Poliovirus arrays and use thereof

Abstract

Arrays comprising probes from at least two polioviruses selected from poliovirus type 1, poliovirus type 2 and poliovirus type 3 are provided. Arrays comprising a plurality of probes comprising short peptide probes from at least one poliovirus are also provided. Methods of using the arrays, as well as kits and systems comprising the arrays are also provided.

Description

POLIOVIRUS ARRAYS AND USE THEREOFREFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0001] The contents of the electronic sequence listing (BGU-NIBN-P-0139-PCT.xml; Size: 555,025 bytes; and Date of Creation: August 27, 2025) is herein incorporated by reference in its entirety.CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 688,315, filed on August 29, 2024, the contents of which are all incorporated herein by reference in their entirety.FIELD OF INVENTION

[0003] The present invention is in the field of polio diagnostics and array design.BACKGROUND OF THE INVENTION

[0004] Poliomyelitis, also termed polio, is an infectious disease caused by the poliovirus. Poliovirus is transmitted between humans through fecal-oral and oral-oral routes, and the fecal-oral route is the most common transmission route. Following infection through the mouth, the poliovirus is replicated in the intestinal tract, and usually secreted in the stool for 2-4 weeks and in some cases several weeks longer. The virus also replicates in the upper respiratory tract, and therefore can be detected in throat swab samples and secretions during the early phase of infection. Early-phase infection symptoms may include upper respiratory tract infection, gastrointestinal disturbances, and influenza-like illness. In severe cases, the virus enters the central nervous system and the patient develops nonparalytic aseptic meningitis, with symptoms of headache, neck, back, abdominal and extremity pain, fever, vomiting, lethargy, and irritability. In 0.5% of cases irreversible paralysis is developed, that rarely lead to death.

[0005] Two types of vaccines were developed and are used worldwide against poliovirus infections. The first type is the inactivated polio vaccine (IPV) developed by Jonas Salk thatwas first introduced in the US in 1955. The Salk vaccine (IPV) includes the following poliovirus strains: Mahoney (type 1), MEF-I (type 2) and Saukett (type 3). The second type is the live attenuated oral poliovirus vaccine (OPV) developed by Albert Sabin. Sabin’s OPV consists of three live attenuated (virulent) Sabin poliovirus strains: Pl / Lsc,2ab (Sabin type 1), P2 / P712,Ch,2ab (Sabin type 2) and P3 / Leon,12alb (Sabin type 3). The effectiveness of poliovirus vaccines relies on inducing the production of neutralizing antibodies.

[0006] There are three serotypes of polioviruses: types 1, 2 and 3. Two of them, types 2 and 3, were recently declared by the world health organization (WHO) as eradicated, and the use of infectious type 2 viruses in live attenuated vaccines was prohibited. Furthermore, the use of replicating type 2 polioviruses was limited to designated poliovirus essential facilities only. Type 3 poliovirus will be the next strain to be withdrawn globally. However, global vaccination by inactivated vaccines will probably continue for all the three types for at least 10 years after eradication due to the uncertainty that all silent circulation had in fact ceased and the probability of polio reemergence due to Vaccine Derived Polio Viruses (VDPVs) or accidental release of virus from vaccine production facilities, a polio laboratory, or a bioterrorism event. Therefore, it is imperative to be able to continue to demonstrate high population immunity to poliovirus during this time.

[0007] The three poliovirus serotypes are distinguished from the other enteroviruses by their susceptibility to neutralization with serotype- specific antisera, use of CD155 as cell surface receptor, and the propensity to cause irreversible paralytic illness. Therefore, the gold- standard assay to measure the level of protective antibodies to poliovirus is the microneutralization assay. However, neutralization requires the use of replicating polioviruses, and therefore the use of this assay for type 2 now and type 3 in the future is currently limited to a small number of poliovirus essential facilities only. Therefore, new assays that do not require the use of replicating polioviruses are required for testing vaccine efficacy and waning, and for population serological screening for exposures.

[0008] The protein shell of the poliovirus is composed of 60 copies of the capsid proteins VP1, VP2, VP3, and VP4, that are arranged on an icosahedral surface. Most of the capsid surface amino acids are VP1 residues. VP2 and VP3 are also partially exposed to the surface and VP4 is internal. Insertion of the N terminus of VP1 may facilitate cell entry either by disrupting a membrane or by forming a pore in a membrane. Several antigenic sites in the VP1 protein of poliovirus type 1 Mahoney strain, were previously identified, some of which are bound by neutralizing antibodies, others are known as binding non-neutralizing antibodies, and the rest are known as binding antibodies without knowledge about theirneutralization potential. Some neutralizing antibodies against poliovirus type 1 VP1 protein, which abolish the ability of the virus to infect, are known as serotype specific with a little cross reactivity with other poliovirus types. A few antigenic sites were also identified as binding antibodies in VP2 and VP3 proteins of type 1 poliovirus, and in VP1, VP2 and VP3 proteins of poliovirus type 2. Both neutralizing and binding antigenic sites were also identified in protein VP1, VP2 and VP3 of poliovirus type 3. However, no assay that detects binding of antibodies to non-infectious poliovirus antigens has been developed to estimate protection from poliovirus infection. A simple, rapid, highly sensitive assay to assess the presence of poliovirus neutralizing antibodies, that does not make use of live, replicating poliovirus is greatly needed.SUMMARY OF THE INVENTION

[0009] The present invention provides arrays comprising probes from at least two polioviruses selected from poliovirus type 1, poliovirus type 2 and poliovirus type 3. Arrays comprising a plurality of probes comprising short peptide probes from at least one poliovirus are also provided. Methods of using the arrays, as well as kits and systems comprising the arrays are also provided.

[0010] According to a first aspect, there is provided an array comprising a plurality of probes each immobilized at a discrete location on the array, wherein the plurality of probes comprises at least two short peptide probes of between 5 and 100 consecutive amino acids from at least one of poliovirus type 1, poliovirus type 2 and poliovirus type 3.[Oi l] According to some embodiments, the plurality of probes comprises at least two short peptide probes of between 5 and 100 consecutive amino acids from the at least one poliovirus.

[0012] According to some embodiments, the plurality of probes comprises a probe from poliovirus type 1, poliovirus type 2 and poliovirus type 3.

[0013] According to some embodiments, the poliovirus type 1 is selected from Sabin type 1, Mahoney type 1, SoAS type 1 and Brunhilde type 1, the poliovirus type 2 is selected from Lansing type 2, Sabin type 2, and MEF-I type 2, and the poliovirus type 3 is selected from Sabin type 3, Leon type 3 and Saukett type 3.

[0014] According to some embodiments, the array comprises probes from Sabin type 1, Sabin type 2, Mahoney type 1, MEF-I type 2 and SoAS type 1.

[0015] According to some embodiments, the plurality of probes further comprises at least one probe selected from a whole virus, a lysed virus, and a virus-like particle (VLP).

[0016] According to some embodiments, the plurality of probes comprises a short peptide from the at least one poliovirus, and wherein the short peptide consists of 10 to 60 amino acids.

[0017] According to some embodiments, the peptide is a sub-sequence from capsid polypeptide VP1, VP2 or VP3.

[0018] According to some embodiments, the peptide comprises between 10-60 consecutive amino acids from a poliovirus protein.

[0019] According to some embodiments, the plurality of probes further comprises at least one of: a. an inactive form of the at least one poliovirus; and b. a VLP of the at least one poliovirus.

[0020] According to some embodiments, the plurality of probes comprises probes comprising at least two poliovirus type 1 antigenic sites selected from those provided in Table 13.

[0021] According to some embodiments, the plurality of probes comprises probes comprising all of the poliovirus type 1 antigenic sites provided in Table 13.

[0022] According to some embodiments, the plurality of probes comprises probes comprising at least two poliovirus type 2 antigenic sites selected from those provided in Table 14.

[0023] According to some embodiments, the plurality of probes comprises probes comprising all of the poliovirus type 2 antigenic sites provided in Table 14.

[0024] According to some embodiments, the plurality of probes comprises probes comprising at least two poliovirus type 3 antigenic sites selected from those provided in Table 15.

[0025] According to some embodiments, the plurality of probes comprises probes comprising all of the poliovirus type 3 antigenic sites provided in Table 15.

[0026] According to some embodiments, the plurality of probes comprises the probes provided in Table 10.

[0027] According to some embodiments, the array comprises serial dilutions of at least one probe, wherein each dilution is immobilized at a discrete location on the array.

[0028] According to some embodiments, the peptide probe is selected from those provided in Table 9.

[0029] According to some embodiments, the plurality of probes comprises the probes provided in Table 1.

[0030] According to some embodiments, the array comprises probes against polio virus type 1, probes against polio virus type 2 and probes against polio virus type 3, wherein the probes against poliovirus type 1 comprise SEQ ID NO: 182, 185, 187, 197, 200, 202, 204, 215, 217, 219, 222-223, 226-228, the probes against poliovirus type 2 comprise SEQ ID NO: 49, 52, 53, 69, 87, 95, 104, 122, 130, 158 and the probes against poliovirus type 3 comprises SEQ ID NO: 260-261, 276-280, 283, 288, 292, 298-299, 302, 309, 312, 315-317, 319, 324-325, 331, 337, 350-355, 358, 361, 366, 369, 372, 374, 378, 381.

[0031] According to some embodiments, the array is for use in determining the presence of neutralizing antibodies against a poliovirus in a sample from a subject.

[0032] According to another aspect, there is provided a method of identifying a subject as having neutralizing antibodies against a poliovirus, the method comprising: a. providing a biological sample from the subject comprising antibodies; b. contacting the sample to an array of the invention in conditions sufficient for antibody binding to the probes; and c. detecting the binding of the antibodies to discrete locations on the array indicating the presence in the sample of antibodies to probes located at the detected discrete locations, wherein a level of binding beyond a predetermined threshold indicates the subject possesses neutralizing antibodies against the poliovirus; thereby identifying a subject as having neutralizing antibodies against a poliovirus.

[0033] According to another aspect, there is provided a method of identifying a subject as having neutralizing antibodies against a poliovirus, the method comprising: a. providing a biological sample from the subject comprising antibodies; b. contacting the sample to an array of the invention in conditions sufficient for antibody binding to the probes;c. detecting the binding of the antibodies to discrete locations on the array indicating the presence in the sample of antibodies to probes located at the detected discrete locations; d. producing an output comprising the level of antibody binding at each discrete location; and e. applying a trained machine learning algorithm to the produced output wherein the trained machine learning algorithm produces a diagnosis indicating the subject does or does not possess neutralizing antibodies against the poliovirus or produces a score indicating the likelihood the subject possesses neutralizing antibodies against the poliovirus; thereby identifying a subject as having neutralizing antibodies against a poliovirus.

[0034] According to some embodiments, a subject not indicated as possessing neutralizing antibodies is further tested by the micro-neutralization assay with live poliovirus to determine if the subject possesses neutralizing antibodies against a poliovirus.

[0035] According to some embodiments, the method is a method of detecting neutralizing antibodies against all of poliovirus type 1, poliovirus type 2 and poliovirus type 3.

[0036] According to some embodiments, the biological sample is a peripheral blood sample, a plasma sample or a serum sample.

[0037] According to some embodiments, the detecting comprises contacting the array with bound antibodies with labeled secondary antibodies against the antibodies in the biological sample.

[0038] According to some embodiments, the detecting further comprises scanning the array with a detector configured to detect the labeled secondary antibodies and producing an output of the discrete locations where antibody was detected.

[0039] According to some embodiments, the detecting is detecting binding to discrete locations comprising probes comprising at least one poliovirus type 1 antigenic sites selected from those provided in Table 13, at least one poliovirus type 2 antigenic sites selected from those provided in Table 14 or at least one poliovirus type 3 antigenic sites selected from those provided in Table 15.

[0040] According to some embodiments, the detecting is detecting binding to discrete locations comprising probes comprising at least two poliovirus antigenic sites selected from those provided in Tables 13-15.

[0041] According to some embodiments, the detecting is detecting binding to discrete locations comprising all provided in Tables 13-15.

[0042] According to some embodiments, the detecting is detecting binding to discrete locations comprising probes comprising those provided in Table 1 and SEQ ID NO: 49, 52, 53, 69, 87, 95, 104, 122, 130, 158, 182, 185, 187, 197, 200, 202, 204, 215, 217, 219, 222- 223, 226-228, 260-261, 276-280, 283, 288, 292, 298-299, 302, 309, 312, 315-317, 319, 324- 325, 331, 337, 350-355, 358, 361, 366, 369, 372, 374, 378, and 381.

[0043] According to some embodiments, the output is a scan of the array indicating intensity of binding at each discrete location.

[0044] According to some embodiments, the output is a summary of the level of binding at each discrete location.

[0045] According to some embodiments, the machine learning algorithm is trained on a training set comprising: a. produced outputs from samples from subjects with known levels of neutralizing antibodies against the poliovirus contacted with the array; and b. labels indicating a sample is from a subject with or without levels of neutralizing antibodies against the poliovirus.

[0046] According to some embodiments, the method is a method of predicting risk of the subject to spread poliovirus following exposure, predicting risk of symptomatic infection of the subject or predicting recent infection of the subject, wherein the presence of neutralizing antibodies indicates the subject is not a risk for spreading poliovirus, is not at risk for symptomatic infection or was recently infected.

[0047] According to another aspect, there is provide a kit comprising the array the invention, and a labeled secondary antibody configured for detection of antibodies bound to the array.

[0048] According to another aspect, there is provided a system comprising the array of the invention, and a detector configured to detect binding of antibodies to probes immobilized on the array.

[0049] According to some embodiments, the detector is configured to detect labeled secondary antibodies.

[0050] According to another aspect, there is provided a method for producing an optimized poliovirus peptide array, the method comprising:a. providing a poliovirus peptide array comprising a plurality of synthetic peptide probes selected from those provided in Table 9; b. contacting the peptide array with a biological sample from a plurality of subjects with known neutralizing antibody status against at least one of poliovirus types 1-3 under conditions sufficient for antibody binding to the probes; c. detecting binding of antibodies to discrete locations on the array indicating the presence in the sample of antibodies to probes located at the detected discrete locations; d. selecting peptides whose detected binding by antibodies correlates with neutralizing antibody status against at least one of the poliovirus types; and e. producing an optimized poliovirus peptide array containing the selected peptides; thereby producing an optimized poliovirus array.

[0051] According to some embodiments, the plurality of subjects is from a common population, wherein a common population comprises a common vaccination history or history of exposure to poliovirus and wherein the array is optimized for the common population, optionally wherein the common population is a population of subjects from the same country.

[0052] According to some embodiments, the provided poliovirus peptide array comprises the probes provided in Table 9.

[0053] According to some embodiments, the selecting comprises applying a trained machine learning algorithm to the detected binding to predict the peptides most important to differentiate between neutralizing antibody status, wherein the machine learning algorithm is trained on a training set comprising binding results from subjects with known neutralizing antibody status, optionally wherein the machine learning model is a Random Forest algorithm.

[0054] According to some embodiments, the selecting further comprises clustering peptides with similar amino acid sequences and selecting only 1 peptide from a cluster, optionally wherein the clustering is by a silhouette clustering method.

[0055] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferredembodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figures 1A-B: Example for polio peptides microarrays. Synthetic 16-20-aa peptides (n=234) were designed to cover selected regions of VP1, VP2 and VP3 capsid proteins of 5 poliovirus strains from types 1 and 2, as reported previously [15-21, 23, 24], and the full sequences of VP1, VP2 and VP3 proteins of Sabin type 2 strain. Similar microarrays were spotted with 152 additional peptides, including additional peptides of VP1 proteins of type 2 MEF strain, as well as peptides covering the full sequence of VP1 type 3 Sabin strain, and selected regions of VP1, VP2 and VP3 capsid proteins of poliovirus type 3 Sabin and Saukett strains (n=152 additional peptides). All peptides were spotted at 1 mg / ml in X-100 triton buffers (8 identical arrays per slide; each array contains 2 blocks, dashed white lines were added to separate the two blocks of the same array). Two arrays that were spotted with 234 type 1 and 2 peptides and hybridized with serum samples of two different individuals (1A and IB) are presented as an example. The sera were diluted 1:1000, and IgG binding to the spotted peptides were detected by anti-human IgG pAb conjugated with Alexa-647. The slides were scanned by 635 nm laser using Genepix 4400A. White spots are saturated. Similar arrays were detected using ant-human IgA pAb conjugated with Alexa- 647 (not shown).

[0057] Figures 2A-B: Calibration of microarrays spotted with inactivated viral antigens of viral-like particles (VLP) of polioviruses. (2A) Left: Image of a slide spotted with four identical viruses / VLP calibration microarrays. Dashed white lines were added to separate between arrays. Each antigen was spotted in 3 different buffers (0.0025% triton X- 100, PBS, SciSpot DI) and in serial dilutions. Right: Image of a representative viruses / VLP calibration microarray, hybridized with a pool of rabbit anti-polio serum samples. The antigens were spotted in serial dilutions from right to left in triplicates (only VLP type 2 was spotted in a single concentration, since the stock concentration was low). Due to the large number of combinations of antigen dilutions and spotting buffers, only four large arrays were spotted on each slide. (2B) An example of calibration array analysis. Poliovirus type 1 antigen (08 / 168) was spotted in 10 serial dilutions (0.1-50 Dag / ml) using two spotting buffers (triton X-100 or PBS). Spotting combinations that generated high MFIs forpoliovirus type 1 antigens that decayed with increased dilutions of the sample, as expected, and lower MFI for types 2 and 3 antigens, were selected for the optimized arrays. MFI = Median Fluorescence Intensity.

[0058] Figures 3A-B: Image of an optimized viruses / VLP antigen microarray (16 small identical arrays on a 25x75 mm slide). (3A) A slide with 16 identical viruses / VLP arrays hybridized with different samples. (3B) Two arrays were hybridized with two different sheep polyclonal control anti-poliovirus antibodies: anti-type 1 (bottom) and anti-type 2 (upper).

[0059] Figure 4: Binding of IgG antibodies to Sabin type 2 poliovirus peptides showing significant differences between vaccination groups of toddlers (two examples). Median Fluorescence Intensity (MFI) staining of serum IgG antibodies binding to poliovirus peptides showing difference between a group of toddlers that were vaccinated with IPV vaccine only (IPV, n = 50) and a group of children that were vaccinated with both IPV and bOPV (IPV + bOPV, n = 100). bOPV included only types 1 and 3 strains, while IPV included strains of the three types. Horizontal lines represent the median values and the box limits correspond to the 25th and the 75th percentiles. The whiskers represent 1.5 times the interquartile range. P-values were adjusted using the Benjamini-Hochberg false discovery rate (FDR) method with an FDR cutoff of q < 0.2. Adjusted p values and q values are presented in the table below the box plots. IPV - inactivated poliovirus vaccine including the 3 types; bOPV - oral attenuated poliovirus vaccine including only type 1 and 3 polioviruses.

[0060] Figure 5: Different levels of IgG antibodies to poliovirus peptides VP1_7 of poliovirus type 1 Mahoney and wildtype SoAS strains in differently vaccinated toddlers. Boxplots represented MFI of serum IgG binding to poliovirus type 1 peptides showing difference between samples from the group that was vaccinated with IPV vaccine only (n = 50) or the group that was vaccinated with both IPV and bOPV (n = 100). The IgG levels to the equivalent Sabin type 1 VP1_7 peptide (SRSESSIESFFARGACVAII; SEQ ID NO: 190, OPV1_VP1_7) were not different (not shown). The sequence of poliovirus type 1 VP1 peptide 7 is conserved, and the only variable amino acid is labeled in purple bold letters. P-values were adjusted using the Benjamini-Hochberg false discovery rate (FDR) method with an FDR cutoff of q < 0.2. Adjusted p values and q values are presented in the table below the box plots. Horizontal lines represent the median values and the box limits correspond to the 25th and the 75th percentiles. The whiskers represent 1.5 times the interquartile range.

