Antigenic neuron-specific enolase peptides for diagnosis and treatment

Abstract

The present disclosure provides peptides that specifically bind to maternal autoantibodies produced in the mother or potential mother against the endogenous polypeptide antigen neuron-specific enolase (NSE) protein. The peptides described herein are useful for determining the risk of an offspring developing autism spectrum disorder (ASD) by detecting the presence of maternal autoantibodies in a biological sample from the mother or potential mother. The peptides or mimotopes thereof can also be administered to the mother or potential mother to block binding between the maternal autoantibodies and their antigens, thereby neutralizing the maternal autoantibodies.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Application No. 62 / 940,175, filed November 25, 2019, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0002] STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT This invention was made with government support under Grant No. 2P01ES011269-11 awarded by the National Institutes of Health (NIH). The government has certain rights in this invention. [Background technology]

[0003] Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders diagnosed in early childhood and characterized by impairments in social interaction and communication, as well as the presence of repetitive and restricted interests and behaviors. In 2018, the CDC estimated that ASD affected 1 in 59 children in the United States, posing a significant health concern and a substantial socioeconomic burden for affected families and the healthcare system. Current therapeutic interventions available for ASD are behaviorally oriented or symptom-based pharmacological treatments applied only after diagnosis. Little is known about the causes of ASD, and although certain treatment approaches applied after early diagnosis have shown promise, preventative alternatives currently lack.

[0004] What is known is that activation of the maternal immune system during early fetal development can adversely affect brain development. For reasons that are unclear, some pregnant women's immune systems produce autoantibodies (proteins produced by the immune system in response to components of one's own tissues) that mistake parts of the fetal brain for foreign bodies. As a result, prenatal exposure to these maternal autoantibodies can lead to altered neurogenesis characteristic of ASD. In fact, 23% of mothers who give birth to children with ASD, in contrast to only 1% of mothers who give birth to otherwise normal children, have circulating autoantibodies against seven proteins highly expressed in the developing brain. Each protein is known to play a critical role in neurogenesis, and interference with the level or function of two or more of them may act synergistically to alter the trajectory of brain development. See U.S. Patent No. 8,383,360. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent No. 8,383,360 Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, there is a need for early, non-genetic, epitope-specific biomarkers to determine a mother's risk of having a child with ASD. There is also a compelling need to address the causes and treatment of ASD, as well as associated symptoms, by creating highly specific therapeutic and / or intervention tools. Early identification of these maternal autoantibodies in affected mothers would allow early medical intervention to limit fetal exposure to autoantibodies and the resulting risk that the child will develop ASD, thereby reducing the prevalence of ASD and improving the quality of life of otherwise affected children and their families. The present disclosure meets these needs and provides related advantages. [Means for solving the problem]

[0007] The present disclosure provides peptides (e.g., peptide epitopes) that specifically bind to maternal autoantibodies produced in the mother or potential mother against neuron-specific enolase (NSE) polypeptide. The peptides described herein are useful for determining the risk of an offspring developing autism spectrum disorder (ASD) by detecting the presence of maternal autoantibodies in a biological sample from the mother or potential mother. The peptides can also be administered to the mother or potential mother to block binding between maternal autoantibodies and their antigens, thereby neutralizing the maternal autoantibodies. Furthermore, the peptides can be used for immunoadsorption to remove circulating autoantibodies from maternal plasma.

[0008] In a first aspect, provided herein is an isolated peptide having at least about 80% sequence identity to any one of SEQ ID NOs: 1-6. In some embodiments, the peptide comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive amino acids of any one of SEQ ID NOs: 1-6. In some embodiments, the peptide binds to a parent antibody that binds to the neuron-specific enolase (NSE) protein.

[0009] In some embodiments, the peptide is about 15 to about 30 amino acids in length. In some embodiments, the peptide is up to about 25 amino acids in length. In certain embodiments, the peptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-6.

[0010] In some embodiments, the peptide is a mimotope. In some embodiments, the mimotope comprises D-amino acids. In other embodiments, the mimotope comprises one or more amino acid modifications (e.g., substitutions) compared to any one of SEQ ID NOs: 1-6.

