Cell bank for characterizing the efficacy of vaccine formulations

Abstract

To provide compositions for determining the efficacy and potency of a vaccine preparation.SOLUTION: Splenocytes from immunized animals are isolated and frozen. Upon thawing aliquots, these cells are activated by exposure to a series of dilutions of a vaccine preparation being tested and a series of dilutions of a reference vaccine with known characteristics. Cells secreting immunogen-specific antibody and cells secreting nonspecific antibody are counted, as is the amount of immunogen-specific and nonspecific antibody produced. Comparison between the results from the vaccine preparations provides a measure of relative vaccine efficacy and / or potency.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] This application claims the interests of U.S. Provisional Application No. 62 / 799,457 filed on 31 January 2019, U.S. Provisional Application No. 62 / 734,866 filed on 21 September 2018, and U.S. Provisional Application No. 62 / 659,592 filed on 18 April 2018. These and all other external reference materials are incorporated herein by reference as a whole. If any definition or use of a term in an incorporated reference is inconsistent with or contradicts a definition of that term provided herein, the definition provided herein shall be deemed dominant.

[0002] The field of the present invention is a method for determining the immunogenicity and / or efficacy of vaccines, specifically prophylactic and / or preventive vaccine formulations. [Background technology]

[0003] The background art description contains information that may be useful in understanding the present invention. It is not acknowledged that any information provided herein constitutes prior art, or is related to the claimed invention, or that any publication specifically or implicitly referenced constitutes prior art.

[0004] Despite careful handling during manufacturing, the potency of vaccine formulations can vary from batch to batch. Therefore, it is necessary to determine the potency of individual batches to ensure that the vaccine can provide the required immune response. Traditionally, this has been determined using immunodiffusion assays. In these assays, a gel matrix (typically agarose mixed with antibodies against the vaccine antigen) is prepared and cast onto a support. A portion of the gel matrix is ​​removed to create test wells, into which samples of the vaccine formulation and quantified standards are placed. Antigen diffusion proceeds outward from these test wells, and if the ratio of antigen to antibody is accurate, a visible precipitate is formed. This distance of the precipitate from the test well is proportional to the antigen concentration of the sample placed in the well. While effective in identifying antibody-responsive substances in vaccine formulations, such an approach to quantifying potency can be relatively slow and somewhat subjective. Furthermore, this approach only helps in characterizing the antigen targeted by the specific antibody used and does not provide a direct indicator of the immunogenicity of the vaccine formulation.

[0005] Other approaches to assess vaccine efficacy have been suggested. U.S. Patent No. 9,057,716 (Balocchi et al.) (Patent Document 1) and U.S. Patent Application Publication No. 2015 / 0301051 (Giuliani and Mori) (Patent Document 2) describe characterizing the efficacy of meningococcal vaccines using an enzyme immunoassay incorporating bactericidal antibodies targeting meningococcal antigens. All publications herein are incorporated by reference to the same extent as individual publications or patent applications are specifically and individually indicated to be incorporated by reference. If a definition or use of a term in an incorporated reference conflicts with or contradicts a definition of that term provided herein, the definition provided herein shall apply, and the definition of that term in the reference shall not apply. However, such approaches only quantify the amount of these specific antigens in the vaccine preparation and cannot directly measure the immunogenicity of an intact preparation.

[0006] International Publication No. 2017 / 005880 (Wen et al.) (Patent Document 3) describes an attempt to characterize the immunogenicity of influenza vaccines based on the difference in sensitivity between HA antigens denatured during processing and those retaining their original conformation to trypsin digestion. However, the described method still relies on immunodiffusion, leaving limitations to the approach. Furthermore, it is not clear whether this approach can be used for a wide variety of vaccines.

[0007] U.S. Patent Application Publication 2014 / 0141455 (Weidanz) (Patent Document 4) describes a method for determining vaccine efficacy by visualizing the density of peptide-HLA complexes on the surface of antigen-presenting cells using highly specific antibodies targeting peptide-HLA complexes. However, such an approach relies on the ability to produce the necessary antibodies and can only provide a measure of the degree of antigen presentation, rather than being able to measure the efficacy of an untreated vaccine formulation. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] U.S. Patent No. 9,057,716 [Patent Document 2] U.S. Patent Application Publication No. 2015 / 0301051 [Patent Document 3] International Publication No. 2017 / 005880 [Patent Document 4] U.S. Patent Application Publication No. 2014 / 0141455 [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] Therefore, there is still a need for methods to directly evaluate the immunogenicity and / or efficacy of vaccine formulations. [Means for solving the problem]

[0010] (Summary of the invention) The subject of this invention is to provide compositions and methods for determining the relative efficacy of vaccine compositions. Splenocytes are collected from immunized mice and optionally depleted of CD138 before freezing. To determine the efficacy of the vaccine formulation, a portion of the frozen splenocytes are thawed and then activated using serial dilutions of the test vaccine formulation and a known efficacy control vaccine formulation. The number of cells expressing antibodies specific to the immunized antigen is determined for each dilution (and optionally, relative to the total number of antibody-producing cells), and the efficacy of the test vaccine is determined using the resulting dose-response curve.

