human phospholipase C-etalb, Proteoglycan-4 precursor, Filamin-A, Laminin gamma- 1 sub-unit, Platelet Basic Protein, and Alpha 1-Acid Glycoprotein 1.

In another aspect, the invention provides methods for detecting, quantitating, or detecting and quantitating potential immune response to influenza vaccine prior to the administration of the vaccine, comprising detecting and/or quantitating at least one biomarker in a biological sample from a subject and correlating said detection and/or quantitation with a determination of likelihood of immune response to influenza vaccine, said at least one biomarker comprising one or any combination of a plurality of the proteins selected from the group consisting of: Apolipoprotein A-I, Alpha 2-HS Glycoprotein, Serum Amyloid A protein, and Alpha 1 -Acid Glycoprotein 1. In another aspect, the invention provides methods for detecting, quantitating, or detecting and quantitating potential immune response to influenza vaccine prior to the administration of the vaccine, comprising detecting and/or quantitating at least one biomarker in a biological sample from a subject and correlating said detection and/or quantitation with a determination of likelihood of immune response to influenza vaccine, said at least one biomarker comprising one or any combination of a plurality of the proteins selected from the group consisting of: Apolipoprotein A-I, Vitronectin, Filamin-A, and Laminin gamma- 1 sub- unit.

In another aspect, the invention provides methods for detecting, quantitating, or detecting and quantitating potential immune response to influenza vaccine prior to the administration of the vaccine is provided for subjects who are very young. Because every vaccination carries with it an inherent risk of adverse effects, and because such adverse effects may result in especially severe reactions and medical problems in babies, infants, and young children, a method of determining whether a particular child's immune system will respond to an influenza vaccine prior to the administration of that vaccine would reduce the number of flu immunizations that are needlessly given to non-responder children, thus reducing the overall number of adverse reactions in children to the flu vaccine.

In another aspect, the invention provides methods for detecting, quantitating, or detecting and quantitating immune response to influenza vaccine following the administration of the vaccine, comprising detecting and/or quantitating at least one biomarker in a biological sample from a subject and correlating said detection and/or quantitation with a determination of immune response to said influenza vaccine, said at least one biomarker comprising one or any combination of a plurality of the proteins selected from the group consisting of: Fibrinogen alpha chain, Fibrinogen gamma chain, Apolipoprotein A-IV, Fibrinogen beta chain, Apolipoprotein A-I, Alpha 2-HS Glycoprotein, Kininogen-1, Histidine rich glycoprotein, Vitronectin, Human cGMP inhibited 3 '5 '-cyclic phosphodiesterase, Serum Amyloid A protein, Complement C3, Platelet Basic Protein, and Alpha 1-Acid Glycoprotein 1.

In another aspect, the invention provides methods for detecting, quantitating, or detecting and quantitating immune response to influenza vaccine following the administration of the vaccine, comprising detecting and/or quantitating at least one biomarker in a biological sample from a subject and correlating said detection and/or quantitation with a determination of immune response to said influenza vaccine, said at least one biomarker comprising one or any combination of a plurality of the proteins selected from the group consisting of: Fibrinogen alpha chain, Fibrinogen beta chain, Apoliprotein A-I, and Histidine rich glycoprotein.

In another aspect, the invention provides kits comprising a substrate comprising an adsorbent attached thereto, wherein the adsorbent is capable of retaining at least one biomarker, and instructions to detect the at least one biomarker by contacting a biological sample with the adsorbent and detecting the at least one biomarker retained by the adsorbent. In another aspect, the invention provides methods comprising detecting at least one biomarker from a group of biomarkers by mass spectrometry, immunoassay, iTRAQ analysis, ELISA, microsphere-based immunoassay, or with lateral flow test strips.