[0011] In some embodiments, the peptide further comprises a label, such as biotin, a fluorescent label, a chemiluminescent label, and a radioactive label. In other embodiments, the label is attached (e.g., covalently attached) to the peptide.

[0012] In another aspect, the present disclosure provides compositions comprising a peptide or peptides described herein. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In certain embodiments, the peptide or peptides in the composition are selected from the group consisting of SEQ ID NOs: 1-6. In certain embodiments, the plurality of peptides comprises at least 2, 3, 4, 5, or 6 different peptides. In some embodiments, the different peptides bind to the same parent antibody, e.g., an antibody against NSE.

[0013] In another aspect, the present disclosure provides kits comprising a peptide or peptides described herein and a solid support. In some embodiments, the solid support is a multiwell plate, an ELISA plate, a microarray, a chip, a bead, a porous strip, or a nitrocellulose filter. In some embodiments, the peptide or peptides are immobilized (e.g., covalently attached) to the solid support. In certain embodiments, the peptide or peptides are selected from the group consisting of SEQ ID NOs: 1-6. In certain embodiments, the plurality of peptides comprises at least 2, 3, 4, 5, or 6 different peptides. In some embodiments, the different peptides bind to the same parent antibody, e.g., an antibody against NSE.

[0014] In some embodiments, the kit further comprises instructions for use. In some instances, the instructions include instructions for contacting the solid support with a biological sample from the mother or potential mother. In other instances, the instructions include instructions for correlating the presence of maternal antibodies that bind to one or more peptides with an increased risk of the offspring (e.g., fetus or child) developing ASD. In other embodiments, the kit further comprises a labeled secondary antibody for detecting the presence of maternal antibodies that bind to one or more peptides.

[0015] In other embodiments, the kit further comprises negative and positive control samples. In some instances, the negative control sample is obtained from a mother with a typically developing (TD) child. In other instances, the biological sample and / or the control sample are reactive to full-length NSE. In yet other instances, neither the biological sample nor the control sample are reactive to full-length NSE. In further embodiments, the kit further comprises a secondary antibody directly or indirectly labeled with a detectable moiety.

[0016] In another aspect, the disclosure provides a method for determining the risk of an offspring to develop an autism spectrum disorder (ASD), the method comprising detecting the presence or absence of maternal antibodies that bind to a peptide or a plurality thereof described herein in a biological sample from the mother or potential mother of the offspring, wherein the presence of maternal antibodies that bind to the peptide or a plurality thereof indicates an increased risk of the offspring to develop an ASD.

[0017] In some embodiments of the method, the method further comprises obtaining a sample from the mother or potential mother. In certain embodiments, the sample is selected from the group consisting of blood, serum, plasma, amniotic fluid, breast milk, and saliva. In some embodiments, the peptide or peptides are selected from the group consisting of SEQ ID NOs: 1-6. In some embodiments of the method, the peptide or peptides comprise at least 2, 3, 4, 5, or 6 different peptides. In certain embodiments, the different peptides bind to the same maternal antibody, e.g., an antibody against NSE. In some embodiments of the method, the peptide or peptides are attached to a solid support, e.g., a multiwell plate, an ELISA plate, a microarray, a chip, beads, a porous strip, or a nitrocellulose filter.

[0018] In some embodiments of the method, the maternal antibodies are detected by techniques such as, for example, Western blot, dot blot, ELISA, radioimmunoassay, immunoprecipitation, electrochemiluminescence, immunofluorescence, FACS analysis, or multiplex bead assay.

[0019] In some embodiments of the method, the presence of maternal antibodies in a test sample (i.e., a biological sample from the mother or potential mother) is detected without comparing the test sample to a control sample. In other embodiments, the test sample is compared to a positive or negative control sample. In some examples, the test sample and / or the control sample are reactive with full-length NSE. In other examples, neither the test sample nor the control sample is reactive with full-length NSE. In yet other examples, the negative control is obtained from a mother of a TD child.