[0011] One embodiment of the concept of the present invention is a method for characterizing the efficacy of a vaccine formulation by inoculating an animal with a reference vaccine, collecting spleen cells or B cells from the animal, exposing a portion of the spleen cells or B cells to a serial dilution of a first set of reference vaccines, exposing another portion of the spleen cells or B cells to a serial dilution of a second set of vaccine formulations, determining the number of cells from the portion exposed to the reference vaccine that express antibodies specific to the antigen of the vaccine formulation against the dilution of the reference vaccine, determining the number of cells from the portion exposed to the reference vaccine that express antibodies nonspecific to the dilution of the reference vaccine, determining the number of cells from the portion exposed to the test vaccine that express antibodies specific to the antigen against the dilution of the test vaccine, determining the number of cells in the portion exposed to the test vaccine that express antibodies nonspecific to the dilution of the test vaccine, and comparing the antigen-specific results from the cells exposed to the reference vaccine and the test vaccine. In some embodiments, the results for nonspecific antibodies from cells exposed to the reference vaccine and the test vaccine are also compared. In some embodiments, the results for antigen-specific antibodies and nonspecific antibodies are determined on the same test surface. In such embodiments, localized, separable labels may be used in the method used to determine antigen-specific and nonspecific antibodies. In some embodiments, the ratio of antigen-specific antibodies to nonspecific antibodies for two or more sets of serial dilutions of cells exposed to the reference vaccine and cells exposed to the test vaccine may be used as the basis for comparison. The relationship between a function (e.g., a first slope) describing the relationship between such ratios relative to the corresponding reference vaccine dilution and a different function (e.g., a second slope) describing the relationship between such ratios relative to the corresponding test vaccine dilution may be used as the basis for comparison.

[0012] In some embodiments of the concept of the present invention, the vaccine formulation to be characterized may contain a second vaccine antigen. In such embodiments, a portion of the collected spleen cells or B cells is exposed to a third set of serial dilutions of the reference vaccine, and another portion of the spleen cells or B cells is exposed to a fourth set of serial dilutions of the vaccine formulation. Not only is the number of cells from the portion exposed to the reference vaccine that express nonspecific antibodies with respect to the dilution of the reference vaccine determined, but the number of cells from the portion exposed to the reference vaccine that express antibodies specific to the second antigen of the vaccine formulation with respect to the dilution of the reference vaccine is also determined. Similarly, not only is the number of cells in the portion exposed to the test vaccine that express nonspecific antibodies with respect to the dilution of the test vaccine determined, but the number of cells from the portion exposed to the test vaccine that express antibodies specific to the second antigen with respect to the dilution of the test vaccine is also determined. The second antigen-specific results from the cells exposed to the reference vaccine and the test vaccine are then compared. In some embodiments, the results for nonspecific antibodies from cells exposed to the reference vaccine and the test vaccine are also compared. In some embodiments, the results for a second antigen-specific antibody and a nonspecific antibody are determined on the same test surface. In such embodiments, localized, separable labels may be used in the method used to determine the second antigen-specific and nonspecific antibodies. In some embodiments, tests for a first antigen-specific antibody, a second antigen-specific antibody, and a nonspecific antibody are performed on the same test surface. In such embodiments, mutually separable and localizable labels can be used to distinguish between the results for the first antigen-specific antibody, the second antigen-specific antibody, and the nonspecific antibody. In some embodiments, the ratio of the second antigen-specific antibody to the nonspecific antibody for two or more sets of serial dilutions of cells exposed to the reference vaccine and cells exposed to the test vaccine may be used as the basis for comparison.The relationship between a function describing the relationship between such ratios relative to the corresponding reference vaccine dilution (e.g., the third slope) and a different function describing the relationship between such ratios relative to the corresponding test vaccine dilution (e.g., the fourth slope) can be used as the basis for comparison.

[0013] In some embodiments of the concept of the present invention, the vaccine formulation includes a prophylactic vaccine. Such a prophylactic vaccine may target adenovirus, anthrax, botulism, cholera, diphtheria, hepatitis A, hepatitis B, hepatitis C, Haemophilus influenzae type b, human papillomavirus, seasonal influenza, Japanese encephalitis, measles, meningococcus, mumps, pertussis, pneumococcus, polio, rabies, rotavirus, rubella, herpes zoster, smallpox, tetanus, tuberculosis, typhoid fever, varicella, and / or yellow fever. In some embodiments, the vaccine formulation includes a therapeutic vaccine. Such a therapeutic vaccine may target gliablastoma, cervical cancer, skin cancer, lung cancer, breast cancer, head and neck cancer, pancreatic cancer, celiac disease, and / or vulvovaginal candidiasis.

[0014] The present invention provides the following:

[0015] A method for determining the efficacy of a vaccine preparation, The process of vaccinating animals with a reference vaccine, A step of collecting spleen cells or B cells from the aforementioned animal, A step of exposing the first portion of the spleen cells or the B cells to a serial dilution of the first set of the reference vaccine, A step of exposing the second portion of the spleen cells or the B cells to a second set of serial dilutions of the vaccine preparation, A step of determining a first number of cells in the first portion that express antibodies specific to the first antigen of the vaccine preparation against the serial dilution of the first set, and a second number of cells in the first portion that express antibodies nonspecific to the serial dilution of the first set. Determining a third number of cells in the second portion that express an antibody specific to the first antigen with respect to the serially diluted samples of the second set, and a fourth number of cells in the second portion that express an antibody non-specific to the serially diluted samples of the second set, and comparing the first number with the third number, in a method comprising: preferably further comprising comparing the second number with the fourth number; preferably, the first number and the second number are determined using a first common test surface; preferably, the third number and the fourth number are determined using a second common test surface; preferably, the first number is determined using a first immunoassay comprising a first label, and the second number is determined using a second immunoassay comprising a second label, wherein the first label and the second label are localized and distinguishable; preferably, the first immunoassay is an antibody plaque assay; preferably, the third number is determined using a third immunoassay comprising a third label, and the fourth number is determined using a fourth immunoassay comprising a fourth label, wherein the third label and the fourth label are localized and distinguishable; preferably, the comparing step comprises: determining a first series of ratios between the first number and the second number for two or more members of the first set of serially diluted samples; determining a second series of ratios between the third number and the fourth number for two or more members of the second set of serially diluted samples; determining a first function of the first series of ratios for each dilution; determining a second function of the second series of ratios for each dilution, and comparing the first function and the second function; preferably, both the first function and the second function are gradient functions; preferably, the vaccine formulation comprises a second vaccine antigen. Preferably, a step of determining a fifth number of cells in the first portion that express antibodies specific to the second vaccine antigen of the vaccine formulation, A step of determining the sixth number of cells in the second portion that express antibodies specific to the second vaccine antigen, and The process further includes comparing a third ratio between the fifth number and the second number for at least one of the first set of serial dilutions with a sixth ratio between the sixth number and the fourth number for at least one of the second set of serial dilutions. Preferably, the fifth number and the second number are determined using a first common test surface. Preferably, the sixth number and the fourth number are determined using a second common test surface. Preferably, the fifth number is determined using a fifth immunoassay including a fifth label, and the second number is determined using a second immunoassay including a second label. Here, the fifth marker and the second marker are localized and identifiable. Preferably, the fifth immunoassay is an antibody plaque assay. Preferably, the sixth number is determined using a sixth immunoassay including a sixth label, and the fourth number is determined using a fourth immunoassay including a fourth label, where the sixth and fourth labels are localized and identifiable. Preferably, the vaccine formulation includes a vaccine species targeting at least one of the group consisting of adenovirus, anthrax, botulism, cholera, diphtheria, hepatitis A, hepatitis B, hepatitis C, Haemophilus influenzae type b, human papillomavirus, seasonal influenza, Japanese encephalitis, measles, meningococcal disease, mumps, pertussis, pneumococcal disease, polio, rabies, rotavirus, rubella, herpes zoster, smallpox, tetanus, tuberculosis, typhoid fever, varicella, and yellow fever. Preferably, the vaccine preparation includes a therapeutic vaccine. Preferably, the vaccine formulation includes a vaccine species that targets at least one of the group consisting of gliablastoma, cervical cancer, skin cancer, lung cancer, breast cancer, head and neck cancer, pancreatic cancer, celiac disease, and vulvovaginal candidiasis.

[0016] Various objects, features, aspects and advantages of the subject matter of the present invention will become more apparent from the following detailed description of preferred embodiments, as well as from the accompanying drawings in which similar figures represent similar components. [Brief explanation of the drawing]

[0017] [Figure 1] A schematic diagram of a conventional antibody plaque immunoassay is shown below. [Figure 2] Typical results from conventional antibody plaque immunoassays are shown. [Figure 3] An exemplary protocol flowchart for determining vaccine efficacy using spleen cells is shown. [Figure 4A] This is a scan image of an HcR / A antigen antibody plaque immunoassay plate from a spleen cell assay based on the concept of the present invention. Spleen cells are coated with either 5 × 10⁴ cells (upper left quadrant), 2.5 × 10⁴ cells (upper right quadrant), 10⁴ cells (lower left quadrant), or 5 × 10³ cells (lower right quadrant) per well. [Figure 4B] Figure 4A shows a typical dose-response curve generated from data obtained from the plates shown. TS = HcR / A preparation test sample, SC = HcR / A preparation control sample. [Figure 4C] Examples of the relative efficacy of tests and reference antigen preparations as determined by the method of the present invention are shown. [Figure 5A] An example of a test antibody plaque immunoassay plate layout for use in the method of the present invention is shown. [Figure 5B] Figure 5A shows a scan image of an antibody plaque immunoassay plate prepared as shown, in which IgG secretion by stimulated spleen cells is visualized. [Figure 5C]Figure 5A shows a scan image of an antibody plaque immunoassay plate prepared as shown, visualizing the secretion of HcR / A-specific antibodies by stimulated spleen cells. [Figure 5D] A magnified view of a typical individual well from an antibody plaque immunoassay plate is shown. [Figure 5E] Figure 5A shows the dose-response curve of IgG secretion from spleen cells in an ELISpot plate prepared as shown, the calculated EC50, and the calculated relative potency. [Figure 5F] Figure 5A shows the dose-response curve, calculated EC50, and calculated relative potency of HcR / A-specific antibody secretion from spleen cells in an antibody plaque immunoassay plate prepared as shown. [Figure 6A] An example of a test antibody plaque immunoassay plate layout for use in a spleen cell vaccine efficacy assay based on the concept of the present invention is shown. [Figure 6B] Figure 6A shows a scan image of an antibody plaque immunoassay plate prepared as shown, in which IgG secretion by stimulated spleen cells is visualized. [Figure 6C] Figure 6A shows a scan image of an antibody plaque immunoassay plate prepared as shown, visualizing the secretion of HcR / A-specific antibodies by stimulated spleen cells. [Figure 6D] Figure 6A shows the dose-response curve, calculated EC50, and calculated relative potency for IgG secretion from spleen cells in an antibody plaque immunoassay plate prepared as shown. [Figure 6E] Figure 6A shows the dose-response curve, calculated EC50, and calculated relative potency for HcR / A-specific antibody secretion from spleen cells in an antibody plaque immunoassay plate prepared as shown. [Figure 7A] An example of a test microwell plate layout for use in determining vaccine efficacy using the spleen cell assay concept of the present invention is shown. [Figure 7B]Figure 7A shows a scan image of an antibody plaque immunoassay plate prepared as shown, in which IgG secretion by stimulated spleen cells is visualized. [Figure 7C] Figure 7A shows a scan image of an antibody plaque immunoassay plate prepared as shown, visualizing the secretion of HcR / A-specific antibodies by stimulated spleen cells. [Figure 7D] Figure 7A shows dose-response curves, calculated EC50, and calculated relative potency for IgG secretion from spleen cells activated at various cell densities in ELISpot plates prepared as shown. [Figure 7E] Figure 7A shows dose-response curves, calculated EC50, and calculated relative potency for HcR / A-specific antibody secretion from splenic cells activated at various cell densities in antibody plaque immunoassay plates prepared as shown. [Modes for carrying out the invention]

[0018] (Detailed explanation) The following description contains information that may be useful in understanding the present invention. It is not an assertion that any information contained herein is prior art or relating to the claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0019] The following considerations provide numerous exemplary embodiments of the subject matter of the present invention. Each embodiment represents a single combination of the elements of the present invention, but the subject matter of the present invention is considered to include all possible combinations of the disclosed elements. Thus, if one embodiment includes elements A, B, and C, and a second embodiment includes elements B and D, the subject matter of the present invention is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.