In another aspect, the invention provides a method for detecting and quantitating immune response to influenza vaccination in a subject, comprising detecting and quantitating at least one biomarker in a biological sample from said subject following the administration of influenza vaccine; and correlating said detection and quantitation with a determination of immune response in said subject to said vaccine. The method may comprise detecting and quantitating a plurality of said biomarkers. The plurality of biomarkers may comprise at least two biomarkers, at least three biomarkers, at least four biomarkers or may comprise more than four biomarkers. In another aspect, the method may comprise determining the absence of any of said biomarkers or of a plurality of said biomarkers.

The method of detecting and quantitating at least one biomarker in a biological sample from a subject is, in one aspect, performed according to the invention by mass spectrometry, for example, wherein the mass spectrometry is laser desorption mass spectrometry, for example, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. In an aspect, the laser desorption/ionization mass spectrometry comprises the steps of separating biomarkers from other proteins in the biological sample using chromatography; and detecting the biomarkers with a MALDI-TOF mass spectrometer. In another aspect, the laser desorption/ionization mass spectrometry comprises the steps of providing a substrate comprising an adsorbent attached thereto, contacting the biological sample with the adsorbent, desorbing and ionizing the biomarkers from the substrate, and detecting the desorbed/ionized biomarkers with a MALDI-TOF mass spectrometer. The adsorbent may be a cation exchange adsorbent. In another aspect, the mass spectrometry may be tandem mass spectrometry, and may preferably comprise detecting the sequences and quantities of the biomarkers using Isobaric Tag for Relative and Absolute Quantitation (iTRAQ) analysis.

The method of detecting and quantitating at least one biomarker in a biological sample from a subject is, in one aspect, performed according to the invention by an immunoassay. The immunoassay of the invention may take any form known in the art, including RadioImmunoAssay (RIA), or Enzyme Linked Immunosorbent Assay (ELISA), with the latter being presently preferred. The biological sample may be any biological sample, including without limitation whole blood, serum, and plasma.

In an aspect, the method of detecting and quantitating at least one biomarker in a biological sample from a subject according to the invention further comprises managing subject treatment based on the determination of immune response in the subject. The invention may further comprise detecting and quantitating the at least one biomarker after subject treatment.

In yet another aspect of the invention is provided a method for detecting potential immune response to influenza vaccination in a subject, comprising detecting at least one biomarker in a biological sample from said subject prior to the administration of influenza vaccine; and correlating said detection with a determination of potential immune response in said subject to said vaccine. The method may comprise detecting a plurality of said biomarkers. The plurality of biomarkers may comprise at least two biomarkers, at least three biomarkers, at least four biomarkers, or may comprise more than four biomarkers. In another aspect, the method may comprise determining the absence of any of said biomarkers or of a plurality of said biomarkers.

The method of detecting at least one biomarker in a biological sample from a subject is, in one aspect, performed according to the invention by mass spectrometry, for example, wherein the mass spectrometry is laser desorption mass spectrometry, for example, matrix- assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. In an aspect, the laser desorption/ionization mass spectrometry comprises the steps of separating biomarkers from other proteins in the biological sample using chromatography; and detecting the biomarkers with a MALDI-TOF mass spectrometer. In another aspect, the laser desorption/ionization mass spectrometry comprises the steps of providing a substrate comprising an adsorbent attached thereto, contacting the biological sample with the adsorbent, desorbing and ionizing the biomarkers from the substrate, and detecting the des orbed/ionized biomarkers with a MALDI-TOF mass spectrometer. The adsorbent may be a cation exchange adsorbent. In another aspect, the mass spectrometry may be tandem mass spectrometry, and may preferably comprise detecting the sequences and quantities of the biomarkers using Isobaric Tag for Relative and Absolute Quantitation (iTRAQ) analysis.

The method of detecting at least one biomarker in a biological sample from a subject is, in one aspect, performed according to the invention by an immunoassay. The immunoassay of the invention may take any form known in the art, including

RadioImmunoAssay (RIA), or Enzyme Linked Immunosorbent Assay (ELISA), with the latter being presently preferred. The biological sample may be any biological sample, including without limitation whole blood, serum, and plasma.