[0061] Figures 6A-B: Higher MFI levels of IgG antibodies to IPV viral antigens in the group vaccinated with IPV alone (n=50) as compared with IPV+bOPV groups (n=100). Boxplots represent MFI response to (6A) standard mixtures (NIBSC) of the three inactivated viruses of the IPV (12 / 104, left) and trivalent OPV (17 / 160, right) vaccines, (6B) VLP antigens and Horizontal lines represent the median values and the box limits correspond to the 25th and the 75th percentiles. The whiskers represent 1.5 times the interquartile range. P-value was computed by Wilcoxon rank-sum test. The antigens are described in Table 1.

[0062] Figures 7A-B: Higher levels of IgG antibodies to poliovirus type 1 peptides in those vaccinated by both IPV and bOPV and also possibly exposed to type 1 SoASl wildtype strain (IPV + bOPV + possible Exposure, n=26) compared to those vaccinated with the IPV vaccine only (IPV, n=50). (7A) SoAS VP1 peptide 7. This peptide bound higher levels of antibodies also in IPV+bOPV vaccine groups compared with IPV alone (see Fig. 5). (7B) Higher IgG levels to additional peptides in the IPV+bOPV+possible exposure group that did not bind significantly higher IgG levels in the IPV+bOPV vaccine groups. The sequences of overlapping peptides are written below the box plots. The sequence of poliovirus type 1 VP1 peptide 29 is conserved, and the variable amino acid is labeled by purple bold letters. Peptides 1 and 2 of VP3 protein of Sabin type 1 poliovirus are partially parallel, and the common sequence is underlined. Peptide 1 of Sabin type 1 VP3 was also trending to bind higher IgG level in unexposed IPV+bOPV vaccine group compared to IPV only vaccine group. MFI of IgG binding to poliovirus peptides is shown. Horizontal lines represent the median values and the box limits correspond to the 25th and the 75th percentiles. The whiskers represent 1.5 times the interquartile range. P-values were adjusted using the Benjamini-Hochberg false discovery rate (FDR) method with an FDR cutoff of q < 0.2. Adjusted p values and q values are presented.

[0063] Figures 8A-B: Different levels of IgG antibodies to poliovirus type 2 peptides in the IPV+bOPV+pos.Exposure group (n=26) compared with IPV alone (n=50). Box plots of (8A) Higher or (8B) lower IgG levels to poliovirus type 2 Sabin or MEF peptides in the IPV+bOPV+Exposure group. Horizontal lines represent the median values and the box limits correspond to the 25th and the 75th percentiles. The whiskers represent 1.5 times the interquartile range. P-values were adjusted using the Benjamini-Hochberg false discovery rate (FDR) method with an FDR cutoff of q < 0.2. Adjusted p values and q values are presented.

[0064] Figure 9: Lower levels of IgG antibodies to VLP antigens in the IPV+bOPV+pos.Exposure group (n=26) compared with IPV alone (n=50). MFI of type1 VLP and Type 2 VLP. Lower level of IgG in the IPV+bOPV+possible exposure group compared with IPV alone. Horizontal lines represent the median values and the box limits correspond to the 25th and the 75th percentiles. The whiskers represent 1.5 times the interquartile range. P-value was computed by Wilcoxon rank-sum.

[0065] Figure 10: IgG binding to poliovirus peptides showing differences between vaccination groups IPV + IX bOPV vs. IPV + 2X bOPV. IPV was always given before OPV. Box plots represent the MFI response to poliovirus peptides comparing children that were vaccinated with IPV vaccine and a single dose of bOPV (n=50) and children that were vaccinated with IPV and two doses of bOPV (n=50). (First and second rows) Poliovirus type 1 peptides. (Bottom row) Poliovirus type 2 peptides. Toddlers that were vaccinated with two bOPV doses had lower IgG levels to these peptides compared with toddlers that were vaccinated with only a single bOPV dose. Horizontal lines represent the median MFI and the box limits correspond to the 25th and the 75th percentiles. The whiskers represent 1.5 times the interquartile range. P-values were computed using the Wilcoxon rank-sum test. P- values were adjusted using the Benjamini Hochberg false discovery rate, using a cutoff of 0.2.

[0066] Figure 11: Correlation between neutralization titers of different poliovirus (PV) type, as measured by microneutralization assay. One was added to each titer value, to avoid zero values. The log2 of (titer+1) was calculated for each poliovirus type and each individual separately. Each dot represents an individual, the X value is the log2 (titer+1) of one PV type, and the Y value is the log2 (titer+1) of another PV type. Correlation between neutralization titers of poliovirus types 1 (PV1) and 3 (PV3) was the highest, and the correlation between types 1 (PV1) and 2 (PV2) was the lowest.

[0067] Figures 12A-D: IgG antibodies binding to specific sets of viral and peptide antigens were associated with type-specific poliovirus neutralization in toddlers (n=176). (12A-B) IgG gmean magnitude to peptides of VP3 (Fig. 17A) and VP1 (Fig. 17B) envelope proteins of poliovirus type 1 OPV1 strain were associated with protection from poliovirus 1. (12C) IgG binding to poliovirus type 2 VLP2 was associated with protection from poliovirus type 2. (12D) Gmean magnitude of IgG binding to the peptides covering antigenic site 1 on poliovirus type 2 MEF strain was associated with protection from poliovirus type 2. Protection was defined as neutralization titer>l:8. Horizontal lines represent the median values and the box limits correspond to the 25th and the 75th percentiles. The whiskers represent 1.5 times the interquartile range. P-values were computed by Wilcoxon rank-sum.

[0068] Figure 13: Higher levels of IgA binding to viral antigens are associated with protection from poliovirus type 2 in toddlers (n=176). Poliovirus viral antigens from different serotypes and strains, as well as mixtures of inactivated trivalent IPV and OPV vaccine strains, were associated with poliovirus type 2 protection. Horizontal lines represent the median values and the box limits correspond to the 25th and the 75th percentiles. The whiskers represent 1.5 times the interquartile range. P-values were computed by Wilcoxon rank- sum.

[0069] Figure 14: High IgA antibodies binding to two antigenic sites of the wildtype poliovirus serotype 1 SoAS strain are associated with lack of protection from serotype 3 polioviruses (PV3) in toddlers (n=176). Protection was defined as neutralization titer >=8 for PV3. Horizontal lines represent the median values and the box limits correspond to the 25th and the 75th percentiles. The whiskers represent 1.5 times the interquartile range. P- values were computed by Wilcoxon rank-sum.

[0070] Figure 15: Venn diagram of individuals that lack protection from one or more poliovirus serotypes. Each circle denotes the number of individuals that lack protection for each individual serotypes. Shared discs represent individuals that lack protection from more than one serotype. Neutralization is defined by neutralization titer < 1:16.

[0071] Figures 16A-B: Tables of (16A) antigenic sites of polioviruses types 1 and 2 covered by peptides in the extended PAM assay and (16B) antigenic sites of polioviruses type 3 covered by peptides in the extended PAM assay.

[0072] Figures 17A-B: Associations of serum IgG to viral antigens with poliovirus protection in Israeli adults. (17A) Each plot includes swarmplots of the IgG binding to a specific viral antigen, comparing protected individuals (blue) and unprotected individuals (orange) from each of the three polio serotypes (PV1 - type 2, PV2 - type 2, PV3 - type 3; n=223). Each viral antigen was spotted in serial dilutions and the antibody binding to this antigen was defined as the area under the curve (AUC) of the MFI as the function of antigen concentration. Groups were compared using the Wilcoxon ranksum test. Significant associations are marked. * p < 0.05, ** p < 0.01 *** p < 0.001, **** p < 0.00001. (17B) Spider plots of the mean IgG AUC to VLPs and polio viral antigens comparing protected individuals (blue) vs. unprotected individuals (orange). Each spoke represents an individual antigen, and frame colors denote the antigen type (n=223).

[0073] Figures 18A-B: Associations of serum IgA of Israeli adults to viral antigens with poliovirus protection. (18A) Each plot includes swarplots of the IgA binding to a specificviral antigen, comparing protected individuals (blue) and unprotected individuals (orange) from each of the three polio serotypes (PV1 - type 2, PV2 - type 2, PV3 - type 3; n=223). Each viral antigen was spotted in serial dilutions and the antibody binding to this antigen was defined as the area under the curve (AUC) of the MFI as the function of antigen concentration. Groups were compared using the Wilcoxon ranksum test. Significant associations are marked. * p < 0.05, ** p < 0.01 *** p < 0.001, **** p < 0.00001. (18B) Spider plots of the mean IgA AUC to VLPs and polio viral antigens comparing protected individuals (blue) vs. unprotected individuals (orange). Each spoke represents an individual antigen, and frame colors denote the antigen type (n=223).

[0074] Figures 19A-C: Associations between serum IgG binding to linear peptide antigens spanning poliovirus antigenic sites with protection of Israeli adults from polioviruses. (19A) Associations between IgG binding to linear peptide antigens spanning types 1 and 2 poliovirus antigenic sites with protection from the three poliovirus types. Each plot includes swarmplots of the IgG GMM to linear peptides that span a specific antigenic site from poliovirus types 1 and 2 comparing protected individuals (blue) and unprotected individuals (orange) to each of the three polio serotypes (n=223). Groups were compared using the Wilcoxon ranksum test. Significant associations are marked. * p < 0.05, ** p < 0.01 ***, p < 0.001, **** p < 0.00001. (19B) Spider plots of the mean IgG GMM to all the tested antigenic sites of types 1 and 2 comparing protected individuals (blue) vs. unprotected individuals (orange). Each spoke represents an individual antigenic site, and colors denote the antigen type: blue - type 1 antigenic sites that were previously reported as binding neutralizing mAbs, purple - type 1 antigenic sites that were previously reported as binding non-neutralizing mAbs, green - type 2 antigenic sites that their ability to bind neutralizing mAbs has not been characterized (n= 223). (19C) An association between IgG binding to linear peptides spanning a specific type 3 antigenic site with protection from type 3 poliovirus. IgG GMM to linear peptides that span a specific antigenic site from poliovirus type 3 comparing protected individuals (blue) and unprotected individuals (orange) from each of the three polio serotypes (n= 60). Groups were compared using the Wilcoxon ranksum test. Significant associations are marked. * p < 0.05, ** p < 0.01 ***, p < 0.001, **** p < 0.00001.

[0075] Figures 20A-E: Associations between serum IgA binding to linear peptide antigens spanning poliovirus antigenic sites with protection of Israeli adults from the three poliovirus types. (20A) Each plot includes swarmplots of the IgA GMM to linear peptides that span a specific antigenic site from poliovirus types 1 and 2 comparing protectedindividuals (blue) and unprotected individuals (orange) from each of the three polio serotypes (n=223). Groups were compared using the Wilcoxon ranksum test. Significant associations are marked. * p < 0.05, ** p < 0.01 ***, p < 0.001, **** p < 0.00001. (20B) Spider plots of the mean IgA GMM to all the tested antigenic sites of types 1 and 2 comparing protected individuals (blue) vs. unprotected individuals (orange). Each spoke represents an individual antigenic site, and colors denote the antigen type: blue - type 1 antigenic sites that were previously reported as binding neutralizing mAbs, purple - type 1 antigenic sites that were previously reported as binding non-neutralizing mAbs, green - type 2 antigenic sites that their ability to bind neutralizing mAbs has not been characterized (n= 223). (20C) Each plot includes swarmplots of the IgA GMM to linear peptides that span a specific antigenic site from poliovirus type 3 comparing protected individuals (blue) and unprotected individuals (orange) from each of the three polio serotypes (n= 60). Groups were compared using the Wilcoxon ranksum test. Significant associations are marked. * p < 0.05, ** p < 0.01 ***, p < 0.001, **** p < 0.00001. (20D) A summary for all of the associations between IgG or IgA GMM binding to types 1 and 2 antigenic sites and neutralizing titers of the three types. (20E) A summary for all of the associations between IgG or IgA GMM binding to type 3 antigenic sites and neutralizing titers of the three types.

[0076] Figures 21A-I: Associations between serum IgG or IgA binding to linear peptide antigens spanning poliovirus antigenic sites with protection of DRC children from the three poliovirus types. (21A) Summary of neutralization status of antibodies from the DRC cohort (n=1507) and in the selected subset of 320 samples selected for profiling by PAM microarrays. (21B-G) Representative charts for the DRC cohort associations between (21B, 21D, 21F) IgG and (21C, 21E, 21G) IgA binding to linear peptide antigens spanning (21B- C) types 1, (21D-E) type 2 and (21F-G) type 3 poliovirus antigenic site types. Each plot includes swarmplots of the IgG or IgA GMM to linear peptides that span a specific antigenic site comparing protected individuals (blue) and unprotected individuals (orange) from each of the three polio serotypes. Groups were compared using the Wilcoxon ranksum test. Significant associations are marked. * p < 0.05, ** p < 0.01 ***, p < 0.001, **** p < 0.00001. (21H-I) Summary tables for all of the associations between IgG or IgA binding to (21H) types 1, 2 (211) and type 3 antigenic sites and neutralizing titers of the three types.

[0077] Figure 22: Performance of logistic regression models for predicting protection from polioviruses using IgA levels to VLP and viral antigens. The inventors trained logistic regression models to predict protection from each individual polio serotype using the antibody AUC scores for each of the individual VLP and viral antigens as input features.Protection was defined as neutralizing titers < 1:16. Models were trained using a leave-one- out cross validation framework. Each panel presents the area under the curve of the receiver operating curve (AUC-ROC).

[0078] Figure 23: IgG and IgA peptide measurements are not correlated to each other, but correlated among themselves. Spearman’s correlation coefficients measure the relationship between the IgG and IgA peptide data. The inventors computed the pairwise correlations between the antibody profiles of individual peptides across all individuals for each polio serotype separately. The triangular matrix presents the Spearman correlation coefficient between every pair of features, including both IgG and IgA.

[0079] Figure 24: Silhouette score for selecting number of clusters for type 1 peptides. The optimal number of clusters was determined by identifying the cluster count that yields the highest average silhouette score.

[0080] Figure 25: Comparison of the performance of PAM based prediction models for poliovirus neutralization. Precision recall curves comparing three different peptide based antigen sets for training random forest models: (1) Complete data (green) - a profile of the antibody reactivity to the complete set of peptides from each given polio serotype; (2) Collinearity corrected data (orange) - a subset of peptide antigens selected solely after adjusting multi-collinearity and feature selection using nested cross-validation random forest model; and (3) Clustering based feature selection (blue) - a subset of peptide antigens selected using a combination of clustering and feature selection using a nested cross- validation random forest model. All models were evaluated for predicting polio neutralization for each polio serotype: PV1 - type 1 neutralization titer (NT), PV2 - type 2 NT, PV3 - type 3 NT.

[0081] Figures 26A-C: Optimal number of peptides to predict protection from the same poliovirus type. Precision scores for different subset sizes of peptides averaged scores across different random seeds. (26A) Fifteen (15) type-1 peptides are sufficient to optimally predict protection for serotype 1 poliovirus, with prediction accuracy of 93.8%. (26B) Ten (10) type-2 peptides are sufficient to optimally predict protection for serotype 2 poliovirus, with prediction accuracy of 82.0%. (26C) Thirty-five (35) type-3 peptides are required to optimally predict protection for serotype 3 poliovirus, with prediction accuracy of 87.0%.

[0082] Figures 27A-F: Performance of neutralization prediction of each poliovirus serotype, based on the model composed of the selected peptides from the same type. (27A, 27C, 27E) The confusion matrix of (27A) type 1 prediction, (27C) type 2 prediction,or (27E) type 3 prediction, where x-axis counts the predicted values and y-axis counts the actual values. Top left corner of the matrix (0,1) is the False positive and bottom left comer (1,0) is the False negative (n=184 samples). (27B, 27D, 27F) The precision-recall curve for different classification thresholds for (8B) type 1, (27D) type 2, or (27F) type 3 prediction by the type-specific models. (27A-B) The performance of a type 1 prediction model trained on 15 optimally selected type-1 peptides achieved 93% precision and an AUC score of 93.9%. (27C-D) The performance of a type 2 prediction model trained on 10 optimally selected type-2 peptides achieved 80% precision and a P-R AUC score of 86.0%. (27E-F) The performance of a type 3 prediction model trained on 35 optimally selected type-3 peptides achieved 87% precision and a P-R AUC score of 83.1%.DETAILED DESCRIPTION OF THE INVENTION

[0083] The present invention, in some embodiments, provides arrays comprising probes from at least two polioviruses selected from: poliovirus type 1, poliovirus type 2 and poliovirus type 3. Arrays comprising a plurality of probes comprising short peptide probes from at least one poliovirus are also provided. Methods of using the arrays, analyzing the arrays, selecting probes for the arrays as well as kits and systems comprising the arrays are also provided.

[0084] Polio arrays

[0085] By a first aspect, there is provided an array comprising a plurality of probes each immobilized at a discrete location on the array, wherein the plurality of probes comprises a probe from a first poliovirus and a probe from a second poliovirus.

[0086] By another aspect, there is provided an array comprising a plurality of probes each immobilized at a discrete location on the array, wherein the plurality of probes comprises a peptide probe from at least one poliovims.

[0087] In some embodiments, the array is a solid support. In some embodiments, the solid support is a chip. As used herein, the term “array” refers to a solid support with regularly spaced probes attached to distinct and defined locations. In some embodiments, an array is an array of probes. In some embodiments, the support or array comprises probes at known locations. Thus, the location of each probe is known and so binding to a given probe can be correlated to the probe itself based on its position on the support or array. In some embodiments, an array is a single solid support with probes arrayed thereupon. In someembodiments, an array is a plurality of solid supports with probes arrayed thereupon. In some embodiments, each probe is on a separate solid support. In some embodiments, an array is an array of beads. In some embodiments, an array is an array of solid supports. Methods of making arrays and in particular protein and peptide arrays are well known in the art. Any method of making an array such as described herein may be employed. One such method is provided hereinbelow in the Materials and Methods section. Non-limiting examples of methods of producing protein / peptide arrays include U.S. Pat. No. 5,143,854, U.S. Patent Application Publication Nos. 2007 / 0154946, 2007 / 0122841, 2007 / 0122842, and 2008 / 0108149 and International Patent Application Publication No. WO / 2000 / 003307.

[0088] The solid support, or support, refers to a material or group of materials having a rigid or semi-rigid surface or surfaces. In some embodiments, at least one surface of the solid support will be substantially flat, although in some embodiments it may be desirable to physically separate synthesis regions for different molecules with, for example, wells, raised regions, beads, pins, etched trenches, or the like. In certain embodiments, the solid support may be porous. In some embodiments, the solid support is glass. In some embodiments, the solid support is coated. In some embodiments, the solid support is uncoated. In some embodiments, the coating adheres amines. In some embodiments, the coating adheres lysine residues. In some embodiments, the coating adheres amino termini of proteins. In some embodiments, the coating adheres 6-His sequences. In some embodiments, the coating adheres biotin residues.

[0089] Support materials useful in embodiments of the present invention include, for example, silicon, bio-compatible polymers such as, for example poly(methyl methacrylate) (PMMA) and polydimethylsiloxane (PDMS), glass, SiO2 (such as, for example, a thermal oxide silicon wafer such as that used by the semiconductor industry), quartz, silicon nitride, functionalized glass, gold, platinum, and aluminum. Functionalized surfaces include for example, amino-functionalized glass, carboxy functionalized glass, and hydroxy functionalized glass. Additionally, a support may optionally be coated with one or more layers to provide a surface for molecular attachment or functionalization, increased or decreased reactivity, binding detection, or other specialized application. Support materials and or layer(s) may be porous or non-porous. For example, a support may be comprised of porous silicon. Additionally, the support may be a silicon wafer or chip such as those used in the semiconductor device fabrication industry. In the case of a wafer or chip, a plurality of arrays may be synthesized on the wafer. A person skilled in the art would know how to select an appropriate support material.