[0020] The grouping of alternative elements or embodiments of the present invention disclosed herein should not be construed as limiting. Each group member may be referenced and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group may be included in or removed from a group for convenience and / or patentability reasons. In the event of such inclusion or removal, the specification shall be deemed to include the modified group and thus satisfy the description of all Markush groups used in the appended claims.

[0021] The subject of this invention is to provide compositions and methods for vaccinating mice or similar test animals with a reference batch of a vaccine known to be immunogenic and having efficacy that meets a standard. Spleen cells and / or B cells are obtained from the vaccinated animals, and in some embodiments, the B cell population of such a sample can be enriched or depleted (e.g., depletion of existing plasma cells). Suitable cells can be obtained by a variety of means, including dissection and / or perfusion of the spleen, dissection of one or more lymph nodes, and / or collection from circulation. Spleen cells or B cells can be isolated using antibody-coated magnetic beads, fluorescence-activated cell sorting, or any suitable method. After isolation, the cells can be used immediately or replicated before use. In some embodiments, the isolated cells can be stored before use (e.g., stored at about -80°C, or in liquid nitrogen in liquid or gas phase).

[0022] For the test, cells are plated to two sets of test sites or surfaces, one of which is used to characterize the secretion of antibodies against the vaccine antigen, and the other is used to characterize the secretion of nonspecific (i.e., non-targeting) antibodies. Suitable test surfaces include slides, the surfaces of wells in microwell plates, beads, and / or the surfaces of microfluidic devices.

[0023] Serial dilutions of the vaccine under test are applied to one set of cells, while serial dilutions of the reference vaccine are applied to a second set of cells. In some embodiments, cells pre-sensitized by exposure to the vaccine formulation (either in vivo or in vitro) are used for testing. In such embodiments, these pre-sensitized cells may be reactivated by exposure to the test and / or control vaccine formulation to determine vaccine batch efficacy. In some embodiments, such reactivation may be performed before applying the cells to a method for detecting antibody secretion (e.g., using an antibody plaque assay such as ELISpot®). In other embodiments, such reactivation may be performed during at least part of the implementation of the method for detecting antibody secretion. The number of cells secreting vaccine antigen-specific antibodies is characterized (e.g., using a sandwich assay targeting vaccine antigen-specific antibodies that produce a localization signal), and the number of cells secreting non-specific antibodies is characterized (e.g., using a sandwich assay targeting species-specific antibodies that produce a localization signal).

[0024] The slope of the titration curve of the percentage of cells secreting vaccine antigen-specific antibodies (relative to the response produced by cells stimulated with the reference lot of vaccine) allows for the direct measurement of vaccine efficacy (i.e., its ability to induce an antigen-specific antibody response). For example, by comparing dose-response curves created by titrating a test vaccine sample with a reference vaccine sample, vaccine efficacy can be measured relatively by comparing dilutions that provide a midpoint response (EC50, instant midpoint slope, etc.) in each titration curve. By comparing such values ​​with those of the reference vaccine, the suitability of the test vaccine for use can be assessed.

[0025] In some embodiments, both specific and non-antigen-specific antibodies produced in response to exposure to the vaccine formulation can be evaluated. In such embodiments, the ability of the test vaccine to elicit an antigen-specific response can be compared to a reference vaccine. The degree of non-specific antibody response induced by the vaccine formulation provides information related to the efficacy of the non-antigen component (such as an adjuvant) of the vaccine formulation. For example, determining the ratio of specific antibody responses to non-specific antibody responses in the test vaccine can be compared to determining that ratio in the reference vaccine to determine efficacy.

[0026] It should be understood that the disclosed technology offers many beneficial technical advantages, including the ability to directly measure vaccine efficacy using a relatively small number of test animals. For example, spleen cells isolated from a single immunized mouse can be preserved and used for multiple determinations. Furthermore, while the described approach allows for the measurement of immunogenicity / efficacy of untreated vaccine formulations (e.g., including vehicle and adjuvant), prior art approaches have characterized only specific components (e.g., vaccine antigens). The applicant notes that this approach is highly adaptable and not limited in terms of the range of vaccines or antigen species.

[0027] As described above, immunoassays can be used to count and / or characterize B cells that secrete antibodies with desired specificity in response to exposure to a vaccine formulation. For example, the secretion of antibodies against a desired antigen can be detected using a surface (e.g., a membrane) proximal to such cells coated with an anti-species antibody to capture the secreted immunoglobulin, in what is commonly called an ELISpot® or antibody plaque assay. The captured immunoglobulin may then play a role in capturing a specific antigen (or its labeled analog). Such captured specific antigens can be visualized using a second antigen-specific antibody (e.g., in a sandwich assay) or by visualizing a label attached to the captured antigen analog. The secretion of non-specific antibodies can be detected in a similar manner using a pair of species-specific antibodies. Alternatively, the amount of antigen-specific and / or non-specific antibodies secreted can be characterized, for example, by an immunoassay from a sample of cell culture medium exposed to stimulated spleen cells.