In an aspect, the method of detecting at least one biomarker in a biological sample from a subject according to the invention further comprises managing subject treatment based on the determination of immune response in the subject. The invention may further comprise detecting the at least one biomarker after subject treatment.

In another aspect, the invention provides a method for predicting whether or not a subject is or is not likely to have an immune response to an influenza vaccination, comprising detecting or quantitating at least one biomarker in a biological sample from said subject prior to the administration of influenza vaccine, and correlating said detection or quantitation with a determination of immune response or lack thereof in said subject to said vaccine. The method may comprise detecting or quantitating one or a plurality of biomarkers. The plurality of biomarkers may comprise at least two biomarkers, at least three biomarkers, at least four biomarkers or may comprise more than four biomarkers. In another aspect, the method may comprise determining the absence of any of said biomarkers or of a plurality of said biomarkers. In yet another aspect, the presence or absence of one or more biomarkers may be detected, while one or more other biomarkers may be quantitiated. In a further aspect, the method of the invention further comprises managing subject treatment based on the prediction of immune response to vaccine.

EXAMPLES

Biomarkers of Vaccine Responders and Non-Responders

Materials and Methods

Two cohorts were used in this study. In one study from 2004-2005, 114 healthy elderly (ages 60-94) and 60 healthy young adults (ages 21-40) were recruited. Plasma samples were collected on days 0, 7, 14 and 28 after trivalent split influenza vaccination. Sera was obtained on day 0 (pre-vaccination) and on day 28 (post vaccination) from the same individual for antibody response by Hemagglutination assay. Based on antibody response towards the vaccine and age, four sub-groups (n=10 per group) were selected: young responders, elderly responders, young non-responders and elderly non-responders. In a second study, healthy elderly (n=43, ages >_65) and healthy young adults (n=48, ages 21-40) were recruited between 2005-2006. Serum samples were collected on days 0, 4, 7, 14 and 21 after trivalent split influenza vaccination. Immune response to vaccination was based on the four-fold rise in antibody titers based on Hemagglutination assays. The entire sample set was analyzed by MALDI-TOF analysis using WCX bead capture as described in the next section.

Each serum/plasma sample was incubated with weak cation (WCX) beads (Bruker Daltonics). Proteins bound to the beads were eluted, mixed with alpha-cyano-4- hydroxycinnamic acid (HCCA) matrix (1: 1) and 1 microliter was robotically spotted onto a pre-structured sample support (600 um AnchorChip™ target, Bruker Daltonics) and allowed to air dry at room temperature. The samples were analyzed using a UltraFlex MALDI- TOF/TOF mass spectrometer (Bruker Daltonics) equipped with a pulsed ion extraction ion source. Each spectrum was detected in linear positive mode and was externally calibrated using a mixture of peptide standards. For iTRAQ analysis, four plasma sub-groups (n=10 per group) were used: young responders, elderly responders, young non-responders and elderly non-responders. WCX bound protein eluates were labeled with iTRAQ reagents as per manufacturers instructions (Applied Biosystems). Sequencing and quantitation of isobarically tagged peptides were done using a Thermo LTQ LC(C18)-ESI tandem mass spectrometer.

Plasma and Serum MALDI-TOF and Data Analysis

All selected serum or plasma samples were processed in duplicate, along with 16 quality control serum samples as reference controls For each analysis, 20 μl of serum was incubated with 10 μl of MB-WCX (weak-cation) magnetic beads for 10 minutes on the