[0090] In some embodiments, the plurality of probes is immobilized on the array. In some embodiments, the plurality of probes is linked to the array. In some embodiments, the immobilization is via linkage. In some embodiments, the plurality of probes is directly linked to the array. In some embodiments, the plurality of probes is indirectly linked to the array. In some embodiments, the linking is via a linker. In some embodiments, the linker is an amino acid linker. In some embodiments, the linker is at least one lysine residue. In some embodiments, the linker is a plurality of lysine residues. In some embodiments, the linker is two lysine residues. In some embodiments, the linker is KK. In some embodiments, the linker is at least one histidine residue. In some embodiments, the linker is a plurality of histidine residues. In some embodiments, the linker is six histidine residues. In some embodiments, the linker is a biotin residue. In some embodiments, the linker is an N-terminal linker. In some embodiments, the linker is a C-terminal linker. In some embodiments, the linker is an N-terminal or C-terminal linker.

[0091] The peptides, proteins, virus like particles (VLPs) and viruses present on the array may be linked covalently or non-covalently to the array and can be attached to the array support (e.g., silicon or other relatively flat material) by cleavable linkers. A linker molecule can be a molecule inserted between the support and peptide that is being synthesized, and a linker molecule may not necessarily convey functionality to the resulting peptide, such as molecular recognition functionality, but instead elongates the distance between the support surface and the peptide functionality to enhance the exposure of the peptide functionality on the surface of the support. Preferably a linker should be about 4 to about 120 atoms long to provide exposure. In some embodiments, a linker is at least 40 atoms long. In some embodiments, a linker is at least 48 atoms long. In some embodiments, a linker is between 40 and 150 atoms long. In some embodiments, a linker is about 48 atoms long. In some embodiments, a linker is about 120 atoms long. The linker molecules may be, for example, aryl acetylene, ethylene glycol oligomers containing 2-10 monomer units (PEGs), diamines, diacids, amino acids, biotin, among others, and combinations thereof. A person skilled in the art would know how to design appropriate linkers. In some embodiments, a probe is immobilized on the array but not linked. In some embodiments, a virus is immobilized but not linked. In some embodiments, a VLP is immobilized but not linked. In some embodiments, a peptide is immobilized but not linked. In some embodiments, a link is reversible. In some embodiments, linking is printing the probe on the array. In some embodiments, a peptide is printed. In some embodiments, a recombinant protein is printed.

[0092] In some embodiments, each probe is located at a discrete location on a support. In some embodiments, each probe is located at a discrete location on an array. In some embodiments, each probe is immobilized at a discrete location. In some embodiments, each probe is distinctly immobilized. It will be understood by a skilled artisan that each probe must be able to be uniquely detected such that upon reading / scanning the array, the precise probe bound by an antibody can be determined. In some embodiments, each probe is immobilized on a separate support. In some embodiments, each probe is immobilized in a separate region of a support or array. In some embodiments, each probe is located or immobilized such that they can be uniquely measured or detected. In some embodiments, each probe is located or immobilized such that an antibody binding to the probe can be uniquely measured or detected.

[0093] In some embodiments, the plurality of probes comprises at least one probe from a first poliovirus. In some embodiments, the plurality of probes comprises at least one probe from a second poliovirus. In some embodiments, the plurality of probes comprises at least one probe from a third poliovirus. In some embodiments, the plurality of probes comprises at least one probe from at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 poliovirus. Each possibility represents a separate embodiment of the invention. In some embodiments, the first and second polioviruses are different polioviruses. In some embodiments, the first and second polioviruses are different types of polioviruses. In some embodiments, the first and second poliovirus are different strains of poliovirus. In some embodiments, the first and second polioviruses are the same type of poliovirus and different strains of poliovirus. As used herein a “type” refers to a specific poliovirus defined by the proteins expressed by the poliovirus. There are three types of poliovirus, poliovirus type 1, poliovirus type 2 and poliovirus type 3. In some embodiments, the array comprises probes from at least one of poliovirus type 1, poliovirus type 2 and poliovirus type 3. In some embodiments, the array comprises probes from at least two of poliovirus type 1, poliovirus type 2 and poliovirus type 3. In some embodiments, the array comprises probes from poliovirus type 1 and poliovirus type 2. In some embodiments, the array comprises probes from poliovirus type 2 and poliovirus type 3. In some embodiments, the array further comprises provided from poliovirus type 3. In some embodiments, the array comprises probes from poliovirus type 1, poliovirus type 2 and poliovirus type 3.

[0094] As used herein, a “strain” refers to a mutational genetic variant. In some embodiments, a strain is from a given year. In some embodiments, a strain is from a given location. In some embodiments, poliovirus type 1 is selected from Sabin type 1, Mahoneytype 1, SoAS type 1 and Brunhilde type 1. In some embodiments, poliovirus type 2 is selected from Lansing type 2, Sabin type 2, and MEF-1 type 2. In some embodiments, poliovirus type 3 is selected from Sabin type 3, Leon type 3 and Saukett type 3. In some embodiments, the array comprises probes from Sabin type 1, Sabin type 2, Mahoney type 1, MEF-I type 2 and SoAS type 1.

[0095] As used herein the term “probe” refers to a part from a virus or a whole virus that contains at least one epitope that can be bound by an antibody. In some embodiments, a probe is a whole virus. In some embodiments, the probe is a virus-like particle (VLP). As used herein, the term “virus-like particle” refers to a synthetic multiprotein structure that mimics the organization and conformation of authentic poliovirus. VLPs, however, lack a viral genome. In some embodiments, the VLP comprises three poliovirus capsid proteins. In some embodiments, the VLP comprises three poliovirus envelope proteins. In some embodiments, the three envelope proteins are VP1, VP2 and VP3. In some embodiments, the three capsid proteins are VP1, VP2 and VP3. In some embodiments, the VLP further comprises protein 3CD. In some embodiments, a probe is a lysed virus. In some embodiments, a probe is a fraction from a lysed virus. In some embodiments, a probe is a whole protein. In some embodiments, the protein is a recombinant protein. In some embodiments, a probe is a portion of a protein. In some embodiments, the portion comprises a functional domain. In some embodiments, the protein is a peptide. In some embodiments, the probe comprises amino acids. In some embodiments, the probe comprises viral protein. In some embodiments, the probe comprises a viral epitope. In some embodiments, the epitope is an immunological epitope. In some embodiments, the probe is selected from a whole virus, a lysed virus, a VLP, a whole recombinant protein and a peptide. In some embodiments, the probe is selected from a whole virus and a recombinant protein. In some embodiments, a recombinant protein is a whole protein. In some embodiments, the virus is inactivated virus.

[0096] As used herein, the terms “peptide”, and " polypeptide " are used interchangeably to refer to a polymer of amino acid residues. In another embodiment, the terms "peptide", "polypeptide" and "protein" as used herein encompass native peptides, peptidomimetics (typically including non-peptide bonds or other synthetic modifications) and the peptide analogues peptoids and semipeptoids or any combination thereof. In another embodiment, the peptides polypeptides and proteins described have modifications rendering them more stable while in the body or more capable of penetrating into cells. In one embodiment, the terms “peptide”, "polypeptide" and "protein" apply to naturally occurring amino acidpolymers. In another embodiment, the terms “peptide”, "polypeptide" and "protein" apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid. In some embodiments, the probe comprises a peptide. It will be understood that even a full virus will inherently comprise a peptide and it will comprise an amino acid. Similarly, a VLP and a recombinant protein must comprise a peptide.

[0097] In some embodiments, the peptide is a purified peptide. In some embodiments, the peptide is an isolated peptide. In some embodiments, the peptide is a recombinant peptide. In some embodiments, the peptide is a synthetic peptide. As used herein, the term "isolated peptide" refers to a peptide that is essentially free from contaminating cellular components, such as carbohydrate, lipid, or other proteinaceous impurities associated with the peptide in nature. Typically, a preparation of isolated peptide contains the peptide in a highly purified form, i.e., at least about 80% pure, at least about 90% pure, at least about 95% pure, greater than 95% pure, or greater than 99% pure. In some embodiments, a synthetic peptide is at least 99% pure. In some embodiments, a synthetic peptide is about 100% pure.

[0098] In some embodiments, the peptide is a fragment of a poliovirus protein. In some embodiments, the peptide is a protein fragment that retains an immunogenic epitope. In some embodiments, the peptide is a linear peptide. In some embodiments, the peptide is a conformational peptide. In some embodiments, the peptide contains three-dimensional structure. As used herein, the term “conformational peptide” refers to a peptide that forms a three-dimensional structure and a “linear peptide” refers to a peptide that does not from a three-dimensional structure but rather remains linear. A linear peptide is generally a short peptide. Similarly, a “conformational epitope” refers to a 3D epitope that can be found on long peptides, whole recombinant proteins, VLPs and whole virus. A “linear epitope” is a non-3D epitope that remains essentially linear. Linear epitopes are present in the array as short linear peptides.

[0099] In some embodiments, the peptide comprises a domain from a poliovirus protein. In some embodiments, the peptide comprises a functional domain from an poliovirus protein. In some embodiments, the peptide comprises a motif from an poliovirus protein. In some embodiments, the peptide comprises sufficient amino acids to retain a secondary structure found in an intact protein. In some embodiments, the secondary structure is a three- dimensional structure. In some embodiments, the peptide comprises a functional fragment of the protein. In some embodiments, the probe is functional. In some embodiments, the probe comprises a 3D functional epitope. In some embodiments, the probe comprises aconformational epitope. In some embodiments, the probe comprises a linear epitope. In some embodiments, a linear epitope does not comprise secondary structure / 3D structure / a conformational epitope.

[0100] In some embodiments, the probe is a full protein. In some embodiments, the protein is a recombinant protein. In some embodiments, the protein is an isolated protein. In some embodiments, the protein comprises a post-translational modification. In some embodiments, the post-translational modification is glycosylation. In some embodiments, the probe is a full virus. In some embodiments, the probe is a lysed virus. In some embodiments, the virus is an inactivated virus. It will be understood by a skilled artisan that full proteins and full viruses are likely to be properly folded and thus provide 3D, conformational epitopes, while long peptides may or may not have conformational epitopes and not just linear epitopes and short peptides are likely to just have linear epitopes.

[0101] In some embodiments, a peptide is a protein. In some embodiments, a peptide is a part of a protein. In some embodiments, a peptide comprises a functional domain of a protein. In some embodiments, a peptide is a complete protein. In some embodiments, a complete protein is a whole protein. In some embodiments, a complete protein comprises a signal peptide. In some embodiments, a complete protein lacks a signal peptide. In some embodiments, the protein is a recombinant protein. In some embodiments, the probe is a complete protein. Recombinant proteins, VLPs and inactivated viruses can be produced by any method known in the art, or can be purchased for commercial supplies, such as for example NIBSC and The Native Antigen Company.

[0102] In some embodiments, the peptide is a short peptide. In some embodiments, a peptide comprises at least 5, 7, 10, 12, 14, 15, 16, 18, 20 or 25 amino acids. Each possibility represents a separate embodiment of the invention. In some embodiments, a peptide comprises at least 10 amino acids. In some embodiments, a peptide comprises at least 14 amino acids. In some embodiments, a peptide comprises at least 20 amino acids. In some embodiments, a peptide is not a complete protein. In some embodiments, a peptide comprises between 5 and 200, 5 and 150, 5 and 100, 5 and 90, 5 and 90, 5 and 70, 5 and 60, 5 and 58, 5 and 50, 5 and 40, 5 and 30, 5 and 25, 5 and 22, 10 and 200, 10 and 150, 10 and 100, 10 and 90, 10 and 90, 10 and 70, 10 and 60, 10 and 58, 10 and 50, 10 and 40, 10 and 30, 10 and 25, 10 and 22, 12 and 200, 12 and 150, 12 and 100, 12 and 90, 12 and 90, 12 and 70, 12 and 60, 12 and 58, 12 and 50, 12 and 40, 12 and 30, 12 and 25, 12 and 22, 14 and 200, 14 and 150, 14 and 100, 14 and 90, 14 and 90, 14 and 70, 14 and 60, 14 and 58, 14 and 50, 14 and 40, 14 and 30, 14 and 25, 14 and 22, 15 and 200, 15 and 150, 15 and 100, 15 and 90,15 and 90, 15 and 70, 15 and 60, 15 and 58, 15 and 50, 15 and 40, 15 and 30, 15 and 25 or 15 and 22 amino acids. Each possibility represents a separate embodiment of the invention. In some embodiments, a peptide comprises between 5 and 60 amino acids. In some embodiments, a peptide comprises between 10 and 60 amino acids. In some embodiments, a peptide comprises between 14 and 60. In some embodiments, a peptide comprises between 5 and 58 amino acids. In some embodiments, a peptide comprises between 10 and 58 amino acids. In some embodiments, a peptide comprises between 14 and 58. In some embodiments, a peptide comprises between 5 and 30 amino acids. In some embodiments, a peptide comprises between 10 and 30 amino acids. In some embodiments, a peptide comprises between 14 and 30. In some embodiments, a peptide comprises between 5 and 25 amino acids. In some embodiments, a peptide comprises between 10 and 25 amino acids. In some embodiments, a peptide comprises between 14 and 25. In some embodiments, a peptide comprises at most 22, 25, 30, 35, 40, 45, 50, 58, 60, 70, 80, 90, 100, 125, 150, 175, 200 or 250 amino acids. Each possibility represents a separate embodiment of the invention. In some embodiments, a peptide comprises at most 60 amino acids. In some embodiments, a peptide comprises at most 100 amino acids. In some embodiments, a peptide comprises at most 22 amino acids. In some embodiments, a peptide comprises at most 25 amino acids. In some embodiments, a peptide comprises between 18 and 22 amino acids. In some embodiments, the amino acids are consecutive amino acids from a poliovirus protein.

[0103] In some embodiments, the plurality of probes comprises a probe comprising an amino acid sequence of a poliovirus protein. In some embodiments, the plurality of probes comprises a probe consisting of an amino acid sequence of a poliovirus protein. In some embodiments, the plurality of probes comprises a protein probe from a first poliovirus. In some embodiments, the plurality of probes comprises a protein probe from a second poliovirus. In some embodiments, the protein is a surface protein. In some embodiments, the protein is a capsid protein. In some embodiments, a capsid protein is a capsid polypeptide. In some embodiments, the protein is a VP1 protein. In some embodiments, the protein is a VP2 protein. In some embodiments, the protein is a VP3 protein. In some embodiments, the protein is a recombinant protein. In some embodiments, the protein is a secreted protein. It will be understood by a skilled artisan that by using a protein, secondary structures and intramolecular bonds and interactions will be preserved. In some embodiments, a probe comprises a whole poliovirus protein. In some embodiments, a probe consists of a whole poliovirus protein. In some embodiments, the plurality of probes comprises a first probe that consists of a whole protein and a second probe that consists of a whole protein. In someembodiments, each probe is present in both the first poliovirus and the second poliovirus. In some embodiments, each probe is present in all polioviruses of the array.

[0104] In some embodiments, the plurality of probes comprises a linear epitope probe and a conformational epitope probe. In some embodiments, the plurality of probes comprises a short peptide probe and a whole virus probe. In some embodiments, the plurality of probes comprises a short peptide probe and a recombinant protein probe. In some embodiments, the plurality of probes comprises a short peptide probe and a recombinant VLP capsid. In some embodiments, the plurality of probes comprises a short peptide probe and a VLP. In some embodiments, the recombinant protein probe is the whole protein. In some embodiments, the recombinant protein probe is a VLP composed of at least the 3 viral surface proteins. In some embodiments, the 3 viral surface proteins are VP1, VP2 and VP3. In some embodiments, the recombinant protein probe is a fragment of the protein comprising a structural domain. In some embodiments, the plurality of probes comprises a short peptide probe and at least one of a recombinant protein probe and a whole virus probe. In some embodiments, a short peptide comprises 10-60 consecutive amino acids from the poliovirus protein. In some embodiments, a short peptide consists of 10-60 consecutive amino acids from the poliovirus protein. In some embodiments, a short peptide comprises 18-22 consecutive amino acids from the poliovirus protein. In some embodiments, a short peptide consists of 18-22 consecutive amino acids from the poliovirus protein. It will be understood that when a short peptide is said to comprise a specific range of amino acids, it means that peptides with larger numbers of amino acid are excluded and the range is the minimum and maximum number of amino acids that can be part of the probe. In some embodiments, each of the first and second polioviruses comprise a plurality of probes comprising a short peptide probe and at least one of a recombinant protein probe and a whole virus probe.

[0105] In some embodiments, the plurality of probes comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 peptides from a poliovirus. Each possibility represents a separate embodiment of the invention. In some embodiments, the plurality of probes comprises at least 2 peptides from a poliovirus. In some embodiments, the plurality of probes comprises 2 peptides from a poliovirus. In some embodiments, the plurality of probes comprises probes from at least 2 different poliovirus proteins. In some embodiments, the two proteins are selected from VP1, VP2 and VP3. In some embodiments, the two proteins are VP1 and VP2. In some embodiments, the two proteins are VP1 and VP3. In some embodiments, the two proteins are VP2 and VP3. In some embodiments, the plurality of probes comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 peptides from each poliovirus. Each possibility represents a separateembodiment of the invention. In some embodiments, the plurality of probes comprises at least 2 peptides from each poliovirus. In some embodiments, the plurality of probes comprises 2 peptides from each poliovirus. In some embodiments, at least 2 is 2. In some embodiments, the plurality of probes comprises at least a peptide from VP1 from each poliovirus. In some embodiments, the plurality of probes comprises at least a peptide from VP2 from each poliovirus. In some embodiments, the plurality of probes comprises at least a peptide from VP3 from each poliovirus. In some embodiments, the plurality of probes comprises at least the VP1 protein from each poliovirus. In some embodiments, the plurality of probes comprises at least the VP2 protein from each poliovirus. In some embodiments, the plurality of probes comprises at least the VP3 protein from each poliovirus. In some embodiments, the plurality of probes comprises the whole inactivated poliovirus type 1. In some embodiments, the plurality of probes comprises the whole inactivated poliovirus type 2. In some embodiments, the plurality of probes comprises the whole inactivated poliovirus type 3.

[0106] In some embodiments, an array comprises probes to at least 2 polioviruses. In some embodiments, an array comprises probes to at least 3 polioviruses. In some embodiments, an array comprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550 or 600 probes. Each possibility represents a separate embodiment of the invention. In some embodiments, an array comprises at least 10 probes. In some embodiments, an array comprises at least 50 probes. In some embodiments, an array comprises at least 90 probes. In some embodiments, an array comprises at most 250, 300, 400, 500, 600, 700, 750, 800, 900 or 1000 probes. Each possibility represents a separate embodiment of the invention. In some embodiments, an array comprises at most 600 probes. In some embodiments, an array comprises 2-800, 10-800, 50-800, 90-800, 100-800, 200- 800, 2-700, 10-700, 50-700, 90-700, 100-700, 200-70, 2-650, 10-650, 50-650, 90-650, 100- 650, 200-650, 2-600, 10-600, 50-600, 90-600, 100-600, 200-600, 2-550, 10-550, 50-550, 90- 550, 100-550, 200-550, 2-500, 10-500, 50-500, 90-500, 100-500, 200-500, 2-400, 10-400, 50-400, 90-400, 100-400, 200-400, 2-300, 10-300, 50-300, 90-300, 100-300, 200-300, 2- 250, 10-250, 50-250, 90-250, 100-250, or 200-250. Each possibility represents a separate embodiment of the invention. In some embodiments, an array comprises 90-600 probes. In some embodiments, an array comprises 90-250 probes.

[0107] In some embodiments, a peptide is a protein. In some embodiments, a peptide is a complete protein. In some embodiments, a complete protein comprises a signal peptide. In some embodiments, a complete protein lacks a signal peptide. In some embodiments, theplurality of probes comprises an amino acid sequence of an poliovirus protein. In some embodiments, the plurality of probes comprises a protein probe from a first poliovirus. In some embodiments, the plurality of probes comprises a protein probe from a second poliovirus. In some embodiments, the protein is a surface protein. In some embodiments, the protein is a poliovirus envelope protein. In some embodiments, the protein is capsid protein. In some embodiments, the protein is selected from VP1, VP2 and VP3. In some embodiments, the protein is a recombinant protein. In some embodiments, the protein is a VLP. It will be understood by a skilled artisan that by using a protein secondary structures and intramolecular bonds and interactions will be preserved.