[0020] In some embodiments of the methods, the mother or potential mother has a child with ASD. In some embodiments, the mother or potential mother has a family history of ASD or autoimmune disease.

[0021] In another aspect, the disclosure provides a method for preventing or reducing the risk of an offspring developing an autism spectrum disorder (ASD), comprising administering a therapeutically effective amount of a peptide or peptides described herein to the mother or potential mother of the offspring, wherein the peptide or peptides bind to maternal antibodies circulating in the mother or potential mother to form a neutralizing complex, thereby preventing or reducing the risk of the offspring developing ASD.

[0022] In some embodiments of the above methods, the method further comprises removing neutralizing complexes from the mother or potential mother. In some embodiments of the method, the neutralizing complexes are removed by affinity plasmapheresis.

[0023] In some embodiments of this method, the peptide or peptides are administered intravenously. In some embodiments, the peptide or peptides are selected from the group consisting of SEQ ID NOs: 1-6. In certain embodiments, the plurality of peptides comprises at least 2, 3, 4, 5, or 6 different peptides. In some embodiments, the different peptides bind to the same parent antibody, e.g., an antibody against NSE.

[0024] Other objects, features and advantages of the present disclosure will become apparent to those skilled in the art from the following detailed description. [Brief explanation of the drawings]

[0025] [Figure 1] Figures 1A-1D. Western blots (WB) of fetal monkey brain (FMB) probed with maternal plasma. (Figure 1A) Ponceau-stained nitrocellulose membrane containing the first and then every tenth fraction collected from preparative cell separation of FMB. (Figure 1B) WB of the replicate membrane shown in Figure 1A probed with a pool of maternal plasma reactive to the 37 kDa (LDH), 39 kDa (YBX1), 44 kDa (GDA), and 73 kDa (STIP1, CRMP1 / 2) antigens. (Figure 1C) Preparative cell fractions containing proteins between 39 and 42 kDa. Fraction 12 was used for 2D gel electrophoresis. (Figure 1D) WB of FMB fraction 12 probed with maternal plasma unreactive to LDHA-B, GDA, and YBX1. Lane 1: secondary-only antibody control; Lanes 2-4: maternal plasma reactive to LDHA-B (green arrow), YBX1 (blue arrow), and GDA (black arrow); Lanes 5-8: maternal plasma pool 1 with band reactivity for proteins around 39 kDa; Lanes 10-14: maternal plasma pool 2 with band reactivity for two proteins around 37 and 39 kDa; Lane 9: plasma sample negative control for FMB antigen. Abbreviations: FMB, fetal monkey brain; LDH A and B, lactate dehydrogenase A and B; YBX1, Y-box binding protein 1; GDA, guanine deaminase; CRMP1 and CRMP2, collapsin response mediator 1 and 2; and STIP1, stress-inducible phosphoprotein 1. [Figure 2] Figures 2A-2E. Two-dimensional (2-D) gel electrophoresis and antigen selection for mass spectrometry. Figure 2A shows protein alignment with FMB fraction number 12 (Figure 2B) and an anti-IgG stained gel of membrane-blotted plasma pool 1 and plasma pool 2 (Figure 2C). Figure 2D shows a merged image of Figures 2B and 2C. (Figure 2E) WB of proteins bound by maternal IgG antibodies (plasma pools 1 and 2), each labeled with a spot number. A total of 27 protein spots were collected and subsequently analyzed by mass spectrometry. [Figure 3]Heatmap of sequences with an average reactivity (FI) greater than 50 from ELISA-positive samples. Samples were considered positive if their FI was >200. Red letters indicate amino acid residues that are part of the major epitope of ES293-297, and ES408-410 indicates an amino acid sequence recognized only by the ASD group. On the right, the histogram represents FI reactivity. Abbreviations: ES, epitope sequence; ASD, autism spectrum disorder; TD, neurotypical; FI, fluorescence intensity. [Figure 4] Workflow representation of the method used in the Examples. The first three steps were to identify a 37-45 kDa protein that was reactive with maternal plasma from mothers whose children had ASD. The fourth step led to the identification of target antibodies (including NSE). The next step shows the characterization of the antigen in relation to the MAR ASD biomarker. [Figure 5] Heatmap of sequences with an average reactivity (FI) greater than 50 from ELISA-negative samples. Samples were considered positive if their FI was >200. Amino acid residues that are part of the major epitope are highlighted in red. On the right, the histogram represents FI reactivity. Abbreviations: ES, epitope sequence; ASD, autism spectrum disorder; TD, neurotypical; FI, fluorescence intensity. DETAILED DESCRIPTION OF THE INVENTION