ClinProt robotic platform as per the manufacturers' instructions (Bruker Daltonics, Billerica, MA). Unbound proteins were discarded, and each sample washed twice in binding buffer. Bound proteins were eluted as per the manufacturers instructions, and spotted in duplicate on an AnchorChip sample target platform (384 spots), mixed 1 : 10 with CHCA (alpha-4-cyano- hydroxycinnamic acid) in an acetone ethanol mixture of 1 :2. Samples were assayed randomly, and blinded to the operator. Profile spectra were acquired from an average of 400 laser shots in the linear mode on an Ultraflex MALDI-TOF instrument (Bruker Daltonics). ClinProt software version 2.0 (Bruker Daltonics) was used to baseline subtract, normalize spectra (using total ion current) and determine peak m/z values and intensities in the mass range of 2-15kDa. To align the spectra a mass window of 0.5% was employed. A k nearest neighbor genetic algorithm contained in this software suite was used to identify statistically significant differences in protein peaks in the groups analyzed. After each model was generated, a leave-one out cross validation process was done within the software. The cross- validated values were used for determining the sensitivity and specificity of the classifications.

Plasma and Serum MALDI-TOF Analysis Using Trypsin Bead Digest

The sample sets were fractionated using weak cation exchange magnetic beads (WCX-MB) manually on a magnetic bead separator. For each set, 20 μl of plasma was mixed with 40 μl MB-WCX along with 40 μl binding buffer for 5 minutes. To remove unbound proteins beads were washed three times with 200 μl WCX-MB wash solution. The bound proteins were eluted by using 10 μl elution solution and diluted with 8 μl HPLC grade water. The eluted proteins were then trypsinized using paramagnetic immobilized trypsin, ENZYBEADS (Agro-Bio Corp.). For each sample, an equivalent amount of protein (8 μg) was measured by Bradford protein assay. The samples were reduced (using DTT) and alkylated (using iodoacetamide) at 60° C for 60 minutes and then digested using the paramagnetic trypsin beads for 30 minutes at 37° C. Samples were tapped after an interval of 10 minutes during the 30 minute incubation period at 37° C. The digested peptides were recaptured and concentrated with HIC-C 18 paramagnetic beads as per the manufacturer's instructions. The unbound proteins were removed by washing the beads with wash solution twice. The bound proteins were eluted using 50% acetonitrile (elution buffer) and tryptic peptides were then spotted in duplicate on a target (600 um ANCHORCHIP Plate, Bruker Daltonics). The tryptic peptides were mixed with alpha-cyano-4-hydroxycinnamic acid (CHCA) matrix before spotting. The matrix was comprised of 0.002 g CHCA in 1 ml Ethanol and 2 ml Acetone. Tryptic peptides were mixed in 1 :2 ratios with the matrix and lμl was robotically spotted onto a pre-structured sample support (600 um ANCHORCHIP target, Bruker Daltonics). The peptides were analyzed by an ULTRAFLEX III MALDI-TOF/TOF instrument (Bruker Daltonics, Germany) in linear and reflectron modes. The peptides were identified using the MASCOT search engine after fragmentation using LIFT mode of the ULTRAFLEX III MALDI-TOF/TOF.

Comparative iTRAQ analysis of Influenza vaccine responders and non-responders

The method used involved front-end capture of pooled plasma or serum proteins using a weak cation exchange (WCX) resin linked to magnetic beads (supplied by Bruker Daltonics). Four pools of plasma or serum (n = 10/pool) samples from young responders/non-responders (ages 21-40) and elderly responders/non-responders (age >65) were generated based on antibody titers against viral hemagluttinnin. Responders had at least a 4-fold rise in hemagluttinnin antibody titers compared with their baseline pre-vaccination titers. Each of the proteins bound, and subsequently eluted from the WCX bead, were then digested with trypsin, and the resulting peptides were labeled with a specific iTRAQ isotope tag (114/115/116/117 isobaric mass tags) as per the manufacturers directions (Applied Biosystems). These four tagged peptide groups were mixed together, and then bound to a strong-cation exchange (SCX) resin to remove unreacted mass tags and organics. The peptides were released from the SCX resin in 800 mM ammonium formate. For tandem mass spectrometry analysis, each sample was diluted 1: 10 in an acetonitrile/TFA solution and separated on a Cl 8 column with a 90 min elution gradient, spraying directly into a Thermo LTQ ion-trap mass spectrometer. The most prevalent peptide ions are trapped in the first quadropole, then dissociated by high energy to their amino acid constituents in a second step. This step allows sequence determinations of the peptides, and also results in the fragmentation of the iTRAQ reporter ions to allow quantitation per peptide. A typical MS/MS run resulted in greater than 4000 individual ion determinations. Each spectral scan was submitted to the SEQUEST database search algorithm, which has recently been upgraded to accommodate iTRAQ data for protein ID's and reporter ratio determinations. Also, each spectral scan was submitted to the MASCOT database search algorithm, and a MASCOT score was assigned to each identified protein as an indicator of the likelihood of the actual existence of said protein.