[0108] In some embodiments, the plurality of probes comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 peptides from a poliovirus. Each possibility represents a separate embodiment of the invention. In some embodiments, the plurality of probes comprises at least 2 peptides from a poliovirus. In some embodiments, the plurality of probes comprises 2 peptides from a poliovirus. In some embodiments, the two proteins are VP1 and VP2. In some embodiments, the two proteins are VP1 and VP3. In some embodiments, the two proteins are VP2 and VP3. In some embodiments, the plurality of probes comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 peptides from each poliovirus. Each possibility represents a separate embodiment of the invention. In some embodiments, the plurality of probes comprises at least 2 peptides from each poliovirus. In some embodiments, the plurality of probes comprises 2 peptides from each poliovirus. In some embodiments, the plurality of probes comprises at least 2 peptides from each type of poliovirus. In some embodiments, the plurality of probes comprises at least 2 peptides from each strain of poliovirus. In some embodiments, at least 2 is 2. In some embodiments, the plurality of probes comprises at least a peptide from VP1 from each type. In some embodiments, the plurality of probes comprises at least a peptide from VP2 from each type. In some embodiments, the plurality of probes comprises at least a peptide from VP3 from each type. In some embodiments, the plurality of probes comprises at least a peptide from VP1 from each strain. In some embodiments, the plurality of probes comprises at least a peptide from VP2 from each strain. In some embodiments, the plurality of probes comprises at least a peptide from VP3 from each strain. In some embodiments, the plurality of probes comprises at least the VP1 protein from each type. In some embodiments, the plurality of probes comprises at least the VP2 protein from each type. In some embodiments, the plurality of probes comprises at least the VP3 protein from each type. In some embodiments, the plurality of probes comprises at least the VP1 protein from each strain. In some embodiments, the plurality of probes comprises at least the VP2 protein from eachstrain. In some embodiments, the plurality of probes comprises at least the VP3 protein from each strain.

[0109] In some embodiments, the plurality of probes comprises probes from polioviruses from different years. In some embodiments, the plurality of probes comprises a probe from a poliovirus from a first year. In some embodiments, the plurality of probes comprises a probe from an poliovirus from a second year. In some embodiments, the first and second years are different years. In some embodiments, the plurality of probes comprises the same probe from different years. In some embodiments, the plurality of probes comprises a probe from a given strain from a first year, and a probe from the same strain from a second year. In some embodiments, different strains are by definition from different years. In some embodiments, the plurality of probes comprises a probe from a given type from a first year, and a probe from the same type from a second year. In some embodiments, the probe from different years are the same region of a peptide or protein but comprise different amino acid sequences. In some embodiments, it is the same protein from different years. It will be understood that in different years due to genetic drift there will be introduced mutations into a given amino acids sequence. Thus, the same peptide from one year to another, may be recognizable as the same peptide even though the sequence may be altered. Similarly, a given protein may be recognized as the same protein even if mutations have been generated in the amino acid sequence.

[0110] In some embodiments, the plurality of probes further comprises a poliovirus. In some embodiments, the poliovirus is an inactivated virus. As used herein, the term “inactivated virus” refers to a virulent virus that has been made non-infectious. In some embodiments, an inactivated virus is a killed virus. In some embodiments, an inactivated virus is a virus comprising a mutation that reduces virulence. In some embodiments, an inactivated virus is a virulent virus some of whose proteins have been transferred to a backbone of a less virulent or non-virulent virus. In some embodiments, the poliovirus is a lysed virus. In some embodiments, the virus is a virion. In some embodiments, the lysed virus is a lysed cell culture infected by the virus. In some embodiments, the lysed virus is media from infected cells containing virus. In some embodiments, the virus is a virus-like particle. In some embodiments, the plurality of probes comprises a virus-like particle (VLP). In some embodiments, the VLP is a poliovirus VLP. As used herein, the term “virus-like particle” refers to a multiprotein structure that mimics the organization and conformation of an authentic native virus but lacks the viral genome. In some embodiments, the plurality of probes comprises a lysate from a cell infected by a poliovirus. In some embodiments, thelysate is mixed with spotting buffer before immobilization on the array or support. It will be appreciated by a skilled artisan that by using whole virus, VLPs or cell lysate, viral epitopes will be provided in their natural confirmation. In some embodiments, the plurality of probes comprises at least two VLP probes. In some embodiments, the plurality of probes comprises at least two lysed virus probes. In some embodiments, the plurality of probes comprises at least two virus probes that are different poliovirus strains. In some embodiments, the plurality of probes comprises at least one mixture of inactivated polioviruses from 3 strains or types.

[0111] In some embodiments, the probes are present on the array or support at a concentration sufficient for antibody binding. In some embodiments, the probes are present on the array or support at a concentration sufficient for detectable antibody binding. In some embodiments, the concentration is at least 0.00001, 0.00005, 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 10, 100, 1000, 10000, 100000, 1000000, 10000000, 100000000, or 1000000000 ng per spot of probe. Each possibility represents a separate embodiment of the invention. In some embodiments, the concentration is at least 0.00001, 0.00005, 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 10, 100, 1000, 10000, 100000, 1000000, 10000000, 100000000, or 1000000000 ng / ml. Each possibility represents a separate embodiment of the invention. In some embodiments, the concentration is at most 0.00001, 0.00005, 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 10, 100, 1000, 10000, 100000, 1000000, 10000000, 100000000, or 1000000000 ng per spot of probe. Each possibility represents a separate embodiment of the invention. In some embodiments, the concentration is at most 0.00001, 0.00005, 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 10, 100, 1000, 10000, 100000, 1000000, 10000000, 100000000, or 1000000000 ng / ml. Each possibility represents a separate embodiment of the invention. In some embodiments, the concentration of whole poliovirus antigen is 10, 20, 40, 50, 100 or 200 DAg / mlln. Each possibility represents a separate embodiment of the invention. In some embodiments, the concentration of peptide is ~1 mg / ml. In some embodiments, the volume of the spot is at least 0.00001, 0.00005, 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 10, 100, 1000, 10000, 100000, 1000000, 10000000, 100000000, or 1000000000 nL. Each possibility represents a separate embodiment of the invention. In some embodiments, the volume of the spot or probe is at most 0.00001, 0.00005, 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 10, 100, 1000, 10000, 100000, 1000000, 10000000, 100000000, or 1000000000 nL. Each possibility represents a separate embodiment of the invention. In some embodiments, the volume of the spot or probe is -370pL. In some embodiments, the value of the spot or probe is from 250-370 pL. In some embodiments, the spotted mass of whole recombinant proteins is between 3-20 picograms. In some embodiments, the spotted mass of peptides is between 300-400 picograms. It will be understood that shorter peptides will tend to have a lower mass and longer peptides a larger mass. In some embodiments, the spotted mass of peptide is -370 picograms.

[0112] In some embodiments, the plurality of probes is selected from the probes provided in Table 1. In some embodiments, the plurality of probes comprises the probes provided in Table 1. In some embodiments, the conformational epitope probes consist of the probes provided in Table 1. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 probes are selected from Table 1. Each possibility represents a separate embodiment of the invention. In some embodiments, the plurality of probes comprises a group of probes selected from the groups provided in Figures 16A-B. In some embodiments, the plurality of probes comprises a group of viral antigens selected from the groups provided in Figures 16A-B. In some embodiments, the plurality of probes comprises at least two groups of viral antigens selected from the groups provided in Figures 16A-B. In some embodiments, the plurality of probes comprises all groups provided in Figures 16A-B.

[0113] In some embodiments, the plurality of probes is selected from the probes provided in Table 3. In some embodiments, the plurality of probes comprises the probes provided in Table 3. In some embodiments, at least 2, 3, 4, 5, or 6 probes are selected from Table 3. Each possibility represents a separate embodiment of the invention.

[0114] In some embodiments, the plurality of probes is selected from the probes provided in Table 9. In some embodiments, the plurality of probes comprises the probes provided in Table 9. In some embodiments, the linear epitope probes consist of the probes provided in Table 9. In some embodiments, the short peptide probes consist of the probes provided in Table 9. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 probes are selected from Table 9. Each possibility represents a separate embodiment of the invention. In some embodiments, the plurality of probes is selected from SEQ ID NO: 1-386. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 probes are selected from SEQ ID NO: 1-386. In some embodiments, at least one probe is any one of: SEQ ID NO: 1-386. Each possibility represents a separate embodiment of the invention. It will be understood that inclusion of each probe from Table 9 in the plurality of probes is considered a separate embodiment of the invention.

[0115] Table 9: All poliovirus peptide probes. (OPVl=Sabin type 1; OPV2=Sabin type 2;S3=Sabin type 3 (OPV3), SoASl=type 1 SoASl (wildtype strain); Mahl=Mahoney type 1; MF=MEF-1 type 2; Sk=Saukett type 3).

[0116] The linear peptide probes can also be selected from a smaller more refined list (e.g.,Table 10). In some embodiments, the plurality of probes is selected from the probes provided in Table 10. In some embodiments, the plurality of probes comprises the probes provided in Table 10. In some embodiments, the linear epitope probes consist of the probes provided in Table 10. In some embodiments, the short peptide probes consist of the probes provided in Table 10. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 probes are selected from Table 10. Each possibility represents a separate embodiment of the invention. It will be understood that inclusion of each probe from Table 10 in the plurality of probes is considered a separate embodiment of the invention. In some embodiments, the plurality of probes comprises all the probes provided in Table 10. In some embodiments, the plurality of probes consists of the probes provided in Table 10.

[0117] In some embodiments, the plurality of probes comprises probes comprising a poliovirus type 1 antigenic site. Tn some embodiments, the type 1 antigenic site is selected from the sites provided in Table 13. In some embodiments, comprising probes to an antigenic site in Table 13 is including in the plurality of probes all probes listed in Table 13 for that site. In some embodiments, at least two poliovirus type 1 antigenic sites. In some embodiments, the at least two sites are selected from those provided in Table 13. In some embodiments, the plurality of probes comprises probes to all the antigenic sites in Table 13. In some embodiments, the plurality of probes comprises all the probes provided in Table 13.

[0118] In some embodiments, the plurality of probes comprises probes comprising a poliovirus type 2 antigenic site. In some embodiments, the type 2 antigenic site is selectedfrom the sites provided in Table 14. In some embodiments, comprising probes to an antigenic site in Table 14 is including in the plurality of probes all probes listed in Table 14 for that site. In some embodiments, at least two poliovirus type 2 antigenic sites. In some embodiments, the at least two sites are selected from those provided in Table 14 In some embodiments, the plurality of probes comprises probes to all the antigenic sites in Table 14. In some embodiments, the plurality of probes comprises all the probes provided in Table 14.

[0119] In some embodiments, the plurality of probes comprises probes comprising a poliovirus type 3 antigenic site. In some embodiments, the type 3 antigenic site is selected from the sites provided in Table 15. In some embodiments, comprising probes to an antigenic site in Table 15 is including in the plurality of probes all probes listed in Table 15 for that site. In some embodiments, at least two poliovirus type 3 antigenic sites. In some embodiments, the at least two sites are selected from those provided in Table 15. In some embodiments, the plurality of probes comprises probes to all the antigenic sites in Table 15. In some embodiments, the plurality of probes comprises all the probes provided in Table 15.

[0120] In some embodiments, the plurality of probes comprises probes comprising a poliovirus type 1 antigenic site, a poliovirus type 2 antigenic site and a poliovirus type 3 antigenic site. In some embodiments, the plurality of probes comprises probes comprising at least two poliovirus type 1 antigenic site, at least two poliovirus type 2 antigenic site and at least two poliovirus type 3 antigenic site. In some embodiments, the plurality of probes comprises probes comprising all of the antigenic sites provided in Table 13. In some embodiments, the plurality of probes comprises probes comprising all of the antigenic sites provided in Table 14. In some embodiments, the plurality of probes comprises probes comprising all of the antigenic sites provided in Table 15. In some embodiments, the plurality of probes comprises probes comprising all of the antigenic sites provided in Tables 13, 14 and 15. In some embodiments, the plurality of probes comprises all the probes provided in Table 13. In some embodiments, the plurality of probes comprises all the probes provided in Table 14. In some embodiments, the plurality of probes comprises all the probes provided in Table 15. In some embodiments, the plurality of probes comprises all the probes provided in Tables 13, 14 and 15. In some embodiments, the plurality of probes comprises probes comprising at least one of the antigenic sites provided in Table 13. In some embodiments, the plurality of probes comprises probes comprising at least one of the antigenic sites provided in Table 14. In some embodiments, the plurality of probes comprises probes comprising at least one of the antigenic sites provided in Table 15. In some embodiments, the plurality of probes comprises probes comprising at least one of theantigenic sites provided in Tables 13, 14 and 15. In some embodiments, the plurality of probes comprises probes comprising at least two of the antigenic sites provided in Table 13. In some embodiments, the plurality of probes comprises probes comprising at least two of the antigenic sites provided in Table 14. In some embodiments, the plurality of probes comprises probes comprising at least two of the antigenic sites provided in Table 15. In some embodiments, the plurality of probes comprises probes comprising at least two of the antigenic sites provided in Tables 13, 14 and 15. In some embodiments, the plurality of probes comprises probes to all of the antigenic sites provided in Table 13. In some embodiments, the plurality of probes comprises probes to all of the antigenic sites provided in Table 14. In some embodiments, the plurality of probes comprises probes to all of the antigenic sites provided in Table 15. In some embodiments, the plurality of probes comprises probes to all of the antigenic sites provided in Tables 13, 14 and 15.

[0121] In some embodiments, the plurality of probes against poliovirus type 1 comprise SEQ ID NO: 182, 185, 187, 197, 200, 202, 204, 215, 217, 219, 222-223, and 226-228. In some embodiments, the plurality of probes against poliovirus type 1 consist of SEQ ID NO: 182, 185, 187, 197, 200, 202, 204, 215, 217, 219, 222-223, and 226-228. In some embodiments, the plurality of probes against poliovirus type 2 comprise SEQ ID NO: 49, 52, 53, 69, 87, 95, 104, 122, 130 and 158. In some embodiments, the plurality of probes against poliovirus type 2 consist of SEQ ID NO: 49, 52, 53, 69, 87, 95, 104, 122, 130 and 158. In some embodiments, the plurality of probes against poliovirus type 3 comprises SEQ ID NO: 260-261. 276-280, 283, 288, 292, 298-300, 302, 309, 312, 315-317, 319, 324-325, 331, 337, 350- 355, 358, 361, 366, 369, 372, 374, 378, and 381. In some embodiments, the plurality of probes against poliovirus type 3 comprises SEQ ID NO: 260-261, 276-280, 283, 288, 292, 298-299, 302, 309, 312, 315-317, 319, 324-325, 331, 337, 350-355, 358, 361, 366, 369, 372, 374, 378, and 381. In some embodiments, the plurality of probes against poliovirus type 3 consist of SEQ ID NO: 260-261, 276-280, 283, 288, 292, 298-300, 302, 309, 312, 315-317, 319, 324-325, 331, 337, 350-355, 358, 361, 366, 369, 372, 374, 378, and 381. In some embodiments, the plurality of probes against poliovirus type 3 consist of SEQ ID NO: 260-261. 276-280, 283, 288, 292, 298-299, 302, 309, 312, 315-317, 319, 324-325, 331, 337, 350- 355, 358, 361, 366, 369, 372, 374, 378, and 381.

[0122] In some embodiments, the array comprises probes against SEQ ID NO: 49, 52, 53, 69, 87, 95, 104, 122, 130, 158, 182, 185, 187, 197, 200, 202, 204, 215, 217, 219, 222-223, 226-228, 260-261, 276-280, 283, 288, 292, 298-300, 302, 309, 312, 315-317, 319, 324-325, 331, 337, 350-355, 358, 361, 366, 369, 372, 374, 378, and 381. In some embodiments, thearray comprises probes against SEQ ID NO: 49, 52, 53, 69, 87, 95, 104, 122, 130, 158, 182, 185, 187, 197, 200, 202, 204, 215, 217, 219, 222-223, 226-228, 260-261, 276-280, 283, 288, 292, 298-299, 302, 309, 312, 315-317, 319, 324-325, 331, 337, 350-355, 358, 361, 366, 369, 372, 374, 378, and 381. In some embodiments, the array comprises probes against SEQ ID NO: 49, 52, 53, 69, 87, 95, 104, 122, 130, 158, 182, 185, 187, 197, 200, 202, 204, 215, 217, 219, 222-223, 226-228, 260-261, 276-280, 283, 288, 292, 298-300, 302, 309, 312, 315-317, 319, 324-325, 331, 337, 350-355, 358, 361, 366, 369, 372, 374, 378, and 381 and the virus probes provided in Table 1. In some embodiments, the array comprises probes against SEQ ID NO: 49, 52, 53, 69, 87, 95, 104, 122, 130, 158, 182, 185, 187, 197, 200, 202, 204, 215, 217, 219, 222-223, 226-228, 260-261, 276-280, 283, 288, 292, 298-299, 302, 309, 312, 315- 317, 319, 324-325, 331, 337, 350-355, 358, 361, 366, 369, 372, 374, 378, and 381 and the virus probes provided in Table 1.

[0123] In some embodiments, the plurality of probes comprises at least 1 peptide probe. In some embodiments, the plurality of probes comprises at least 1 peptide probe and at least one conformational probe. In some embodiments, the plurality of probes comprises at least 1 short peptide probe (e.g., from 10-60 amino acids) and at least one whole virus or recombinant protein. In some embodiments, the plurality of probes comprises at least 1 short peptide probe (e.g., from 10-60 amino acids) and at least one whole virus. In some embodiments, the plurality of probes comprises at least 1 short peptide probe (e.g., from 10- 60 amino acids) and at least one VLP. It will be understood that the inclusion of both conformational epitopes and linear epitopes produces a superior array. In some embodiments, the plurality of probes comprises a peptide probe selected from the probes provided in Table 9 and a conformational probe selected from the viruses and proteins provided in Tables 1. In some embodiments, the plurality of probes comprises a peptide probe selected from the probes provided in Table 10 and a conformational probe selected from the viruses and VLPs provided in Tables 1.

[0124] In some embodiments, the plurality of probes comprises viruses, VLPs or both selected from Table 1. In some embodiments, the plurality of probes consists of virus, VLPs or both selected from Table 1. In some embodiments, the plurality of probes consists of the viruses, VLPs or both of Table 1. In some embodiments, the plurality of probes comprises viruses, wherein the viruses consist of the viruses, VLPs or both of Table 1. In some embodiments, the plurality of probes consists of viruses, VLPs or both selected from Table 1.

[0125] In some embodiments, the support or array consists of the plurality of probes. In some embodiments, the only probes on the array / support are the plurality of probes. In some embodiments, the solid support or array further comprises control probes. In some embodiments, control probes are probes that are bound by known antibodies found in all subjects. In some embodiments, control probes are probes that bind known antibodies found in all subjects. In some embodiments, control probes comprise secondary antibodies to human antibodies. In some embodiments, the secondary antibodies are selected from antihuman IgG, anti-human IgA, anti-human slgA, and anti-human IgM. In some embodiments, control probes are peptides or proteins used to generate a vaccine. In some embodiments, the plurality of probes comprises control probes. In some embodiments, a control is cell lysate from a cell uninfected by a poliovirus.

[0126] According to some embodiments, the plurality of probes comprises an amino acid sequence of a surface protein from a first strain or type of poliovirus and an amino acid sequence of a surface protein from a second strain or type of poliovirus. According to some embodiments, the plurality of probes comprises at least two probes from each strain or type of poliovirus.

[0127] Kits and Systems

[0128] According to another aspect, there is provided a kit comprising an array or support of the invention.

[0129] According to another aspect, there is provided a system comprising an array or support of the invention.