[0028] While antibody plaque assays are described, other techniques can also be used. For example, a reverse hemolytic plaque assay can be used to count antibody-secreting cells and obtain an estimate of the amount of antibody being secreted. Nonspecific antibody secretion and specific antibody secretion can be distinguished by exposure to either an Ig-specific antibody or a specific antigen (or appropriate analog) prior to complement exposure and subsequent negative plaque formation. Alternatively, antibody-producing cells can be identified and counted using flow cytometry by dividing the sample and labeling it with either an anti-Ig antibody (for nonspecific antibody secretion) or an antigen or antigen analog (for antigen-specific antibody secretion) conjugated with a detectable label (e.g., a fluorescent compound). The amount of specific and / or nonspecific antibodies produced in the remaining portion of such divided sample can be quantified using conventional immunoassay techniques. The results of cell counting, and the results of the amount of specific and / or nonspecific antibodies produced, can be evaluated separately or combined to obtain values ​​representing both the number of cells and the amount of immunoglobulin produced.

[0029] In an exemplary embodiment of an assay for detecting secreted antibodies against a specific antigen, wells of a test plate are coated with the antigen of interest. Immunoglobulin-secreting cells (e.g., spleen cells treated with a diluted vaccine formulation) are added to the plate and incubated for a sufficient time to form a complex between the secreted antibody and the portion of the antigen-coated plate adjacent to the cells. After a washing step, a secondary antibody against the secreted antibody species, carrying a detectable label (e.g., horseradish peroxidase, alkaline phosphatase, fluorophore, etc.), is added to the plate and allowed to form a complex with the secreted antibody bound to the plate via the immobilized antigen. After a further washing step, the label can be visualized (e.g., by adding a chromogenic substrate or excitation at an appropriate light frequency), and the sites where specific antibody secretion occurs are counted. Counting can be performed manually or by image recognition software.

[0030] In an exemplary embodiment of an assay for detecting total secretory antibodies (i.e., both antigen-specific and non-specific), wells of a test plate are coated with an antibody against the immunoglobulin species (e.g., anti-mouse IgG). Immunoglobulin-secreting cells (e.g., spleen cells treated with a diluted vaccine formulation) are added to the plate and incubated for a sufficient time for the secreted antibodies to form a complex with the portion of the antibody-coated plate proximal to the cells. After a washing step, a secondary antibody against the secretory immunoglobulin species, carrying a detectable label (e.g., horseradish peroxidase, alkaline phosphatase, fluorophore, etc.), is added to the plate to form a complex with the secretory antibodies bound to the plate via the immobilized anti-species antibody. After a further washing step, the label can be visualized (e.g., by adding a chromogenic substrate or excitation at an appropriate light frequency) and the sites where specific antibody secretion occurs are counted. Counting can be performed manually or by image recognition software.

[0031] The detectable signals generated by such assay systems can be localized to facilitate the counting of secretory cells. Such localized signals can be generated by any suitable means, including fluorescence, phosphorescence, luminescence, emission by radionuclides, and / or enzymatic production of at least partially insoluble products (e.g., chromophores or fluorophores). Localization of signals near cells can be interpreted as the secretion of antibodies having assayed characteristics by those cells. Such cells can be determined manually or using automated systems.

[0032] In some embodiments, the secretion of antigen-specific and non-specific antibodies can be determined using overlapping test surfaces / plates. In other embodiments, the secretion of antigen-specific and non-specific antibodies can be determined using the same test surface by utilizing identifiable labeling. For example, the secretion of antigen-specific antibodies can be characterized using a first fluorophore associated with an antigen-specific assay performed on the test surface, and the secretion of antibodies (antigen-specific or other) can be determined using a second fluorophore, where the second fluorophore is separable from the first fluorophore and associated with a species-specific immunoassay performed on the test surface. Characterization of the luminescence from the first fluorophore allows for the characterization of cells secreting antigen-specific antibodies, and characterization of the luminescence from the second fluorophore allows for the characterization of all antibody-secreting cells. The number of cells secreting non-specific antibodies can be determined by the difference between these values.

[0033] As described above, the method of the present invention is not limited to a specific vaccine species. Suitable vaccines may target viruses, bacteria, fungi, antigens associated with autoimmune diseases, and / or neoplastic antigens. Examples of vaccines that the method of the present invention may target include prophylactic vaccines such as those targeting adenovirus, anthrax, botulism, cholera, diphtheria, hepatitis A, hepatitis B, hepatitis C, Haemophilus influenzae type b, human papillomavirus, seasonal influenza, Japanese encephalitis, measles, meningococcal disease, mumps, pertussis, pneumococcal disease, polio, rabies, rotavirus, rubella, herpes zoster, smallpox, tetanus, tuberculosis, typhoid fever, varicella, and / or yellow fever. Further examples of vaccines that may be targeted by the method of the present invention include therapeutic vaccines such as those targeting gliablastoma, cervical cancer, skin cancer, lung cancer, breast cancer, head and neck cancer, pancreatic cancer, celiac disease, and / or vulvovaginal candidiasis.

[0034] The vaccine formulation under test may be polyvalent (i.e., incorporating more than one vaccine antigen). Examples include pediatric MMR vaccine formulations, botulism vaccines, and vaccines targeting seasonal influenza (typically incorporating viral antigens associated with several different virus strains). In some embodiments, the response of a polyvalent vaccine formulation to each component antigen species may be determined using different test surfaces or sets of test surfaces. In other embodiments, the response of a polyvalent vaccine formulation to different antigens may be characterized using a common test surface or set of test surfaces. For example, when characterizing a polyvalent vaccine formulation incorporating a first vaccine antigen and a second vaccine antigen, a test surface carrying antibodies against both the first and second vaccine antigens may be provided. When the test surface is treated with the first and second antigens (or analogs thereof), first and second antibody:antigen complexes are formed on the test surface. The secretion of antibodies against the first vaccine antigen captures those antibodies proximal to the secreting cells as antibody:first antigen:antibody complexes. Similarly, the secretion of antibodies against a second vaccine antigen captures those antibodies proximal to the secreting cells as antibody:second antigen:antibody complexes. The use of separable labels allows for the distinct identification of such complexes using a common test surface, and enables the identification of a first set of cells secreting antibodies against the first vaccine antigen and a second set of cells secreting antibodies against the second vaccine antigen. In some embodiments, cells secreting nonspecific antibodies may be determined on the same test surface using a third identifiable label.