[0026] I. Introduction Autism spectrum disorder (ASD) is a significant health problem characterized by social and behavioral impairments, along with restricted interests and repetitive behaviors. Previous studies have determined that maternal autoantibody-associated (MAR) autism is thought to be associated with approximately 23% of ASD cases. Seven MAR-specific autoantigens have previously been identified, including CRMP1, CRMP2, GDA, LDHA, LDHB, STIP1, and YBX1 (see, for example, International Publication No. 2016 / 210137, the entire contents of which are incorporated herein by reference). The epitope peptide sequences recognized by maternal autoantibodies against each of the seven ASD-specific autoantigens have also been described.

[0027] This disclosure relates to the identification of additional antigens recognized by ASD-specific maternal autoantibodies and the mapping of unique ASD-specific epitopes using microarray technology. Fetal rhesus macaque brain tissue was separated by molecular weight, and fractions containing bands between 37 and 45 kDa were analyzed using 2-D gel electrophoresis, followed by peptide mass mapping using MALDI-TOF MS and TOF / TOF tandem MS / MS. Using this methodology, neuron-specific enolase (NSE) was identified as the target autoantigen and selected for epitope mapping. The complete NSE sequence was translated into 15-mer peptides with 14 amino acid overlaps on microarray slides and probed with maternal plasma from mothers with ASD children and typically developing (TD) children (27 for ASD and 21 for TD). The resulting data were analyzed by t-test. Using both t-tests and SAM t-tests, 16 ASD-specific NSE peptide sequences were found, with four sequences being statistically significant (p<0.05): DVAASEFYRDGKYDL (SEQ ID NO: 1) (SEQ ID NO: 1) (p=0.047; SAM score 1.49), IEDPFDQDDWAAWSK (SEQ ID NO: 2) (SEQ ID NO: 2) (p=0.049; SAM score 1.49), ERLAKYNQLMRIEEE (SEQ ID NO: 3) (SEQ ID NO: 3) (p=0.045; SAM score 1.57), and RLAKYNQLMRIEEEL (SEQ ID NO: 4) (p=0.017; SAM score 1.82). All ASD-specific NSE peptide sequences had odds ratios (ORs) greater than 3, with SERLAKYNQLMRIEE (SEQ ID NO: 6) (OR 10.1, CI 95% 0.5094-200.7) and ERLAKYNQLMRIEEE (SEQ ID NO: 3) (OR 12.6, CI 95% 0.6408-247.7) being the two epitopes with the highest ORs. Five sequences were found that were recognized by both ASD and TD antibodies, suggesting a large immunodominant epitope (DYPVVSIEDPFDQDDWAAW (SEQ ID NO: 5)). Maternal autoantibodies against NSE proteins are present in both mothers of ASD and TD children, and several ASD-specific epitopes exist that could potentially be used as MAR ASD biomarkers.

[0028] II. Definition As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0029] The terms "autism spectrum disorder," "autism spectrum disorder," "autism," or "ASD" refer to a range of neurodevelopmental disorders characterized by impairments in social interaction and communication accompanied by repetitive and stereotyped behaviors. Autism encompasses a range of social interaction and communication disorders, but the disorders can be broadly categorized as "high-functioning autism" or "low-functioning autism" depending on the degree of social interaction and communication impairment. Individuals diagnosed with "high-functioning autism" have minimal but discernible social interaction and communication impairments (i.e., Asperger's syndrome). Further information on autism spectrum disorders can be found in, for example, Autism Spectrum Disorders: A Research Review for Practitioners, Ozonoff, et al., eds., 2003, American Psychiatric Pub; Gupta, Autistic Spectrum Disorders in Children, 2004, Marcel Dekker Inc; Hollander, Autism Spectrum Disorders, 2003, Marcel Dekker Inc; Handbook of Autism and Developmental Disorders, Volkmar, ed., 2005, John Wiley; Sicile-Kira and Grandin, Autism Spectrum Disorders: The Complete Guide to Understanding Autism, Asperger's Syndrome, Pervasive Developmental Disorder, and Other ASDs, 2004, Perigee Trade; and Duncan, et al., Autism Spectrum Disorders [Two Volumes]: A Handbook for Parents and Professionals, 2007, Praeger.