[0130] In some embodiments, the kit further comprises a detecting agent. In some embodiments, the kit further comprises at least one detecting agent. In some embodiments, the detecting agent is a labeled detecting agent. In some embodiments, the detecting agent is for detecting binding of an antibody to a probe of the array or support. In some embodiments, the detecting agent is for detecting antibodies. In some embodiments, the detecting agent is for detecting antibodies from a subject. In some embodiments, the detecting agent is for detecting human antibodies. In some embodiments, the detecting agent is for detecting IgG, IgA, IgM, sig A or a combination thereof. In some embodiments, the detecting agent is for detecting IgA. In some embodiments, the detecting agent is at least one labeled secondary antibody. In some embodiments, the secondary antibody is configured for detection of antibodies bound to the array or support. In some embodiments, the secondary antibody is an anti-human secondary antibody. In some embodiments, the secondary antibody is an anti-IgG antibody. In some embodiments, the secondary antibody is an anti-IgA antibody. In some embodiments, the secondary antibody is an anti-IgM antibody. In some embodiments, the secondary antibody is an anti-sIgA antibody. In some embodiments, agents for detecting IgM, IgA, slgA and IgG comprise distinct labels. In some embodiments, the secondary antibody is not labeled, and the detecting agent is at least one labeled third antibody. In some embodiments, a third labeled antibody is used to detect the secondary antibody (e.g. if the secondary antibody is unlabeled mouse anti-human slgA, it will be detected by a labeled anti-mouse IgG antibody).

[0131] In some embodiments, the kit comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 secondary antibodies. Each possibility represents a separate embodiment of the invention. In some embodiments, the kit comprises at least 2 secondary antibodies. In some embodiments, the kit comprises an anti-IgG and an anti-IgA antibody. In some embodiments, the kit comprises an anti-IgA antibody. In some embodiments, the kit comprises an anti-IgG. In some embodiments, the kit comprises an anti-IgM antibody, antibody. In some embodiments, the kit comprises an anti-sIgA antibody. In some embodiments, each secondary antibody comprises a uniquely detectable label. As such, the binding of each secondary antibody can be measured separately, or simultaneously but distinctly identified.

[0132] In some embodiments, the label is a fluorescent label. In some embodiments, the label is a radioactive label. Detectable labels are well known in the art and any uniquely detectable label may be used.

[0133] In some embodiments, the system comprises a detector or sensor configured to detect binding of antibodies to probes of the array or support. In some embodiments, the detector or sensor is configured to detect labeled secondary antibodies. In some embodiments, the detector or support is configured to detect fluorescence. In some embodiments, the detector or sensor is configured to detect binding at specific locations on the array or support. In some embodiments, the detector or sense is configured to detect binding of antibodies to probes immobilized on the array or support. In some embodiments, the detector is a laser scanner.

[0134] In some embodiments, the array or support is for use in predicting the risk of symptomatic infection by a poliovirus. In some embodiments, the array or support is for use in predicting spread poliovirus if infected. In some embodiments, the array or support is for use in predicting the risk of symptomatic infection of a subject by a poliovirus. In some embodiments, the array or support is for use in predicting the effectiveness of a poliovirusvaccination of a subject. In some embodiments, the array or support is for use in predicting the effectiveness of an poliovirus vaccine in a subject. In some embodiments, the array or support is for use in detecting previous poliovirus infection of a subject. In some embodiments, the array or support is for use in detecting previous vaccination of a subject against poliovirus. In some embodiments, the array or support is for use in identifying a subject having neutralizing antibodies against poliovirus. In some embodiments, the array or support is for use in a method of the invention.

[0135] In some embodiments, the prediction is for poliovirus type 1. In some embodiments, the prediction is for poliovirus type 2. In some embodiments, the prediction is for poliovirus type 3. In some embodiments, the prediction is for at least two types of poliovirus. In some embodiments, the prediction of all three types of poliovirus. In some embodiments, the array provides a prediction for each poliovirus separately (e.g., a prediction for type 1, a prediction for type 2 and a prediction for type 3). Thus, a subject may be determined to have neutralizing antibodies against type 1, but not type 2 or 3 for example. In some embodiments, a subject determined to lack neutralizing antibodies is administered a vaccine. In some embodiments, a subject determined to lack neutralizing antibodies against a specific type is administered a vaccine against that type.

[0136] Methods of use

[0137] In some embodiments, the array or support is for use in determining suitability of a subject to receive a poliovirus vaccine. In some embodiments, receiving a vaccine is being revaccinated. In some embodiments, receiving a vaccine is receiving a first vaccination. In some embodiments, the array or support is for use in detecting poliovirus vaccination in a subject. In some embodiments, the array or support is for use in determining the strains of poliovirus that a subject had previously been vaccinated against. In some embodiments, the array or support is for use in determining the types of poliovirus that a subject had previously been vaccinated against. In some embodiments, the array or support is for use in determining the potency of a poliovirus vaccine. In some embodiments, the array or support is for use in determining the efficacy of a poliovirus vaccine. In some embodiments, the array or support is for use in determining the efficacy of a poliovirus vaccine in a subject. In some embodiments, the array or support is for use in screening a subject for protection from poliovirus. In some embodiments, screening for protection is screening for being protected from poliovirus. It will be understood by a skilled artisan that any use for which the array or support can be used, so too a kit or system of the invention can also be used. In someembodiments, the kit is for use in a method of the invention. In some embodiments, the system is for use in a method of the invention.

[0138] According to another aspect, there is provided a method of identifying a subject having neutralizing antibodies against poliovirus, the method comprising providing a sample from the subject, contacting the sample to an array or support of the invention, and detecting the binding of an antibody from the sample to a discrete location on the array or support, thereby identifying a subject having neutralizing antibodies against a poliovirus.

[0139] According to another aspect, there is provided a method of identifying a subject having neutralizing antibodies against poliovirus, the method comprising providing a sample from the subject, contacting the sample to an array or support of the invention, detecting the binding of antibodies from the sample to discrete locations on the array or support, and applying a trained machine learning algorithm to the detected binding at discrete locations on the array, thereby identifying a subject as having neutralizing antibodies against a poliovirus.

[0140] According to another aspect, there is provided a method of probing an antibody repertoire of a subject, the method comprising providing a sample from the subject, contacting the sample to an array or support of the invention, and detecting the binding of an antibody from the sample to a discrete location on the array or support, thereby probing the antibody repertoire of a subject.

[0141] In some embodiments, the method is a method of detecting previous vaccination with a poliovirus vaccine. In some embodiments, the method is a method of determining vaccination effectiveness in a subject. In some embodiments, the method is a method of detecting risk of infection with a poliovirus. In some embodiments, the method is a method of detecting protection form poliovirus. In some embodiments, the method is a screening method. In some embodiments, the method is a method of determining risk of a subject to be symptomatically infected by poliovirus. In some embodiments, the method is a method of determining risk of a subject spreading poliovirus. In some embodiments, the identification of neutralizing antibodies indicates the subject is not at risk of infection. In some embodiments, the identification of neutralizing antibodies indicates the subject is not at risk of spreading infection. In some embodiments, lack of the identification of neutralizing antibodies indicates the subject is at risk of infection. In some embodiments, infection is symptomatic infection. In some embodiments, infection can be spread even if not symptomatic. In some embodiments, the identification of neutralizing antibodies indicatesthe vaccine was effective in the subject. In some embodiments, lack of the identification of neutralizing antibodies indicates the vaccine was ineffective in the subject. In some embodiments, the method is an ex vivo method. In some embodiments, the method is an in vitro method. In some embodiments, the method is a diagnostic method. In some embodiments, an antibody repertoire is a repertoire against poliovirus.

[0142] In some embodiments, the subject is a human. In some embodiments, the subject is at risk for developing poliovirus. In some embodiments, the subject is at risk for contracting poliovirus. In some embodiments, the subject has previously been vaccinated against poliovirus. In some embodiments, the subject’s poliovirus vaccination status is unknown. In some embodiments, the subject from a community suffering a poliovirus outbreak. In some embodiments, the subject has been vaccinated against poliovirus, but the effect of the vaccination is unknown. In some embodiments, the subject is an infant. In some embodiments, the subject is a child. In some embodiments, the subject is an adult. In some embodiments, the subject is pregnant or at risk of becoming pregnant.

[0143] In some embodiments, the sample is a biological sample. In some embodiments, the sample is a bodily fluid. In some embodiments, the bodily fluid is selected from: blood, serum, gastric fluid, intestinal fluid, saliva, bile, breast milk, nasal swab, oral swab, urine, interstitial fluid, fecal water and stool. In some embodiments, the bodily fluid is blood. In some embodiments, the bodily fluid is serum. In some embodiments, the bodily fluid is plasma. In some embodiments, the blood is peripheral blood. In some embodiments, the bodily fluid is selected from blood and serum. In some embodiments, the bodily fluid is selected from blood, plasma and serum. In some embodiments, the bodily fluid is saliva. In some embodiments, the bodily fluid is selected from blood, saliva and serum. In some embodiments, the sample is from the subject. In some embodiments, the sample is a sample comprising antibodies. In some embodiments, the sample comprises antibodies from the subject. In some embodiments, the sample comprises polyclonal antibodies from the subject.

[0144] In some embodiments, the sample comprises at least 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 100 microliters of fluid. Each possibility represents a separate embodiment of the invention. In some embodiments, the sample comprises at most 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400 or 500 microliters of fluid. Each possibility represents a separate embodiment of the invention. In some embodiments, the sample comprises sufficient liquid to cover the array or support. In some embodiments, the sample comprises sufficient liquid to cover the plurality of probes. In some embodiments, the sample is diluted in buffer. In some embodiments, the buffer is binding buffer.

[0145] In some embodiments, the contacting is in conditions sufficient for antibody binding to the probes. In some embodiments, the contacting is in conditions sufficient for antibody binding to the plurality of probes. Conditions for antibody binding will be known by one skilled in the art. Further, optimization of binding conditions can be determined by a skilled artisan. In some embodiments, the contacting produces an array or support with bound antibodies. In some embodiments, the contacting produces antibodies bound to the array or support.

[0146] In some embodiments, the detecting comprises detecting binding of an antibody to a probe. In some embodiments, the binding indicates the presence in the sample of an antibody to a probe located at the detected location. In some embodiments, the binding indicates the presence in the sample of an antibody to a given peptide, protein, VLP or virus. The identity of the virus, peptide, protein or VLP to which there are antibodies in the sample is determined by the known locations of each probe.

[0147] In some embodiments, the detecting comprises contacting the array or support with bound antibodies with a labeled detecting agent. In some embodiments, the detecting agent is a secondary antibody. In some embodiments, the detecting agent detects antibodies from the sample. In some embodiments, the detecting agent detects binding of antibodies to a target. In some embodiments, the detecting agent detects binding of antibody from the sample to a probe of the array or support.

[0148] In some embodiments, a level of binding beyond a predetermined threshold indicates the subject possesses neutralizing antibodies against the poliovirus. In some embodiments, the level of binding to a specific probe indicates the subject possesses neutralizing antibodies. In some embodiments, the level of binding to a plurality of probes indicates the subject possesses neutralizing antibodies. In some embodiments, a plurality is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 25, 30, 35, 40, 45, 50, 55 or 60 probes. Each possibility represents a separate embodiment of the invention. In some embodiments, the magnitude of binding to a set of probes is measured. In some embodiments, a magnitude of binding beyond a predetermined threshold indicates the subject possesses neutralizing antibodies. In some embodiments, the magnitude of binding of a plurality of sets of probes is combined and a combined magnitude beyond a predetermined threshold is indicative of a subject possessing neutralizing antibodies. In some embodiments, the magnitude is the sum of the antibody binding levels to each probe in a set of probes. In some embodiments, the magnitude is the geometric mean of antibody binding levels to each probe in a set of probes. In some embodiments, the combined magnitude calculated by a pre-defined formula that summarizesthe magnitudes of several sets of probes. In some embodiments, the set of probes is all probes that comprise an epitope. In some embodiments, the set of probes is at least one probe that comprises an epitope. In some embodiments, the set of probes is at least two probes that comprise an epitope. In some embodiments, the epitope is a target epitope. In some embodiments, the magnitude of binding is calculated for the epitopes provided in Table 13. In some embodiments, the magnitude of binding is calculated for the epitopes provided in Table 14. In some embodiments, the magnitude of binding is calculated for the epitopes provided in Table 15. In some embodiments, the magnitude of binding is calculated for the epitopes provided in Tables 13, 14 and 15. In some embodiments, the epitope is a target epitope. In some embodiments, the magnitude of binding is calculated for the groups of probes provided in Figures 14A-B. It will be understood that the magnitude is calculated for each group.

[0149] In some embodiments, all VP1 peptide binding magnitudes are combined. In some embodiments, all VP2 peptide binding magnitudes are combined. In some embodiments, all VP3 peptide binding magnitudes are combined. In some embodiments, binding magnitudes of all VP1 peptides of one type are combined. In some embodiments, binding magnitudes of all VP2 peptides of one type are combined. In some embodiments, binding magnitudes of all VP3 peptides of one type are combined. In some embodiments, binding magnitudes of all VP1 peptides of one strain are combined. In some embodiments, binding magnitudes of all VP2 peptides of one strain are combined. In some embodiments, binding magnitudes of all VP3 peptides of one strain are combined. In some embodiments, all MEF1 VP2 peptide binding magnitudes are combined. In some embodiments, the combined magnitude produces a binding score. In some embodiments, a binding score beyond a predetermined threshold indicates the subject possesses neutralizing antibodies.

[0150] In some embodiments, the method further comprises producing a poliovirus antibody score. In some embodiments, an antibody score is an array score. In some embodiments, the antibody score is determined based on the binding to the array. In some embodiments, the antibody score is determined based on the binding to a subset of probes in the array. In some embodiments, the score is determined based on the binding to groups of antigens in the array. In some embodiments, a score beyond a predetermined threshold indicates the presence of neutralizing antibodies. In some embodiments, beyond is above. In some embodiments, the score is determined for a specific poliovirus subtype. In some embodiments, a poliovirus type 1 score is produced. In some embodiments, a poliovirus type 2 score is produced. In some embodiments, a poliovirus type 3 score is produced. In some embodiments, themachine learning model outputs the score. In some embodiments, the score is produced from the magnitudes of binding.

[0151] In some embodiments, the predetermined threshold is the binding magnitudes from a control subject. In some embodiments, a control subject is a healthy subject. In some embodiments, a control subject is a subject that has been vaccinated against poliovirus. In some embodiments, a control subject is a subject that has been vaccinated against poliovirus. In some embodiments, a control subject is a subject that was exposed to a poliovirus and did not develop a symptomatic infection. In some embodiments, a control subject is a subject that has been vaccinated against poliovirus. In some embodiments, a control subject is a subject that was exposed to a poliovirus and did not spread the virus. In some embodiments, spread of the virus comprises detectable virus DNA in a biological sample from the subject. In some embodiments, vaccinated is vaccinated against a specific poliovirus type or strain. In some embodiments, the predetermined threshold is the binding magnitudes from a subject that has been vaccinated. In some embodiments, the predetermined threshold is the binding magnitudes from a subject that has been vaccinated against a specific strain. In some embodiments, the predetermined threshold is the binding magnitudes from a subject that has been vaccinated, exposed to a poliovirus and did not develop a symptomatic infection. In some embodiments, the predetermined threshold is the binding magnitudes from a subject that has been vaccinated. In some embodiments, the predetermined threshold is the binding magnitudes from a subject that has been exposed to a poliovirus and did not spread the virus. In some embodiments, the predetermined threshold is the binding magnitudes from a subject that has been vaccinated. In some embodiments, the predetermined threshold is the binding magnitudes from a subject that has been exposed to a poliovirus and did not secrete detectable poliovirus into a biological sample obtained from the subject.

[0152] In some embodiments, the magnitude of binding is measured by mean fluorescence intensity (MFI), when a fluorescent secondary agent is used. In some embodiments, the threshold for probe binding is an MFI above 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, 1700 or 2000. Each possibility represents a separate embodiment of the invention. In some embodiments, the threshold is 500. In some embodiments, the threshold is 1000. In some embodiments, the probe is a peptide probe and the threshold for probe binding is an MFI above 2000. In some embodiments, the threshold is binding to a predetermined number of probes. In some embodiments, the predetermined number is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or 100 probes. Each possibility represents a separate embodiment of the invention.

[0153] As used herein, the term “symptomatic infection”, “symptomatic disease” and “symptomatic poliovirus” are synonymous and used interchangeably and refer to a disease that is apparent due to the presence of a poliovirus symptom. It will be understood by a skilled artisan that a subject can become infected by poliovirus and would be found positive by a PCR test or other sensitive test, but still be asymptomatic. Poliovirus is commonly known as poliomyelitis or just polio and examples of symptoms include, but are not limited to, fever, fatigue, headache, sore throat, nausea, vomiting, nonparalytic aseptic meningitis, stiff neck / back, muscle pain, paralytic poliomyelitis, loss of reflexes, flaccid paralysis, and respiratory difficulties. In some embodiments, symptomatic infection comprises at least one symptom. In some embodiments, infection comprises the release or poliovirus in a bodily fluid. In some embodiments, symptomatic infection comprises the release or poliovirus in a bodily fluid. In some embodiments, the fluid is feces. Methods of detecting poliovirus in a sample are well known. By determining if poliovirus is passed on one can determine if a person is infectious. In some embodiments, the method is a method of determining if the subject is infectious. In some embodiments, a subject with neutralizing antibodies is not infectious.

[0154] In some embodiments, the trained machine learning algorithm produces a diagnosis indicating said subject does or does not possess neutralizing antibodies against the poliovirus. In some embodiments, the trained machine learning algorithm produces a score indicating the likelihood the subject possesses neutralizing antibodies against the poliovirus. In some embodiments, the trained machine learning algorithm produces a formula that is used to calculate a score from the array results.

[0155] In some embodiments, the machine learning model is trained on binding results from arrays contacted with samples from subjects that do have neutralizing antibodies and subjects that do not have neutralizing antibodies. In some embodiments, subjects that do not have neutralizing antibodies are subjects that have been vaccinated against poliovirus but do not have neutralizing antibodies. In some embodiments, neutralizing antibodies are neutralizing against a particular strain or type. In some embodiments, the subjects have been vaccinated against a first poliovirus and do not have neutralizing antibodies against a second poliovirus. In some embodiments, the trained machine learning model is trained on a training set.

[0156] In some embodiments, images of the array are provided to the trained machine learning model. In some embodiments, the training set comprises images of arrays after contact with samples from subjects that do have neutralizing antibodies and subjects that donot have neutralizing antibodies. In some embodiments, the training set comprises images of the array after contact with samples from subjects with neutralizing antibodies. In some embodiments, the training set comprises images of the array after contact with samples from subjects without neutralizing antibodies. In some embodiments, the training set comprises labels indicating if the image is from an array contacted with a sample from a subject with or without neutralizing antibodies. In some embodiments, the images are after detection with a reagent. In some embodiments, the reagent is fluorescent. In some embodiments, the agent renders the binding of the antibodies detectable.

[0157] In some embodiments, values of binding are provided to the trained machine learning model. The conversion or translation of the array image into numerical values can be done using any method or software known in the art for this purpose. Software for this purpose is commercially available, such as from Genepix and the like. In some embodiments, the values of binding are the locations of binding. In some embodiments, the training set comprises values of binding from arrays after contact with samples from subjects that do have neutralizing antibodies and subjects that do not have neutralizing antibodies. In some embodiments, the values of binding are magnitudes of binding. In some embodiments, the magnitude of binding of each probe of the array is provided to the trained machine learning algorithm. In some embodiments, the training set comprises the magnitude of binding of each probe of arrays after contact with samples from subjects that do have neutralizing antibodies and subjects that do not have neutralizing antibodies. In some embodiments, the training set comprises labels indicating if the values of binding is from an array contacted with a sample from a subject with or without neutralizing antibodies. In some embodiments, the training set comprises labels indicating if the magnitudes of binding are from arrays contacted with a sample from a subject with or without neutralizing antibodies. In some embodiments, the machine learning model is provided a binding score. In some embodiments, the training set comprises binding scores from arrays contacted with a sample from a subject with or without neutralizing antibodies. In some embodiments, the training set comprises labels indicating if the binding scores are from arrays contacted with a sample from a subject with or without neutralizing antibodies. It will be understood that scores / magnitudes / values can be provided in a table or any other computer readable format such that the model can evaluate the provided data.