[0035] The antigenicity and / or efficacy of a vaccine formulation can be determined by comparing the results obtained with a reference vaccine to the results obtained with a test or unknown vaccine formulation using one or more of the above assays. For example, a series of dilutions (e.g., serial dilutions) of the reference vaccine and a similar or identical set of dilutions prepared from the test vaccine can be prepared and applied to spleen cells and / or B cells as described above. The number of cells secreting antigen-specific antibodies versus cells secreting nonspecific antibodies can be determined for one or more of these dilutions of both the reference vaccine and the test vaccine, and the results can be compared. In a preferred embodiment, the ratio between the number of antigen-specific antibody-secreting cells and the number of nonspecific antibody-secreting cells can be used as the basis for such a comparison. Such a comparison can be performed by any suitable method, e.g., parallel line analysis of each dilution:response curve.

[0036] The results between a reference vaccine and one or more test vaccines can be compared by any suitable means, and the correspondence between the results of the reference vaccine and the test vaccines indicates efficacy that meets the criteria. For example, dilutions corresponding to the half-maximal response (EC50, etc.) of the reference vaccine and the test vaccine can be compared. In a preferred embodiment, a function of data from two or more dilutions (e.g., optical density vs. dilution) is obtained for the reference vaccine and the test vaccine, and the functions are compared. For example, a first slope may be determined for a line drawn between two or more data points representing various dilutions of the reference vaccine, and a second slope may be determined for a line drawn between two or more data points representing various dilutions of the test vaccine, and then the first and second slopes are compared to determine the degree to which they are parallel (e.g., by parallel line analysis). Significant deviations from parallelism (e.g., differences in slopes from about 5% to 10%, 15%, 20%, or 25%) may indicate a significant difference in efficacy between the test lot and the reference lot of the vaccine composition. If the difference between the slopes is found to be within an acceptable range, the degree to which they deviate from each other can be considered. For example, if the derived lines of the dose-response curves for the test and reference vaccine compositions are found to be essentially parallel, deviations along the dilution axis (usually the x-axis) of the dose-response curves beyond dilutions of approximately 0.2x, 0.5x, 0.8x, 1x, 1.2x, 1.5x, 1.8x, 2x, and 2.5x or more may indicate a significant difference in potency between the test and reference compositions. Such differences in potency may be used to determine the acceptability and / or dosing dilution of the test vaccine formulation.

[0037] In some embodiments, such dose / response curves may be nonlinear over a portion of the dilutions being tested, in which case alternative functions suitable for the nonlinear data (e.g., instantaneous slope at half of the maximum response via a 4PL or 5PL fit) are available. Alternatively, the minimum dilution of the vaccine formulation that produces a median-maximal response can be selected and used as an acceptance criterion.

[0038] In some embodiments of the present invention, the vaccine efficacy assay may be based on a cell-based assay demonstrating a secondary response to immunization. In such embodiments, many animals (e.g., mice, rabbits, guinea pigs, etc.) are immunized with a vaccine (e.g., a reference vaccine of known efficacy), and spleen cells and / or B cells are isolated from the immunized animals to provide a population of primary sensitized cells. Such primary sensitized cells may be obtained and used directly from living animals. In other embodiments, the primary sensitized cells may be collected and frozen, and then thawed before use. In some embodiments, the B cell population within the collected spleen cells is enriched. In other embodiments, one or more specific cell populations (e.g., plasma cells) may be depleted.

[0039] Such preparations of primary sensitized cells can be treated with a series of dilutions of the test vaccine to elicit specific and / or nonspecific immune responses. In some embodiments, dilutions of a reference vaccine of known potency may be tested in parallel. The immune response can be characterized as described above. For example, specific and nonspecific responses can be determined by performing ELISPOT and similar assays targeting specific antigens and immunoglobulin species of the vaccine preparation, respectively. A typical antibody plaque immunoassay is schematically shown in Figure 1. Typical results of an antibody plaque immunoassay are shown in Figure 2.

[0040] In some embodiments, primary sensitized cells may be treated with the test vaccine before being applied to such assay. In other embodiments, primary sensitized cells may be treated with the test vaccine at the time of or during the assay (e.g., after the distribution of primary sensitized cells to the wells of the antibody plaque immunoassay plate). The efficacy of the test vaccine can be measured by counting specific and nonspecific Ig-secreting cells and subsequently generating dose / response curves. For example, the efficacy of the test vaccine can be measured relatively by comparison with similar dilutions of a reference vaccine of known efficacy. Although not representing a de novo primary response, the inventors believe that the characterized secondary response has a similar correlation with the efficacy of the vaccine.

[0041] Some vaccine formulations are formulated such that some or all of the immunizing antigens are coated or bound to the surface of an adjuvant solid. Aluminum hydroxide hydrogels and CpG oligonucleotides are examples of such adjuvants and are widely used (e.g., in anthrax vaccines). For the purpose of characterizing such vaccine formulations, the immunizing antigens may be eluted or released from the adjuvant before application to the primary sensitized cells in the assay to improve accessibility and / or facilitate diffusion (e.g., through substitution with competing polymers, pH shift, electrolytic elution, etc.). In other embodiments, such vaccine formulations can be tested as formulated.