Differences in protein levels were characterized across the 4 sample groups in the day 0 (pre-vaccination) and day 28 post-vaccination samples. As shown in Figures 52-53, the identities and ratios of the most differentially expressed proteins determined in these two samples sets are listed (114 = young responders; 115 = young non-responders; 116 = elderly responders; 117 = elderly non-responders). The results demonstrate that the biomarkers of the invention distinguish influenza vaccine responders from non-responders.

While the invention has been illustrated and described in the figures and foregoing description, the same is to be considered as illustrative and not restrictive in character, it being understood that only the preferred embodiments have been shown and described and that all changes and modifications that come within the spirit of the invention are desired to be protected. In addition, all references and patents cited herein are indicative of the level of skill in the art and hereby incorporated by reference in their entirety.

Claims

WHAT IS CLAIMED IS;

1. A method for detecting immune response to influenza vaccination in a subject, comprising:

(a) detecting at least one biomarker in a biological sample from said subject following the administration of influenza vaccine; and

(b) correlating said detection with a determination of immune response in said subject to said vaccine.

2. The method of claim 1, comprising detecting a plurality of said biomarkers.

3. The method of claim 2, wherein the plurality comprises at least 3 biomarkers.

4. The method of claim 2, wherein the plurality comprises at least 4 biomarkers.

5. The method of claim 1, wherein said method comprises determining the absence of any of said biomarkers.

6. The method of claim 1, wherein said method comprises determining the absence of a plurality of said biomarkers.

7. The method of claim 1, wherein said detecting at least one biomarker in a biological sample from a subject is performed by mass spectrometry.

8. The method of claim 7, wherein said mass spectrometry is laser desorption mass spectrometry.

9. The method of claim 8, wherein said mass spectrometry is matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry.

10. The method of claim 9, wherein the laser desorption/ionization mass spectrometry comprises the steps of:

(a) separating biomarkers from other proteins in the biological sample using chromatography; and

(b) detecting the biomarkers with a MALDI-TOF mass spectrometer.

11. The method of claim 9, wherein the laser desorption/ionization mass spectrometry comprises the steps of:

(a) providing a substrate comprising an adsorbent attached thereto;

(b) contacting the biological sample with the adsorbent;

(c) desorbing and ionizing the biomarkers from the substrate; and

(d) detecting the des orbed/ionized biomarkers with a MALDI-TOF mass spectrometer.

12. The method of claim 11, wherein the adsorbent is a cation exchange adsorbent.

13. The method of claim 9, wherein said mass spectrometry is tandem mass spectrometry.

14. The method of claim 13, wherein the tandem mass spectrometry comprises detecting the sequences and quantities of the biomarkers using Isobaric Tag for Relative and Absolute Quantitation (iTRAQ) analysis.

15. The method of claim 1, wherein said detecting at least one biomarker in a biological sample from a subject is performed by an immunoassay.

16. The method of claim 1, wherein the biological sample is blood plasma.

17. The method of claim 1, wherein the biological sample is serum.

18. The method of claim 1, further comprising (c) managing subject treatment based on the determination of immune response.

19. The method of claim 18, further comprising (d) detecting the at least one biomarker after subject treatment.