[0158] In some embodiments, any suitable machine learning algorithm or combination of methods may be employed, including, but not limited to:• Support Vector Machine (SVM): A nonparametric model which finds the optimal separating hyperplane that discriminate between different classes. It can perform linear or non-linear classification.• Penalized Logistic Regression (PLR) - a logistic model for regression that imposes a penalty to reduce the impact of certain features.• Generalized linear model (GLM): a generalization of linear regression that unifies statistical models such as linear regression, logistic regression and Poisson regression. GLM extends linear regression by (1) supporting response variables with error distributions other than the normal distribution (2) a non-linear relationship between the predictors and the response variable.• Random forest (RF): involves in the generation of multiple decision trees that consist sequences of decision rules for protein expression values. To avoid over-fitting, these trees may be pruned. Each tree is constructed by randomly selecting different samples.• extreme Gradient Boosting (XGB): a gradient boosted decision trees-based classification and regression algorithm. The decision trees are built one at a time, and each new tree corrects the error of the previously trained decision tree.

[0159] In other embodiments, machine learning model may be trained based on statistical measures, i.e., variance, median, mean, average and the same.

[0160] In some embodiments, the machine learning model is a machine learning algorithm. In some embodiments, the machine learning model is a machine learning classifier. In some embodiments, the machine learning model is a classifier. In some embodiments, the classifier classifies a subject as having or not having neutralizing antibodies against poliovirus or a specific strain or type of poliovirus. In some embodiments, at an inference stage the machine learning model predicts if a subject has or does not have neutralizing antibodies. In some embodiments, at a training stage the machine learning model is trained on the training set.

[0161] In some embodiments, the method further comprises administering a poliovirus vaccine to a subject determined to lack neutralizing antibodies. In some embodiments, the method further comprises administering prophylactic treatment of a subject determined to lack neutralizing antibodies. In some embodiments, the method further comprises instructing the subject determined to lack neutralizing antibodies in methods of avoiding poliovirus infection. In some embodiments, the administered vaccine is against the strain or type forwhich the subject is determined to not have neutralizing antibodies. In some embodiments, the method further comprises performing a micro-neutralization assay on a subject determined not to have antibodies. In some embodiments, the method further comprises performing a micro-neutralization assay on a subject with an undetermined or questionable level of neutralizing antibodies. In some embodiments, the method further comprises performing a micro-neutralization assay on a subject determined to have binding but at a level below the predetermined threshold.

[0162] By another aspect, there is provided a method for producing a poliovirus peptide array, the method comprising: a. providing a poliovirus peptide array comprising a plurality of synthetic peptide probes; b. contacting the peptide array with a sample from a plurality of subjects with known neutralizing antibody status against poliovirus; c. detecting binding of the antibodies to discrete locations on the array indicating the presence in the sample of antibodies to probes located at the detected discrete location; d. selecting peptides whose detected binding by antibodies correlates with neutralizing antibody status against poliovirus; and e. producing a poliovirus peptide array containing the selected peptides; thereby producing a poliovirus array.

[0163] In some embodiments, the method is a method of producing an optimized array. In some embodiments, optimized is optimized for a target population. In some embodiments, the target population is a population from a specific region or country. In some embodiments, a target population is a population of a specific age. In some embodiments, a target population is a population with a specific viral exposure history.

[0164] In some embodiments, the provided poliovirus peptide array comprising a plurality of synthetic peptide probes selected from those provided in Table 9. In some embodiments, the provided poliovirus peptide array comprising a plurality of synthetic peptide probes selected from those provided in Table 9 and a plurality of probes from Table 1. In some embodiments, the provided poliovirus peptide array comprising the synthetic peptide probes provided in Table 9. In some embodiments, the provided poliovirus peptide array comprising the synthetic peptide probes provided in Table 9 and Table 1. In some embodiments, theplurality is at least 2, 5, 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, or 375. Each possibility represents a separate embodiment of the invention. In some embodiments, the plurality is at least 100. In some embodiments, the plurality comprises probes from poliovirus type 1. In some embodiments, the plurality comprises all the probes from poliovirus type 1 in Table 9. In some embodiments, the plurality comprises probes from poliovirus type 2. In some embodiments, the plurality comprises all the probes from poliovirus type 2 in Table 9. In some embodiments, the plurality comprise probes from poliovirus type 3. In some embodiments, the plurality comprises all the probes from poliovirus type 3 in Table 9.

[0165] In some embodiments, the sample is a biological sample. In some embodiments, the sample is a bodily fluid. In some embodiments, the sample comprises antibodies. In some embodiments, the sample is a blood sample. In some embodiments, blood is peripheral blood. In some embodiments, blood is plasma. In some embodiments, blood is serum. In some embodiments, blood is whole blood.

[0166] In some embodiments, the subjects have known neutralizing antibody status against at lest one of poliovirus types 1-3. In some embodiments, the subjects have known neutralizing antibody status against poliovirus type 1. In some embodiments, the subjects have known neutralizing antibody status against poliovirus type 2. In some embodiments, the subjects have known neutralizing antibody status against poliovirus type 3. In some embodiments, the subjects have known neutralizing antibody status against all three poliovirus types.

[0167] In some embodiments, the subjects are from a common population. In some embodiments, a common population comprises a common vaccination history. In some embodiments, a common population comprises a common history of exposure to poliovirus. In some embodiments, a common population is from the same country. In some embodiments, a common population is from the same location. In some embodiments, a common population is a population of a common age. In some embodiments, the age is a child. In some embodiments, a child is a toddler. In some embodiments, the age is an adult. In some embodiments, the age is elderly. In some embodiments, the array is optimized for the common population.

[0168] In some embodiments, the binding correlates with neutralizing antibody status against at least one poliovirus type. In some embodiments, the binding correlates with neutralizing antibody status against at least two poliovirus types. In some embodiments, thebinding correlates with neutralizing antibody status against all three poliovirus types. In some embodiments, the correlation is determined by applying a machine learning (ML) model. In some embodiments, the ML model is a trained ML model. In some embodiments, the model is trained on the binding of a sample from subjects with known neutralizing antibody status. In some embodiments, selecting comprises applying a trained machine learning algorithm to the detected binding to predict the peptides most important to differentiate between neutralizing antibody status. In some embodiments, the machine learning algorithm is trained on a training set comprising binding results from subjects with known neutralizing antibody status. In some embodiments, the machine learning model is a Random Forest (RF) algorithm. The RF algorithm is well known in the art and any other algorithm / model that allows for identifying how much each input contributes to differentiation / classification may also be used. In some embodiments, the selecting further comprises clustering peptides. In some embodiments, peptides with similar amino acid sequences are clustered. In some embodiments, the clustering further comprises selecting only 1 peptide from a cluster. In some embodiments, the clustering is by a silhouette clustering method. Silhouette clustering is well known in the art and any similar clustering method may also be used.

[0169] As used herein, the term "about" when combined with a value refers to plus and minus 10% of the reference value. For example, a length of about 1000 nanometers (nm) refers to a length of 1000 nm+- 100 nm.

[0170] It is noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polynucleotide" includes a plurality of such polynucleotides and reference to "the polypeptide" includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0171] In those instances where a convention analogous to "at least one of A, B, and C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrasepresenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B".

[0172] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0173] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents, unless the context clearly dictates otherwise. The terms “a” (or “an”) as well as the terms “one or more” and “at least one” can be used interchangeably.

[0174] Furthermore, “and / or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” is intended to include A and B, A or B, A (alone), and B (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).

[0175] Wherever embodiments are described with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of’ and / or “consisting essentially of’ are included.

[0176] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[0177] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.EXAMPLES

[0178] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I- III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.Example 1: Polio antigen microarray (PAM) construction

[0179] The inventors set out to design a novel polio antigen microarray for profiling poliospecific antibody responses using several poliovirus preparations: whole inactivated poliovirus, complete recombinant capsid proteins and overlapping peptides of capsid proteins. The microarray is able to profile antibody responses in serum, dried blood spots, oral swabs, buccal swabs and stool suspensions using human samples and identify the presence of not just antibodies, but neutralizing antibodies in the patient samples.

[0180] Two types of polio antigen microarrays were spotted, calibrated and optimized. Each antigen was spotted in triplicate. The two types of microarrays together compose the Polio antigen microarray (PAM).

[0181] 1. Peptide microarrays, spotted with 18-22-mer synthetic peptides that covered the full sequences of proteins VP1, VP2 and VP3 of poliovirus type 2 Sabin strain with a partial overlap of 15aa. In addition, partially overlapping peptides were also designed for the full VP1 protein sequences of type 2 MEF and type 3 Sabin strains, and for selected antigenic and neutralization sites that were previously identified in type 1, type 2 and type 3 polioviruses, in particular sites with variable sequences in different strains from the same type. The peptides for selected sites were designed for VP1, VP2 and VP3 proteins of three poliovirus type 1 strains (Sabin, Mahoney, and the wild type SoAS), two type 2 strains (Sabin and MEF), and two type 3 stains (Sabin and Saukett). Each peptide was designed as a 16-20 amino acids (aa) sequence from the target protein. Two lysine residues (KK) were added at the N-terminus of each peptide, to improve amine-binding to amine-binding hydrogelcoating slides. In total: 386 synthetic peptides (Table 9) were spotted on the arrays. The peptides were spotted in triton X-100 buffer at the concentration of 1 mg / ml. Triton X-100 concentration was adjusted for each peptide individually in the range 0-0.05%. Several types of peptide microarrays were spotted in different sizes. Eight or 16 identical peptide microarrays were spotted on each slide, dependent on the microarray size. Each microarray can be tested with a different serum sample, or another biological sample that contains human antibodies. The sequences of the known antigenic sites that were covered by some of the peptides have been described in the following: Minor et al., “Antigenic structure of polioviruses of serotypes 1, 2 and 3”, J Gen Virol, 1986. 67 (Pt 7): 1283-91; He et al., “Complexes of poliovirus serotypes with their common cellular receptor, CD155”, J Virol, 2003. 77: 4827-35; Huovilainen et al., “Antigenic variation among 173 strains of type 3 poliovirus isolated in Finland during the 1984 to 1985 outbreak”. J Gen Virol, 1988. 69 (Pt 8): 1941-8; and Roivainen et al., “Antigenic regions of poliovirus type 3 / Sabin capsid proteins recognized by human sera in the peptide scanning technique”, Virology, 1991. 180: 99-107. In addition, the inventors designed peptides to cover the full length of Sabin type 2 VP1, VP2 and VP3 proteins, and the full sequences of the VP1 protein of three type 3 strains. Figures 1A-B show, as examples, peptide microarrays that contain type 1 and type 2 peptides and includes 2 blocks (8 identical microarrays per slide).

[0182] 2. Virus microarrays - spotted with whole inactivated viruses or recombinant complete capsid proteins (viral-like particles, VLPs) of polioviruses from types 1, 2 or 3 (Table 1). During calibration experiments, the viral and VLP antigens were spotted in serial dilutions and in three types of buffers, in 4 large arrays on each slide. The arrays were hybridized with positive and negative polyclonal antibodies (obtained from NIBSC) specificto each of the three poliovirus types to select optimal spotting conditions that allow specific and sensitive identification of antibodies to each antigen (Fig. 2A-B). The selected spotting conditions used for profiling human sera are listed in Table 1. Next, the viral and VLP antigens were spotted only in the optimal conditions on smaller arrays. Sixteen identical virus-microarrays (a single block) were spotted on each slide (Fig. 3A-B).

[0183] Table 1: Viral antigens in the PAM microarray

[0184] ** The VLPs are non-infectious virus-like empty particles from the same strains as those used for IPV. MAH61024AFT (Mahoney), MEF162020 (MEF1) and SAU60343AFT (Saukett), contain mutations to stabilize the capsids. The bOPV vaccine given to the toddler cohort did not include type 2 strains.Example 2: PAMs recapitulate micro-neutralization assay in vaccinated toddlers

[0185] Serum samples from 176 children (0.7-3 years old) that were vaccinated with the inactivated poliovirus vaccine (IPV) were provided by the Israel Center for Disease Control (ICDC). Some of the children were also vaccinated with one or two doses of the live oral poliovirus vaccine (bOPV, n=126). The cohort was divided into groups by their history of poliovirus vaccinations and possible exposures (Table 2). The cohort included samples from 26 children that were possibly exposed to wildtype poliovirus type 1 during the sustained asymptomatic outbreak of WPVl-SoAS in Israel in 2013-2014 (group 4). The epicenter of poliovirus infections during this outbreak in Israel was in the Bedouin communities (non- Jewish) in the southern health districts, as determined by quantitative environmental surveillance and by stool surveys. The inclusion criteria for group 4 were non-Jewish children who were of the same age range as in groups 1 to 3 and were living in the southern health districts during the outbreak.

[0186] Groups 1-3 were matched as closely as possible by age, geographic location of their health district throughout Israel, and by the number of Jewish and non-Jewish children. Group 4 was age matched to groups 1-3 but consisted of only non-Jewish children from the Southern Health District. Gender distributions were less well matched (Table 2).

[0187] Table 2: Serum samples

[0188] * The cohort was divided into groups by their history of probable poliovirus vaccinations and possible exposures. IPV - inactivated poliovirus vaccine, including all the 3 types; bOPV - live oral poliovirus vaccine, including only poliovirus types 1 and 3; WPVl-SoAS - an Israeli wildtype poliovirus circulated in Israel in 2013.

[0189] The total protein concentration of each serum sample was measured, and 85 pl of serum diluted to 90 pg / ml total protein in hybridization buffer (PBS containing 1% BSA and 0.025% tween-20) were hybridized with each microarray block for IgG profiling. For IgA profiling, serum was diluted to 450 ug / ml total protein for hybridization. The secondary antibodies used were anti-human IgG- Alexa fluor 647 (Jackson cat# 709-605-149) diluted 1:1000, and anti-human IgA- Alexa fluor 647-(Jackson cat# 109-605-011) diluted 1:6000. The arrays were scanned using a robotic Genepix 4400A laser scanner and annotated using Genepix Pro 7 software. Each antigen in each concentration was spotted in triplicate spots. The mean local background was subtracted from the mean fluorescent intensity of each spot, and the median fluorescent intensity of each triplicate (MFI) was selected for analysis.

[0190] Python tools were created for analysis of the PAM MFI results. The magnitude and breadth of antibodies to selected groups of antigens (e.g. all type 1 viral antigens, or all peptides for a selected antigenic site, or all peptides of a selected protein) were computed for each serum sample. The magnitude was defined as the sum of MFI, and the breadth was defined as the number of antigens with MFI higher than a selected threshold in a group of antigens. The geometric mean of responses to each group of antigens were also computed. The threshold was selected to represent a background non-specific staining, based on the background MFI of array frames that did not include spots. Two-sided hypothesis tests (Wilcoxon rank sum and Fisher’s exact test) were used to test for differences between the distribution of breadth and magnitude scores in groups of samples. In the analysis of single antigens, to increase the statistical power when testing single peptide antigens, the set of peptide antigens to be tested were filtered using a label blinded approach based on the response rate to each antigen and false discovery rate (FDR) when comparing groups of samples. Using this approach, only peptide antigens to which over 40% of the blinded cohort responded were analyzed.

[0191] IgG antibody binding to a few PAM peptide antigens was significantly different in the two vaccine groups: IPV only (group 1, n=50) compared with IPV+bOPV (bOPVincluded only types 1 and 3 polioviruses; groups 2+3, n=100). For this analysis, the MFI of each peptide was compared individually between the groups using Fisher’s exact test after filtering the peptides using label-blinded response rates. P-values were adjusted using the Benjamini-Hochberg false discovery rate (FDR) method with an FDR cutoff of q < 0.2. Only peptides that bound antibodies from at least 40% of the samples were selected. Although the bOPV vaccine did not include type 2 poliovirus, IgG binding to two adjacent peptides from type 2 Sabin VP2 protein (peptide 11: DQPTEPDVAACRFYTLDTVT (SEQ ID NO: 11) and peptide 15: WRKESRGWWWKLPDALKDMG (SEQ ID NO: 15)) was significantly different in the IPV+bOPV group compared with the IPV only group. The IPV+bOPV vaccine group presented significantly higher IgG levels to Sabin type 2 peptide 15, together with marginally significant lower levels of IgG to peptide 11 (Fig. 4). In addition, the IPV+bOPV groups presented a significantly higher level of IgG to VP1 peptide 7 of the Mahoney and SoASl poliovirus type 1 strains (SEQ ID NO: 191 and 192, respectively), but not to the same peptide from the Sabin strain (Fig. 5). Six additional peptides of the Sabin strains types 1 and 2 presented similar, but non-significant trends (Table 3).

[0192] Thus, using peptide antigens, two domains that could distinguish between subjects who received the IPV vaccine and subjects who received both IPV and bOPV vaccines were identified. The first domain is located in poliovirus type 1 VP1 amino acids 71-90. The second domain is located in poliovirus type 2 Sabin strain in VP2 amino acids 51-90. While IgG binding to type 1 VP1 aa 71-90 and type 2 VP2 aa 71-90 IgG binding was higher for the group that received IPV followed by bOPV, type 2 VP2 aa 51-70 bound a higher level of IgG antibodies in the group that was vaccinated with IPV only.

[0193] Table 3: Additional peptides from poliovirus types 1 and 2 (Sabin strains) that bind different IgG levels in the two vaccine groups.

[0194] IgG levels to monovalent IPV and OPV viral antigens were not significantly different in the IPV and IPV+bOPV groups. However, IgG binding to standard IPV mixtures of trivalent IPV and OPV vaccines (12 / 104 and 17 / 160, respectively) was surprisingly higher in the IPV alone group as compared with the IPV+bOPV group (Fig. 6A). IgG levels to the VLP antigens that represent capsids of IPV strains were also significantly higher in the IPV alone group, as compared with the IPV+bOPV group (Fig. 6B).

[0195] These results suggest that when the bOPV vaccine was given after the IPV vaccine, it modified the anti-polio IgG repertoire and reduced IgG binding to IPV antigens. However, it is unclear why bOPV vaccination also reduced IgG binding to the trivalent OPV standard mixture, while binding to monovalent OPV antigens was not changed. Nevertheless, it is clear that the PAM assay can distinguish between groups of children who were vaccinated with IPV only and children who were vaccinated with IPV+bOPV.

[0196] When the group that was vaccinated with IPV only (group 1, n=50) was compared with the group that was vaccinated with both IPV+bOPV and were also possibly exposed to type 1 SoASl poliovirus (IPV+bOPV+pos.Exposure group 4, n=26), IgG binding to peptide VP1_7 of SoASl strain (SEQ ID NO: 192) and peptide VP3_1 of Sabin type 1 strain (SEQ ID NO: 170) was higher in the IPV+bOPV+pos.Exposure group compared with IPV alone group, as well as to the IPV+bOPV group (Fig. 7A, and Fig. 5). A few additional differences in the IgG repertoire to PAM peptides emerged in the IPV+bOPV+ pos. Exposure group. The sera of individuals possibly exposed to SoAS 1 included significantly higher IgG levels to three additional peptides of SoASl: peptides 12 and 29 of VP1 (SEQ ID NO: 207 and 231, respectively), as well as peptide 1 of VP2 (SEQ ID NO: 166). Peptide VP1_29 of the Sabin type 1 strain (SEQ ID NO: 230) was also targeted by higher IgG levels in the possibly exposed group, most probably since this sequence is conserved and differs from the SoAS 1 VP1_29 (SEQ ID NO: 231) by only a single amino acid (Fig. 7B). The sera from possibly exposed group included higher IgG levels not only to Sabin type 1 VP3_1 peptide (SEQ ID NO: 170), but also to the partially overlapping peptide VP3_2 of Sabin type 1 (SEQ ID NO: 172), supporting the probability that this site is targeted by antibodies developed following exposure (Fig. 7B). All these peptides cover known neutralization antigenic sites (nsNag or type 1 serotype- specific neutralization antigenic sites (SNag): VP1 peptide 7 - SNag #1,VP1 peptide 12 - Nag #1, VP1 peptide 29 - SNag #4, VP2 peptide 1 - Nag #4, and VP3 peptides 1 and 2 - Nag #3.