[0042] In one example of the method of the present invention, mice (or guinea pigs) are immunized with a reference batch of anthrax vaccine to generate sensitized spleen cells. The spleen cells are then isolated from the immunized animals, and in some embodiments, a specific B cell population is enriched. The spleen cells (which may be enriched or depleted) are plated into 96-well plates prepared for use in an antibody plaque immunoassay, such as an enzyme-linked immunospot (ELISpot). In some embodiments, the spleen cells are reactivated with a dilution of either the test vaccine or the reference vaccine before being introduced into the antibody plaque immunoassay plate or a similar technique. In other embodiments, the spleen cells are reactivated during or after introduction into the antibody plaque immunoassay plate or a similar technique. A set of plates may be configured to measure antigen-specific immunoglobulin secretion (e.g., by coating with one or more immunizing antigens). The second set of plates may be configured to measure total (specific and nonspecific) immunoglobulin (Ig) secretion (for example, by coating with one or more Ig-specific antibodies). Serial dilutions of the vaccine test sample are then applied to the plate containing the cells, along with serial dilutions of the reference vaccine sample. Alternatively, spleen cells (or isolates) may be incubated with dilutions of the test vaccine and reference vaccine before application to the antibody plaque immunoassay plate. The plate is then incubated for a sufficient time for the vaccine formulation to interact with the spleen cells and activate specific and / or nonspecific immune responses resulting in Ig secretion.

[0043] The number of Ig-secreting cells correlates with the effective dose of the vaccine, and this is also a function of vaccine efficacy. Once the assay is complete, the number of Ig-secreting cells per well, both antigen-specific and non-specific, or none (or not), is counted using manual or automated methods. Since antigen-specific Ig is also identified by the non-specific antibody plaque immunoassay plate, it should be understood that the number of non-specifically activated cells is represented by the difference in numbers between the non-specific antibody plaque immunoassay plate and the antigen-specific antibody plaque immunoassay plate. Dose-response curves (e.g., number of Ig-secreting cells vs. vaccine dose) are prepared for the test sample and the reference sample. The dose-response curves for the test sample and the reference sample can then be modeled with appropriate equations (e.g., 4-PL or 5-PL asymmetric curve fitting), and the relative efficacy of the test can be calculated using the parallel line method. [Examples]

[0044] An exemplary protocol for a spleen cell assay to determine vaccine efficacy is shown in Figure 3. As shown on the left side of Figure 3, in such a test protocol, mice are immunized with the vaccine antigen (e.g., HcR / A), and then spleen cells are isolated from the harvested spleen. CD138-positive cells are depleted from the spleen cells (e.g., using a commercially available kit), and the spleen cells are divided and frozen to generate a cell bank. To perform the assay (as shown on the right side of Figure 3), cells from the cell bank are thawed and then reactivated by exposure to serial dilutions of a reference antigen (e.g., a reference vaccine) and a test antigen (e.g., the vaccine formulation under evaluation). Following reactivation, antigen-specific and all-species-specific antibodies secreted by the cells are characterized. In this example, characterization is performed using antigen-specific and IgG-specific antibody plaque immunoassay plates. The number and / or density of spots in the test wells are recorded, and dose / response curves for the control and test antigen preparations are generated. These dose-response curves can be compared (for example, by parallel line analysis) to measure the efficacy of the test antigen preparation compared to the control antigen preparation.

[0045] Figure 4A shows an image of an ELISpot test plate for HcR / A antigen. Wells in which splenic cells react to HcR / A show discrete, high optical density spots at the location of the cells. These become nearly confluent at high concentrations / low dilutions. The number and / or density of these spots can be quantified and compared to dilutions of HcR / A antigen containing the preparation applied, as shown in Figure 4B. As shown, the dose-response curve yields a larger dynamic range with higher cell densities. Figure 4C shows 5 × 10⁻⁶ 4 The dose-response curve obtained by the cell coating density is shown in more detail, and the calculated EC 50 Furthermore, comparative studies (TS) and reference (SC) antigen preparations can be made regarding relative potency.

[0046] As described above, characterizing the immune response generated by a vaccine formulation can involve characterizing both the production of antigen-specific antibodies and the proportion of antigen-specific antibodies produced relative to the total production of a particular type of antibody. Therefore, to better characterize the efficacy of the vaccine, antigen-specific and antibody-specific antibody plaque immunoassay plates or similar techniques can be applied in conjunction with banked spleen cells. An example of an antibody plaque immunoassay plate layout incorporating various spleen cell coating densities is shown in Figure 5A. Figure 5B shows an image of the antibody plaque immunoassay plate prepared as described above, with IgG secretion from the spleen cells visualized. Figure 5C shows an image of the antibody plaque immunoassay plate prepared as described above, with HcR / A-specific antibody secretion from the spleen cells visualized. Figure 5D shows a 5 × 10 4Figure 5E shows a magnified view of individual wells of an antibody plaque immunoassay plate, in which spleen cells coated with cells / well and stimulated with 100 pM antigen secrete HcR / A-specific antibodies. As described above, dose-response curves can be derived for either or both antigen-specific responses and antibody species responses. Figure 5E shows a dose-response curve obtained from an antibody plaque immunoassay plate prepared as shown in Figure 5A, and characterized for IgG secretion. Figure 5F shows a dose-response curve obtained from an antibody plaque immunoassay plate prepared as shown in Figure 5A, and characterized for HcR / A-specific antibody secretion.

[0047] Other examples of vaccine efficacy assays based on the concept of the present invention are shown in Figures 6A–6E. In these tests, StemCellTechnologies® PE Selection Kit and anti-CD138, PE-labeled antibody were used to deplete RBC-lysed spleen cells, resulting in 7.5 × 10⁶ cells per well of an incubation plate. 5 The antigen preparation was suspended in cells. The antigen preparation was diluted and applied to the plate, which was then incubated at 37°C and 5% CO2 for 48 hours. On day 2, PVDF plates were prepared by coating them overnight at 4°C with 40 μg / mL HcR / A or 20 μg / mL anti-mouse IgG, and then blocked in EL4-B5 medium at 37°C and 5% CO2 for 2 hours. On day 3, activated spleen cells were harvested from the incubation plate, and 10 per mL was used. 6 Resuspend the cells and place them in the blocked PVDF plate, 5 × 10⁶ per well. 4 or 2.5 × 10 4Add to the cells with a pipette and incubate overnight at 37 °C and 5% CO2. On the fourth day, the cells were removed from the PVDF plate by washing with PBS(3×) and PBST(3×). Subsequently, the PVDF plate was incubated with 80 μL / well of 1:5,000 anti-mouse IgG-HRP at 4 °C for 8 hours. After washing the plate with PBS-T(3×) and PBS(3×), 100 μL of AEC precipitating substrate was added to each well and incubated at ambient temperature for 5 minutes. The reaction was stopped by washing the plate with tap water and drying it overnight in the dark before image acquisition and analysis. Figure 6A shows an exemplary plate layout for such a study.

[0048] Figure 6B shows an image of a typical plate used to characterize IgG secretion from activated spleen cells (i.e., coated with anti-mouse IgG), where the contents of the plate are arranged as shown in Figure 6A. Figure 6C shows a similar image obtained for a typical plate used to characterize HcR / A antigen-specific antibody secretion by activated spleen cells from a plate arranged in the same manner.

[0049] As described above, the potencies regarding both IgG responses and antigen-specific responses can be characterized by quantification of the spot number and / or density from such images. Figure 6D shows the dose-response curve for dilutions of the antigen preparation, the calculated EC50, and the calculated relative potency (against the IgG response of activated spleen cells characterized using the plate layout as shown in Figure 6A). Figure 6E shows the dose-response curve for dilutions of the antigen preparation, the calculated EC 50 50, and the calculated relative potency (against the HRC / A-specific antibody response of activated spleen cells characterized using the plate layout as shown in Figure 6A).

[0050] Figures 7A - 7E show that C138-depleted spleen cells are at 10 6 per mL, 7.5×10 5 or 5×10 5Figures 6A-6E show the results of the tests performed as described above, in which cells were activated by antigen dilutions. The plate format used is shown in Figure 7A.

[0051] Figure 7B shows an image of a typical antibody plaque immunoassay plate used to characterize IgG secretion from activated spleen cells (i.e., coated with anti-mouse IgG), with the contents of the plate arranged as shown in Figure 7A. Figure 7C shows a similar image obtained for a typical antibody plaque immunoassay plate used to characterize HcR / A antigen-specific antibody secretion from activated spleen cells from a plate arranged in the same manner.

[0052] As described above, the efficacy with respect to both the IgG response and the antigen-specific response can be characterized by the quantification of the number and / or density of spots from such images. Figure 7D shows the dose-response curves for dilutions of the antigen preparation and the calculated EC 50 Figure 7E shows the calculated relative potency (against the IgG response of spleen cells activated at various spleen cell concentrations and characterized using an antibody plaque immunoassay plate layout as shown in Figure 7A). 50 This also shows the calculated relative potency (against the HcR / A-specific antibody response of spleen cells activated at various concentrations and characterized using an antibody plaque immunoassay plate layout as shown in Figure 7A).

[0053] While the above examples utilized separate antibody plaque immunoassay plates for total IgG secretion and antigen-specific antibody secretion, it should be understood that in some embodiments, such measurements can be obtained from a single antibody plaque immunoassay plate by using immunoglobulin-specific and antigen-specific secondary antibodies in the same test wells combined with different labels. Similarly, while HRP conjugates were used for detection, it should be understood that other detectable labels, including alternative enzymes (e.g., alkaline phosphatase), fluorophores, chromophores, lumiphores, colloidal gold, and / or staining microparticles, may be used.

[0054] It will be apparent to those skilled in the art that many more modifications beyond those already described are possible without departing from the concept of the invention as described herein. Therefore, the subject matter of the invention should not be limited beyond the spirit of the appended claims. Furthermore, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible way that is consistent with the context. In particular, the terms “comprises” and “comprising” should be interpreted non-exclusively as referring to an element, component, or process, indicating that the referenced element, component, or process exists, is utilized, or can be combined with other elements, components, or processes not explicitly referenced. Where a claim in the specification refers to at least one selected from the groups A, B, C.... and N, the text should be interpreted as requiring only one element from the group, rather than A plus N, or B plus N, etc.

Claims

1. A test plate for characterizing the efficacy of a test vaccine, A first cell population including splenocytes isolated from animals immunized with a vaccine antigen, subsequently frozen, and then resensitized by contact with a reference antigen, and A test plate containing a second cell population, including splenocytes isolated from animals immunized with the aforementioned vaccine antigen, subsequently frozen, and then resensitized by contact with the test antigen.

2. The test plate according to claim 1, wherein CD138-positive cells are depleted from spleen cells before freezing.

3. The test plate according to claim 1, wherein the test vaccine is a prophylactic vaccine.

4. The test plate according to claim 3, wherein the vaccine antigen is derived from a pathogen selected from the group consisting of adenovirus, anthrax bacillus, botulinum toxin, cholera bacillus, diphtheria bacillus, hepatitis A virus, hepatitis B virus, hepatitis C virus, Haemophilus influenzae type b, human papillomavirus, seasonal influenza virus, Japanese encephalitis virus, measles virus, meningococcus, mumps virus, Bordetella pertussis, pneumococcal virus, poliovirus, rabies virus, rotavirus, rubella virus, herpes zoster virus, smallpox virus, tetanus bacillus, tuberculosis bacillus, typhi bacillus, varicella virus, and yellow fever virus.

5. The test plate according to claim 1, wherein the test vaccine includes a therapeutic vaccine.

6. The test plate according to claim 5, wherein the vaccine antigen is specific to a disease selected from the group consisting of gliablastoma, cervical cancer, skin cancer, lung cancer, breast cancer, head and neck cancer, pancreatic cancer, celiac disease, and vulvovaginal candidiasis.

7. The test plate according to claim 1, wherein the animal is a mouse.

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