[0197] Although the possible exposure was to a poliovirus type 1, and bOPV did not include poliovirus type 2, the IgG binding of IPV+bOPV+ pos.Exposure group differed from the IPV only group also by serum IgG to a few poliovirus type 2 peptides. The most significant differences are shown in Figures 8A-B. The potential exposure to SoASl induced a significant decrease in IgG binding to peptide VP3_38 of Sabin type 2 (SEQ ID NO: 90), and a significant increase in IgG binding to Sabin type 2 peptides VP1_49, VP3_25 and MEF VP1_2O peptide (SEQ ID NO: 146, 77 and 117, respectively). Of these peptides, only MEF VP1_2O includes a known antigenic site.

[0198] In contrast to the larger number of peptide antigens that distinguish between IPV+bOPV+ pos.Exposure and IPV only groups, compared with IPV+bOPV vs IPV only, the difference in the antibody repertoire to conformational polio antigens was smaller. While higher IgG levels against almost all viral antigens were detected in the IPV alone group compared with IPV+bOPV group (e.g., Fig. 6A-B), the IPV+bOPV+pos. Exposure group only presented lower IgG levels to VLP Types 1 and 2 antigens compared with IPV only group (Fig. 9).

[0199] The IPV+bOPV group (n=100) was divided into two groups: children that received two bOPV vaccines (group 2, n=50), and children that received only a single bOPV dose (group 3, n=50) (Table 2). The group that was vaccinated twice with bOPV had significantly lower median IgG levels to all the twelve peptides that significantly differed between these groups (type 1: 8 peptides, type 2: 4 peptides; Fig. 10).

[0200] The neutralization titers to types 1 and 3 polioviruses in each of the 176 serum samples from children (Table 2) were measured by the Israeli Central Virology Lab (CVL, Sheba Medical Center) using the WHO approved micro-neutralization assay using Sabin 1 and Sabin 3, WHO-certified polio vaccine strains. Micro-neutralization assays for type 2 poliovirus were performed in the NIBSC (UK) using WHO-certified Sabin 2 vaccine strains. A correlation was found between titers of neutralizing antibodies to the three types, with the highest correlation between neutralization titers to types 1 and 3, and lowest correlation between titers to types 1 and 2 (Fig. 11).

[0201] Samples with a neutralization titer of greater than 8 to a given poliovirus type were defined as protected from this type, and samples with a neutralization titer less than or equal to 8 were defined as unprotected. Thus, for each poliovirus type, the 176 serum samples weresorted into two groups: protected (neutralization titer > 8) or unprotected. The PAM MFI results were then sorted between the protected and unprotected groups for each poliovirus type separately. The Wilcoxon rank sum test was used to compare the antibody repertoires of individuals with protective or non-protective neutralization titers.

[0202] To identify subsets of antigens on the PAM array that are associated with protection, the 176 serum samples (Table 2) were sorted to protection groups (separately for each type), based on their neutralization titers:1. Unprotected group - neutralization titer <8 (low responders);2. Protected group - neutralization titer > 8.A total of 9 individuals were found to be low responders for at least one polio type with 2 of them who were low responders to all three polio types (Tables 4-5), although all these toddlers were vaccinated with polio vaccines. In contrast, there were 140 subjects that were high responders to at least one polio type, with 56 of them who were high responders to all three types.

[0203] Table 4: Number of children in the protected and unprotected groups for each poliovirus type according to micro-neutralization assay.

[0204] Table 5: Protection from each poliovirus type of the unprotected subjects.

[0205] The repertoires of both IgG and IgA antibodies to the array antigens were compared between the protected and unprotected groups, using the Wilcoxon ranksum test, for both viral and peptide antigens (see Tables 9-10). The response of an antibody isotype to a set of viral or peptide antigens was computed as the geometric mean magnitude of MFI of all the antigens included in this set by the referred antibody isotype.

[0206] Serum antibody binding to viral antigens was compared both for each antigen individually and also following grouping the viral antigens into sets (e.g. All IPV viral antigens from the 3 types).

[0207] Serum antibody binding to peptide antigens were compared only following grouping to sets. Two types of peptide sets were created: all peptides covering a specific antigenic site, or all peptides included in a specific protein. Since the peptides of the array are partially overlapping (15aa overlap), the antibody response to each antigenic site or protein was defined as the overall geometric mean (gmean) of response of each antibody isotype to all peptides that cover that site. Peptides were initially designed to cover 18 antigenic sites of 5 strains of poliovirus serotypes 1 and 2 (Tables 6-8). Microarrays with type 3 peptides, have also been produced.

[0208] Table 6: Poliovirus type 1 antigenic sites (Mahoney strain sequences). When aa sequence is given in bold, only the bold amino acids are part of the epitope.

[0209] Table 7: Poliovirus type 2 antigenic sites (Sabin strain sequences).

[0210] Table 8: Poliovirus type 3 antigenic sites (Sabin strain sequences).

[0211] In the following Tables 11 and 12, viral antigens or antigen sets associated with protective neutralization titers (Table 11) or lack of protection (Table 12) from type 1, 2, or 3 polioviruses (PV1, PV2, PV3, respectively), are presented. Viral antigens of all three poliovirus types are included, as are peptide antigens of type 1 and type 2. Wilcoxon ranksum was used to compare these measures in serum samples that are protected (neutralization titer>8) or unprotected (neutralization titer<=8) from for each poliovirus type in the toddlers' cohort. Results are shown only for antigens or antigens sets and antibody isotypes that significantly differed between protected and unprotected groups. Nomenclature for peptides sets in Tables 11-12 and Figures 12A-B, 12D and 14:OPV2_Sabin_2_VP3_gmean_magnitude refers to gmean magnitude of IgG binding (measured as MFI) to all the spotted peptides of protein VP3 of OPV2 strain (poliovirus type 2); Sabin l_VPl_nonNag_2_gmean_magnitude refers to gmean magnitude of IgG binding (measured as MFI) to the spotted peptides covering non-neutralizing site 2 in Sabin 1 strain (poliovirus type 1); etc. Each antigenic site is represented by partially overlapping peptides that together cover its full sequence, together with 10-aa from each terminus. The overlapping peptides that can be combined / summed to get a readout for each antigenic site are provided in Table 13-16. Table 13 provides the peptides covering antigenic sites for type 1 poliovirus. Table 14 provides the peptides covering antigenic sites for type 2 poliovirus. Table 15 provides the peptides covering antigenic sites for type 3 poliovirus. The key for the abbreviations in the antigenic sites is as follows: Nag- antigenic site known as binding antibodies, and a mAb bound to it was reported as neutralizing; binding- antigenic site known as binding antibodies, but it is unknown if it is neutralizing or not; nonNag- antigenic site known as binding antibodies, and a mAb bound to it was reported as non-neutralizing; Snag-antigenic site known as binding antibodies that bind only to type 1 poliovirus, and a mAb bound to it was reported as neutralizing.

[0212] Table 13: Peptides for type 1 antigenic sites.

[0213] Table 14: Peptides for type 2 antigenic sites.

[0214] Table 15: Peptides for type 3 antigenic sites.

[0215] Tables 11 and 12 show the binding of serum IgG and IgA to the various probes that delineate each of the various epitopes that were investigated and are provided in Table 13- 16. Nomenclature of sets of viral antigens in Tables 11-12: Tl_IPV_gmean_magnitude refers to gmean magnitude of IgG binding to all type 1 IPV viral antigens; T2_gmean_magnitude refers to gmean magnitude of IgG binding to all type 2 viral antigens (IPV and OPV); All_IPV_gmean_magnitude refers to gmean magnitude of IgG binding to all IPV viral antigens including the 3 types; etc. Nomenclature of sets of peptide antigens in Tables 11-12: OPV2_Sabin_2_VP3_gmean_magnitude refers to gmean magnitude of antibody binding to all the peptides of protein VP3 of Sabin 2 strain; OPV1- non_neutralizing_site_2_gmean_magnitude refers to gmean magnitude of antibody binding to all the peptides included in OPVl-non_neutralizing_site_2_.

[0216] Table 11: Serum IgG and IgA levels to specific viral antigens, sets of viral antigens, or sets of peptides, that are associated with protection from the different types of polioviruses in toddlers.

[0217] Table 12: Serum IgG and IgA levels to specific viral antigens, sets of viral antigens, or sets of peptides, that are associated with lack of protection from the different types of polioviruses in toddlers.

[0218] IgG binding to 15 sets of peptides (14 / 15 are type 1 peptides) was higher in toddlers protected from type 1 polioviruses. In addition, IgG binding to a single type 2 peptides set and a single type 2 viral antigen (T2-VLP) was higher in toddlers protected from type 2 polioviruses. IgG binding to another single type 2 peptides set was higher in toddlers protected from type 3 polioviruses. On the other hand, IgA binding to 5 individual antigens viral antigens or to 6 sets of viral antigens of the three types was higher only in toddlers protected from type 2 polioviruses (Table 11).

[0219] As expected, mainly antigenic sites of type 1 sequences (14 / 15) were associated with protection from type 1 poliovirus. Subjects in the unprotected group failed to generate an IgG response, or generated very weak responses to these sites, while subjects in the protected group generated a significant level of IgG antibodies to these sites (Table 11).

[0220] Some examples for IgG binding to sets of peptides or a viral antigen that was significantly higher in toddlers protected from specific poliovirus types, compared with the unprotected, are presented in Fig. 12. For example, IgG gmean magnitudes to peptides thatcover the antigenic sites of proteins VP1 or VP3 of type 1 Sabin strain (0PV1) were associated with protection from poliovirus type 1 only (Fig. 12A-B). Similarly, IgG gmean magnitude to antigenic site 1 of poliovirus type 2 MEF strain (MEF_VPl_binding_l) was associated with protection from poliovirus type 2 only (Fig. 12D). IgG binding level to poliovirus type 2 VLP viral antigen was also associated with protection from poliovirus type 2 only (Fig. 12C).

[0221] Some examples for IgA binding to individual viral antigens that were significantly higher in toddlers protected from poliovirus type 2 only, compared with the unprotected, are presented in Fig. 13. Examples of 3 IPV strains of different poliovirus serotypes: type 1 VLP, inactivated MEF virus (type 2), and inactivated Saukett virus (type 3); as well as standard mixtures of both trivalent IPV and OPV vaccine strains, showed association with protection against poliovirus type 2 only (Fig. 13).

[0222] Several type 1 and type 2 antigenic sites were identified as binding significantly higher IgA levels in the type 3 unprotected group compared with the type 3 protected group (Table 12). Specifically, gmean magnitudes of IgA binding to 17 sets of peptides from strains of both poliovirus serotypes 1 and 2, were significantly higher in individuals with neutralizing titer < 1:8 for poliovirus serotype 3 only (see also two examples in Fig. 14).

[0223] Therefore, thus far, microarray IgA signatures were associated with protection from poliovirus serotype 2 and lack of protection from poliovirus serotype 3 (compare Tables 11 and 12).

[0224] As expected, binding to three type 1 antigenic sites that are known as neutralizing epitopes was higher in the type 1 protected group (i.e. in toddlers with a neutralization titer>8). However, interestingly, IgG binding to two OPV1 antigenic sites that were previously published as binding non-neutralizing antibodies (see Chow et al., “Synthetic peptides from four separate regions of the poliovirus type 1 capsid protein VP1 induce neutralizing antibodies”, 1985, Proc Natl Acad Sci U S A, 82(3): p. 910-4 and Martin et al., “Characterization of formaldehyde-inactivated poliovirus preparations made from live- attenuated strains”, 2003,. J Gen Virol, 84(Pt 7): p. 1781-8), was found as higher in the type 1 protected group, suggesting that these sites are actually neutralizing. One of these sites ('non-neutralizing' site 4 (Sabl-Mah-SoAS_VPl_nonNag_4): YKDGTLTPLSTKDLTTY; SEQ ID: 399; OPV1_VP1_31) was conserved in all three type 1 strains that were included in the peptides array (Sabin type 1, Mahoney and SoAS). Therefore, the inventors suggestthat the proposed assay can be used to efficiently identify neutralizing antigenic sites, using polyclonal serum samples instead of monoclonal antibodies.Example 3: PAMs recapitulate micro-neutralization assay in Israeli adults

[0225] The inventors obtained a set of 1169 serum samples from Israeli adults with known type 1 and type 3 neutralization titers (measured by CVL): 908 serum samples that were collected in Israel during 2020, and 261 serum samples that were collected in Israel during 2021. These sample sets included 65 individuals with low neutralization titers < 1:8 for at least one poliovirus type, which are considered non-protected subjects (Table 16). Given these low numbers and data suggesting that higher neutralizing titers may be required for preventing a wildtype infection, the inventors also considered a less stringent definition of low titers defined as individuals with titers < 1:32 to select 147 low-titer samples (Table 17).

[0226] Table 16: Adults cohort distribution for non-protected (neutralization titer < 8) poliovirus Type 1 and 3 groups.*Number of samples collected in the same year, along with their respective percentages relative to the total number of samples collected.

[0227] Table 17: Adults cohort distribution for low neutralization titers (< 32) poliovirus Type 1 and 3 groups.*Number of samples collected in the same year, along with their respective percentages relative to the total number of samples collected.

[0228] Based on the neutralization titers of type 1 and type 3 polioviruses obtained, a subset of 223 samples from the 1,169 serum samples was selected for type 2 neutralization measure. This subset included (Table 18):(a) n=148 samples with low NT < 1:32 for at least one poliovirus serotype (1 or 3);(b) n=30 samples with high NT > 1:64 for at least one poliovirus serotype (1 or 3). The selected 223 samples were sent to NIB SC for measuring type 2 neutralization titer (NT).

[0229] Table 18: Distribution of types 1 and 2 neutralization titers in 223 samples that were sent to NIB SC, based on iCVL measures*

[0230] * Samples were grouped according to the neutralization titers for poliovirus serotypes 1 and 3, as measured by iCVL. Samples were selected if they have low or high NT for type 1 or type 3. Therefore, sometimes the same sample is included in two cells of the table.

[0231] Table 19 presents the distribution of low, intermediate, and high neutralization titers in this subgroup of 223 samples for the three poliovirus types, based on both iCVL and NIBSC measures.

[0232] Table 19: Distribution of low, intermediate and high neutralization titers in 223* adult samples for which type 2 neutralization titers were quantified, based on NIBSC results.0233] * The same sample can appear in different groups. Percentages are from all the 223 samples.

[0234] A PAM microarray including 386 peptides from the three types (listed in Table 9) and 14 viral antigens was used to profile IgG and IgA repertoires of the selected 223 serum samples. The 14 viral and VLP antigens were spotted in four serial dilutions, to calculate the area under the curve (AUC) of the median fluorescent intensity (MFI) as a function of the antigen's concentration. The 386 peptides were spotted in a single concentration of 1 mg / ml. In total, the extended array included 400 antigens in triplicate, and 1,326 spots.

[0235] This extended microarray was very large, and ~3 ul of serum sample was required to profile both IgG and IgA. A new analysis was performed, based on toddler samples from the study in Example 1, leading to selection of 2 / 3 of the antigens as necessary and enough for distinguishing between neutralizing and non-neutralizing serum samples, resulting in a 33% reduction in both work time and consumables. Viral antigens were spotted in four serial dilutions for AUC calculation, while peptide antigens were spotted in a single concentration (1 mg / ml). On the next step, the antigens were divided between 4 separate microarrays, to reduce spotting time. All the NT-characterized 223 samples were run on these microarrays for IgG and IgA profiling. These samples included 90 individuals that had neutralizing titers <16 against one or more poliovirus serotypes (Fig. 15).

[0236] The inventors used the geometric mean magnitude (GMM) summary statistic computed across different sets of antigens. For whole virus and VLP antigens which were spotted at multiple serial dilutions, the median area under the curve (AUC) of the triplicate was used for GMM calculation, while for the peptide antigens, which were spotted at a single concentration, the median fluorescent intensity (MFI) measured over triplicates was used. The antigenic sites covered by the peptides are listed in Figures 16A-B, and the peptides covering them are listed in Tables 13-15. For each antigen or set of antigens, significant associations with protection were tested using the Wilcoxon ranksum test. Association with lack of protection to all three polio serotypes (all types) was also tested for.

[0237] The inventors tested for associations between individual viral antigens from each polio serotype including VEPs and vaccine antigens, as well as a mixture of all three polio serotypes. Antibodies to multiple viral antigens were significantly associated with protection against all three polios serotypes. IgG AUC binding to both VLPs and vaccine strains were significantly associated with type 1 and type 3 neutralization, but not with type 2 neutralization (Fig. 17A-B). In contrast, IgA GMM binding was significantly associated across all three polio serotypes (Fig. 18A-B). Overall, associations for type 2 were weaker than for types 1 and 3 across all antigens. This indicates that IgA antibody binding to both poliovirus VLPs and viral antigens is associated with protection across all three polio serotypes.

[0238] As described above, the microarrays also included 20mer peptides from the antigenic sites of three poliovirus serotype 1 strains (Sabin, Mahoney, and the wildtype WPVl-SoAS), two serotype 2 strains (Sabin and MEF-I), and two type 3 strains (Sabin and Saukett), as well as the entire VP1, VP2 and VP3 of the type 2 Sabin strain (Tables 13-15). The inventors looked for associations between IgG GMM to each of these antigenic sites and neutralization of each poliovirus type. While IgG GMM for some antigenic sites was associated with non-protective neutralization titers, IgG GMM to other antigenic sites (e.g. SoAS_VPl_SNag_2 - SoAaS VP1 serotype- specific neutralizing site 1) was associated with protection (e.g. Fig. 19A-C, and summary in Fig. 20D-E). Interestingly, IgG binding to a broad range of types 1 and 2 antigenic sites was associated with lack of neutralization of type 3 virus (Fig. 20D). This indicates that IgG binding to linear peptides spanning antigenic sites is associated with protection against polioviruses.

[0239] Unlike the mixed associations of IgG GMM and protection, it was found that all significant associations between IgA GMM to types 1 and 2 antigenic sites and protection were positive, and all significant associations between IgA GMM to type 3 antigenic sites and protection were negative (Fig. 20A-E). Specifically, IgA binding to most of the antigenic sites of type 1 strains were associated with neutralization of type 1 poliovirus, while IgA binding to most of the antigenic sites of type 3 strains were associated with lack of neutralization of type 3 poliovirus (Fig. 20D-E ). This indicates that IgA binding to linear peptides spanning antigenic sites of type 1 or type 3 strains have opposite associations with protection.Example 4: PAMs recapitulate micro-neutralization assay in DRC's children

[0240] To study if the same peptides are useful in assaying subjects from vastly different regions and ages, the inventors used the same four microarrays described in Example 3 to profile serum IgG and IgA anti-polio antibodies in a cohort of children from the Democratic Republic of Congo (DRC). Children from the DRC have very different history of vaccination and disease exposures compared with Israeli adults. Indeed, PAM's associations with protection were very different compared with the results in Example 3.

[0241] The inventors received 1507 serum samples with known neutralization titers for the three polio serotypes from the DRC, and selected 320 samples with high (>1:32) or low (<1:8) neutralization titers for at least a single poliovirus type. The neutralization status of the 320 samples is summarized in Figure 21A. Same microarrays and analysis were performed as in Example 3. The analysis revealed IgG GMM to 7 / 13 type 1 antigenic sites were significantly associated with protection from types 1 or 2 viruses or susceptibility to type 3 virus (Fig. 21B, representative examples). IgA GMM of 7 / 13 type 1 antigenic sites were significantly associated with protection from types 1 and 2 viruses, and a single site was associated with susceptibility to type 3 virus (Fig. 21C, representative examples). IgG GMM of 4 / 6 type 2 antigenic sites were significantly associated with protection from type 1 virus, or susceptibility to type 3 virus (Fig. 21D, representative examples). IgA GMM ofonly 1 / 6 type 2 antigenic sites was significantly associated with protection from type 2 virus (Fig. 21E, the same site in two different strains are presented). IgG GMM to 10 / 18 type 3 antigenic sites were significantly associated with protection or susceptibility to types 1, 2 and / or 3 viruses (Fig. 21F, representative examples). IgA GMM to all 18 / 18 type 3 antigenic sites were significantly associated with protection from types 1, 2 and / or 3 viruses or susceptibility to type 3 virus only (Fig. 21G, representative examples). All of the sites for type 1 and 2 are summarized in Figure 21H and all of the sites for type 3 are summarized in Figure 211.

[0242] A comparison of the summary tables of the Israeli adults (Fig. 20D-E) and the DRC children (Fig. 21H-I) demonstrate that IgG or IgA binding to very different sites is informative for determining virus protection in the two cohorts, and even the association direction may be different. This emphasizes that different sets of peptides should be used for neutralization prediction in populations from very different environments and immune histories and ages, such as an African country like DRC and a western country like Israel.Example 5: Selection of an optimal subset of peptides antigens to predict poliovirus neutralization in the Israeli adults cohort

[0243] The results of both the Israeli and DRC cohort show that IgG and IgA magnitudes to a single antigenic site or a single VLP or viral antigen are not sufficient to diagnose if a specific sample has a low or high neutralization titer. Even when the GMM for the protected and unprotected groups is significantly different, most of the samples have GMM values that can belong to both protection groups (see the dots in the swarmplots of Examples 3 and 4). Therefore, we developed a multivariate non-linear model that summarizes antibody binding to a set of antigenic sits and viral antigens for diagnosis of protection.

[0244] The PAM assay included a large set of peptide antigens that spanned the full VP1, VP2 and VP3 sequences of serotype 2, as well as the antigenic sites for all three polio serotypes, as detailed hereinabove. In total this set included 140 PV1 peptides, 192 PV2 peptides and 242 PV3 peptides (total: 574 peptides), from which 386 were included in the final arrays. While antigen microarrays can easily include thousands of antigens on a single glass slide, a crucial requirement of the PAM assay was to minimize the number of antigens utilized in the final assay to allow one to spot 16 individual arrays on each slide, which limits the number of antigens to -120 antigens spotted in triplicates. Given that multiple dilutions for spotting VLP and viral antigens were used, it was necessary to minimize the number of peptide antigens to -80. The smaller arrays reduce the cost and work hours when testing a large number of samples, thus significantly increase the throughput of the PAM assay.Smaller arrays also require shorter spotting times, which increase the arrays quality and redundancy. In addition, the results of Examples 3 and 4 required running each sample on 4 different microarrays.

[0245] The inventors employed a combination of clustering and machine learning algorithms to identify a subset of optimal antigens (peptides) for predicting polio virus types 1, 2, and 3 neutralization, using Israeli adult samples to which neutralization titers were known for all the three types of poliovirus. Serum IgG and IgA in these samples were profiled using PAM arrays spotted with all the peptides listed in Table 9 (the results of Example 3). The IgG and IgA datasets were merged. Using hierarchical clustering with Ward's method, the inventors clustered peptides with similar antibody binding. Spearman correlation was used as the distance metric to capture non-linear relationships between antigens. From each cluster, a random antigen was selected. The inventors then applied a Random Forest algorithm to identify the most significant antigens based on feature importance. To ensure robust selection, the inventors used 20 different random seeds when determining feature importance. To determine the best antigens for each poliovirus type, the inventors ran the Random Forest algorithm on subsets of the top 10 to 40 antigens, in increments of 5, based on their feature importance. This process was repeated 50 times using different random seeds to ensure robust results. Precision was used as the primary performance metric, focusing on minimizing false positives to ensure that the selected antigens were truly relevant. This approach systematically ensured the selection of the most promising antigens, with an emphasis on high precision and reducing false positive errors.

[0246] The inventors first trained logistic regression models using the VEP and viral antigens. Predictors were trained using a leave-one-out cross validation framework with parameter tuning performed on each fold using nested cross-validation. The inventors found that while IgG antibody profiles yielded low to mild AUC scores of 0.59 for type 1, 0.24 for type 2, 0.60 for type 3, and 0.62 against all three types, IgA antibody profiles demonstrated stronger predictive power for protection across all serotypes, with AUC scores of 0.79 for type 1, 0.70 for type 2 and 0.74 for type 3, and an AUC of 0.83 against all three types (Fig. 22).

[0247] Data sets

[0248] To compile the complete dataset, the IgG and IgA microarray antibody profiles data were merged vertically (i.e., column-wise), so that each sample was represented by both its IgG and IgA measurements. Samples with neutralization titer > 1:32 were defined as neutralizing, and samples with titers < 1:8 were defined as non-neutralizing. Samples with 8<NT<32 were dropped for modeling. This process was performed individually for eachpolio type dataset. Due to the large number of peptides for type 3, the number of samples used for this type was further limited, resulting in the following datasets: Pvl: 160 samples and 70 peptides; Pv2: 161 samples and 96 peptides; and Pv3: 54 samples and 121 peptides.

[0249] Clustering

[0250] The inventors applied a hierarchal clustering algorithm to group similar peptides together using Ward’s method. The spearman correlation between the peptides was used as the distance metric to capture the non-linear relationship between the peptides. It was found that the correlation between IgG and IgA profiles were significantly lower than the intracorrelation between IgG or IgA features. This further highlights that responses to each of the two isotypes may be independently associated with protection, and therefore combining the two may improve the prediction performance, as demonstrated in Figure 23.

[0251] To select the optimal number of clusters the inventors used silhouette method. For each peptide the silhouette score is calculated as s=b-a / max(a,b) , where a is the mean intracluster distance (cohesion) and b is the mean nearest-cluster distance (separation). The best number of clusters was chosen by the max average silhouette score (see an example in Fig. 24).

[0252] Feature selection

[0253] To select the best peptides from the set of peptides for predicting neutralizing and non-neutralizing samples, the inventors used the random forest (RF) algorithm, which also provides an intuitive measure of the relative importance of each feature for the prediction model. Specifically, the sklearn python package was used to obtain the feature importance for each of the peptides, i.e. to assess how much the given peptide reduces the prediction impurity across all the trees in the forest. This allowed for the selection of the best n peptides. To reduce redundancy from similar peptides the inventors first used clustering over the peptides and selected one representative peptide from each cluster as a feature for model training. The inventors chose the precision as the primary performance metric, focusing on minimizing false positives to ensure that the selected peptides were truly relevant. Together, this yielded the following pipeline for feature selection, which was performed independently for all three serotypes:1. Generate 20 Random Starting conditions: Begin by generating R (R = 20) random numbers within the range of i = 0 to j=200. From each cluster, randomly select a peptide (feature).2. Filter peptide set using one of the following two approaches: a. Collinearity-corrected data: Only peptides exhibiting a correlation of less than 0.9were retained in the dataset for modeling. b. Correlation based clustering data: The inventors randomly selected a peptide from each cluster and used these peptides for modeling.3. Train a Random Forest model with Hyper-parameter Tuning: For every random seed, train the Random Forest (RF) algorithm Employing 10-fold cross-validation to ensure robustness. Fine-tune the number of trees and the maximum depth for each tree. Select the model with the best performance, according to the chosen evaluation metric (precision).4. Feature Subset Size Selection: To determine the optimal subset size for predicting the outcome with high precision, test feature sets ranging in size from 10 to 40, incrementing by 5. A RF model is retrained for each size and seed, employing 10-fold cross-validation to ensure robustness5. Best Subset Selection: The subset size that yields the best performance score, averaged across all random seeds, is chosen as the optimal subset.6. Final Model Training: Using the best-performing subset from the previous stage, the RF model is trained once again, employing 10-fold cross-validation to ensure robustness.7. Repeat process only for the clustering based representative selection filtering method (2b): To further reinforce the reliability of the feature selection, this entire process was repeated 50 times, each time using a new set of random peptides from each of the clusters.

[0254] The inventors applied this approach to all three serotypes and used the precisionrecall curve to assess model performance. To assess whether the feature selection indeed improves performance, it was compared to the simpler method based only on collinearity- corrected data, as well as to a model that used the full set of peptides for each serotype. It was found that the correlation-based clustering data feature- selection based model obtained better performance both compared to the full peptide set model as well as collinearity- corrected data feature- selection model, with AUC scores of 0.94 for poliovirus type 1 (PV1), 0.86 for poliovirus type 2 (PV2), and 0.83 for poliovirus type 3 (PV3). The collinearity- corrected data feature selection model was better for PV3 prediction with an AUC score of 0.88 (Fig. 25). It was found that only 15 type 1 peptides (SEQ ID NO: 182, 185, 187, 197, 200, 202-203, 204, 215, 217, 219, 222, 226-228) were required to predict PV1 protectionwith 93% precision. Interestingly, these features included both IgG and IgA features, and all of the selected type 1 peptides were from neutralizing sites. Similarly, 10 type 2 peptides (SEQ ID NO: 49, 52, 53, 69, 87, 95, 104, 122, 130, 158) were sufficient to predict PV2 protection with 82% precision, and 35 type 3 peptides were sufficient to predict PV3 protection with 87% precision (Fig. 26A-C and 27A-F). Three type 3 peptides were added for a better redundancy, such that 38 type 3 peptides were selected for the miniarray (SEQ ID NO: 260-261, 276-280, 283, 288, 292, 298-300, 302, 309, 312, 315-317, 319, 324-325, 331, 337, 350-355, 358, 361, 366, 369, 372, 374, 378, 381).

[0255] Based on these findings, a final PAM miniaturized miniarray for Israeli adults was generated comprising the 14 viral antigens provided in Table 1 (spotted in 3 serial dilutions), the 15 type 1 peptides, the 10 type 2 peptides and the 38 type 3 peptides. The peptides of this miniarray are listed in Table 10. Table 10 is a subset of the probes provided in Table 9. The actual mini-array also included the viral antigens of Table 1.

[0256] Table 10: Peptide probes from the mini-array.

[0257] The above-described miniaturized microarray that include subsets of both viral and peptide antigens in the same microarray was designed for one adult population. Different miniaturized microarrays are spotted for different populations / goals, but all of them contain a selection of the peptides from Table 9, with or without a subset of the antigens listed in Table 1.

[0258] The PAM microarray includes spotting of small microarrays (8 or 16 identical microarrays per slide) that include both viral and peptide antigens, based on a machinelearning model. These microarrays include subsets of the viral and peptide antigens described above, and are tailored for each population individually, by training the machinelearning model on samples of the target population where their neutralization titers for the three poliovirus types are known. Different combinations of antigens are required for different populations (e.g. toddlers vs adults, or different geographic regions). Similarly, different subsets of peptides are selected for other human cohorts, from other ages or other geographic origin. Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

Claims

CLAIMS:

1. An array comprising a plurality of probes each immobilized at a discrete location on said array, wherein said plurality of probes comprises at least two short peptide probes of between 5 and 100 consecutive amino acids from at least one of poliovirus type 1, poliovirus type 2 and poliovirus type 3.

2. The array of claim 1, wherein said plurality of probes comprises at least two short peptide probes of between 5 and 100 consecutive amino acids from said at least one poliovirus.

3. The array of claim 1 or 2, wherein said plurality of probes comprises a probe from poliovirus type 1, poliovirus type 2 and poliovirus type 3.

4. The array of any one of claims 1 to 3, wherein said poliovirus type 1 is selected from Sabin type 1, Mahoney type 1, SoAS type 1 and Brunhilde type 1, said poliovirus type 2 is selected from Lansing type 2, Sabin type 2, and MEF-I type 2, and said poliovirus type 3 is selected from Sabin type 3, Leon type 3 and Saukett type 3.

5. The array of claim 4, comprising probes from Sabin type 1, Sabin type 2, Mahoney type 1, MEF-I type 2 and SoAS type 1.

6. The array of any one of claims 1 to 5, wherein said plurality of probes further comprises at least one probe selected from a whole virus, a lysed virus, and a viruslike particle (VLP).

7. The array of any one of claims 1 to 6, wherein said plurality of probes comprises a short peptide from said at least one poliovirus, and wherein said short peptide consists of 10 to 60 amino acids.

8. The array of any one of claims 1 to 7, wherein said peptide is a sub-sequence from capsid polypeptide VP1, VP2 or VP3.

9. The array of claim 7 or 8, wherein said peptide comprises between 10-60 consecutive amino acids from a poliovirus protein.

10. The array of any one of claims 7 to 9, wherein said plurality of probes further comprises at least one of: a. an inactive form of said at least one poliovirus; and b. a VLP of said at least one poliovirus.

11. The array of any one of claims 1 to 10, wherein said plurality of probes comprises probes comprising at least two poliovirus type 1 antigenic sites selected from those provided in Table 13.

12. The array of claim 11, wherein said plurality of probes comprises probes comprising all of the poliovirus type 1 antigenic sites provided in Table 13.

13. The array of any one of claims 1 to 12, wherein said plurality of probes comprises probes comprising at least two poliovirus type 2 antigenic sites selected from those provided in Table 14.

14. The array of claim 13, wherein said plurality of probes comprises probes comprising all of the poliovirus type 2 antigenic sites provided in Table 14.

15. The array of any one of claims 1 to 14, wherein said plurality of probes comprises probes comprising at least two poliovirus type 3 antigenic sites selected from those provided in Table 15.

16. The array of claim 15, wherein said plurality of probes comprises probes comprising all of the poliovirus type 3 antigenic sites provided in Table 15.

17. The array of any one of claims 1 to 16, wherein said plurality of probes comprises the probes provided in Table 10.

18. The array of any one of claims 1 to 17, comprising serial dilutions of at least one probe, wherein each dilution is immobilized at a discrete location on said array.

19. The array of any one of claims 1 to 18, wherein said peptide probe is selected from those provided in Table 9.

20. The array of any one of claims 1 to 19, wherein said plurality of probes comprises the probes provided in Table 1.

21. The array of any one of claims 1 to 20, comprising probes against polio virus type 1, probes against polio virus type 2 and probes against polio virus type 3, wherein said probes against poliovirus type 1 comprise SEQ ID NO: 182, 185, 187, 197, 200, 202, 204, 215, 217, 219, 222-223, 226-228, said probes against poliovirus type 2 comprise SEQ ID NO: 49, 52, 53, 69, 87, 95, 104, 122, 130, 158 and said probes against poliovirus type 3 comprises SEQ ID NO: 260-261, 276-280, 283, 288, 292, 298-299, 302, 309, 312, 315-317, 319, 324-325, 331, 337, 350-355, 358, 361, 366, 369, 372, 374, 378, 381.

22. The array of any one of claims 1 to 21, for use in determining the presence of neutralizing antibodies against a poliovirus in a sample from a subject.

23. A method of identifying a subject as having neutralizing antibodies against a poliovirus, the method comprising: a. providing a biological sample from said subject comprising antibodies; b. contacting said sample to an array of any one of claims 1 to 22 in conditions sufficient for antibody binding to said probes; andc. detecting the binding of said antibodies to discrete locations on said array indicating the presence in said sample of antibodies to probes located at said detected discrete locations, wherein a level of binding beyond a predetermined threshold indicates said subject possesses neutralizing antibodies against said poliovirus; thereby identifying a subject as having neutralizing antibodies against a poliovirus.

24. A method of identifying a subject as having neutralizing antibodies against a poliovirus, the method comprising: a. providing a biological sample from said subject comprising antibodies; b. contacting said sample to an array of any one of claims 1 to 22 in conditions sufficient for antibody binding to said probes; c. detecting the binding of said antibodies to discrete locations on said array indicating the presence in said sample of antibodies to probes located at said detected discrete locations; d. producing an output comprising the level of antibody binding at each discrete location; and e. applying a trained machine learning algorithm to said produced output wherein said trained machine learning algorithm produces a diagnosis indicating said subject does or does not possess neutralizing antibodies against said poliovirus or produces a score indicating the likelihood said subject possesses neutralizing antibodies against said poliovirus; thereby identifying a subject as having neutralizing antibodies against a poliovirus.

25. The method of claim 23 or 24, wherein a subject not indicated as possessing neutralizing antibodies is further tested by the micro-neutralization assay with live poliovirus to determine if said subject possesses neutralizing antibodies against a poliovirus.

26. The method of any one of claims 23 to 25, wherein said method is a method of detecting neutralizing antibodies against all of poliovirus type 1, poliovirus type 2 and poliovirus type 3.

27. The method of any one of claims 23 to 26, wherein said biological sample is a peripheral blood sample, a plasma sample or a serum sample.

28. The method of any one of claims 23 to 27, wherein said detecting comprises contacting said array with bound antibodies with labeled secondary antibodies against said antibodies in said biological sample.

29. The method of claim 28, wherein said detecting further comprises scanning said array with a detector configured to detect said labeled secondary antibodies and producing an output of the discrete locations where antibody was detected.

30. The method of any one of claims 23 to 29, wherein said detecting is detecting binding to discrete locations comprising probes comprising at least one poliovirus type 1 antigenic sites selected from those provided in Table 13, at least one poliovirus type 2 antigenic sites selected from those provided in Table 14 or at least one poliovirus type 3 antigenic sites selected from those provided in Table 15.

31. The method of any one of claims 23 to 29, wherein said detecting is detecting binding to discrete locations comprising probes comprising at least two poliovirus antigenic sites selected from those provided in Tables 13-15.

32. The method of claim 30 or 31, wherein said detecting is detecting binding to discrete locations comprising all provided in Tables 13-15.

33. The method of any one of claims 23 to 32, wherein said detecting is detecting binding to discrete locations comprising probes comprising those provided in Table 1 and SEQ ID NO: 49, 52, 53, 69, 87, 95, 104, 122, 130, 158, 182, 185, 187, 197, 200, 202, 204, 215, 217, 219, 222-223, 226-228, 260-261, 276-280, 283, 288, 292, 298-299, 302, 309, 312, 315-317, 319, 324-325, 331, 337, 350-355, 358, 361, 366, 369, 372, 374, 378, and 381.

34. The method of any one of claims 23 to 33, wherein said output is a scan of said array indicating intensity of binding at each discrete location.

35. The method of any one of claims 23 to 34, wherein said output is a summary of the level of binding at each discrete location.

36. The method of any one of claims 23 to 35, wherein said machine learning algorithm is trained on a training set comprising: a. produced outputs from samples from subjects with known levels of neutralizing antibodies against said poliovirus contacted with said array; and b. labels indicating a sample is from a subject with or without levels of neutralizing antibodies against said poliovirus.

37. The method of any one of claims 23 to 36, wherein said method is a method of predicting risk of said subject to spread poliovirus following exposure, predicting risk of symptomatic infection of said subject or predicting recent infection of said subject,wherein the presence of neutralizing antibodies indicates said subject is not a risk for spreading poliovirus, is not at risk for symptomatic infection or was recently infected.

38. A kit comprising the array of any one of claims 1 to 22, and a labeled secondary antibody configured for detection of antibodies bound to said array.

39. A system comprising the array of any one of claims 1 to 22, and a detector configured to detect binding of antibodies to probes immobilized on said array.

40. The system of claim 39, wherein said detector is configured to detect labeled secondary antibodies.

41. A method for producing an optimized poliovirus peptide array, the method comprising: a. providing a poliovirus peptide array comprising a plurality of synthetic peptide probes selected from those provided in Table 9; b. contacting said peptide array with a biological sample from a plurality of subjects with known neutralizing antibody status against at least one of poliovirus types 1-3 under conditions sufficient for antibody binding to said probes; c. detecting binding of antibodies to discrete locations on said array indicating the presence in said sample of antibodies to probes located at said detected discrete locations; d. selecting peptides whose detected binding by antibodies correlates with neutralizing antibody status against at least one of said poliovirus types; and e. producing an optimized poliovirus peptide array containing said selected peptides; thereby producing an optimized poliovirus array.

42. The method of claim 41, wherein said plurality of subjects are from a common population, wherein a common population comprises a common vaccination history or history of exposure to poliovirus and wherein said array is optimized for said common population, optionally wherein said common population is a population of subjects from the same country.

43. The method of claim 41 or 42, wherein said provided poliovirus peptide array comprises the probes provided in Table 9.

44. The method of any one of claims 41 to 43, wherein said selecting comprises applying a trained machine learning algorithm to said detected binding to predict the peptides most important to differentiate between neutralizing antibody status, wherein said machine learning algorithm is trained on a training set comprising binding results fromsubjects with known neutralizing antibody status, optionally wherein said machine learning model is a Random Forest algorithm.

45. The method of claim 44, wherein said selecting further comprises clustering peptides with similar amino acid sequences and selecting only 1 peptide from a cluster, optionally wherein said clustering is by a silhouette clustering method.

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