[0030] The terms "neurotypical" and "TD" refer to subjects who have not been diagnosed with autism spectrum disorder (ASD). Typically, typically developing children do not exhibit the communication skills, social interaction, or repetitive and / or stereotyped behaviors of a severity typically associated with an ASD diagnosis. Although typically developing children may exhibit some behaviors exhibited by children diagnosed with ASD, typically developing children do not exhibit the constellation of behaviors and / or the severity of behaviors that support a diagnosis of ASD.

[0031] The term "isolated," when applied to a nucleic acid or protein, indicates that the nucleic acid or protein is essentially free from other cellular components with which it is naturally associated. It is preferably in a homogeneous state. It can be either a dry solution or an aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified. In particular, an isolated gene is separated from open reading frames that flank the gene and encode proteins other than the gene of interest. The term "purified" indicates that the nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. In particular, this means that the nucleic acid or protein is at least 85% pure, at least 95% pure, or at least 99% pure.

[0032] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0033] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues or a collection of polymers of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, as well as to naturally occurring and non-naturally occurring amino acid polymers.

[0034] The term "amino acid" includes naturally occurring α-amino acids and their stereoisomers, as well as unnatural (non-naturally occurring) amino acids and their stereoisomers. An "amino acid" "stereoisomer" refers to an enantiomer of an amino acid, such as an L-amino acid or a D-amino acid. For example, a stereoisomer of a naturally occurring amino acid refers to an enantiomer of a naturally occurring amino acid, i.e., a D-amino acid.

[0035] Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Naturally occurring α-amino acids include, but are not limited to, alanine (Ala), cysteine ​​(Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Naturally occurring stereoisomers of α-amino acids include, but are not limited to, D-alanine (D-Ala), D-cysteine ​​(D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Tyr), and combinations thereof.

[0036] Non-natural (non-naturally occurring) amino acids include, but are not limited to, amino acid analogs, amino acid mimetics, synthetic amino acids, N-substituted glycines, and N-methyl amino acids in the L- or D-configuration that function in a manner similar to naturally occurring amino acids. For example, an "amino acid analog" is an unnatural amino acid that has the same basic chemical structure as a naturally occurring amino acid, i.e., an alpha carbon bonded to a hydrogen, a carboxyl group, and an amino group, but has a modified R (i.e., side chain) group or a modified peptide backbone, e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. An "amino acid mimetic" refers to a chemical compound that has a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0037] Amino acids may be referred to herein by either their commonly known three letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. For example, L-amino acids may be represented herein by their commonly known three letter symbols (e.g., Arg for L-arginine) or by the uppercase one-letter amino acid symbol (e.g., R for L-arginine). D-amino acids may be represented herein by their commonly known three letter symbols (e.g., D-Arg for D-arginine) or by the lowercase one-letter amino acid symbol (e.g., r for D-arginine).

[0038] With respect to amino acid sequences, those skilled in the art will recognize that individual substitutions, additions, or deletions to a peptide, polypeptide, or protein sequence that alter, add, or delete a single amino acid or a small percentage of amino acids in the encoded sequence are "conservatively modified variants" in that the alteration results in the replacement of an amino acid with a chemically similar amino acid. Chemically similar amino acids include, but are not limited to, naturally occurring amino acids such as L-amino acids, stereoisomers of naturally occurring amino acids such as D-amino acids, and unnatural amino acids such as amino acid analogs, amino acid mimetics, synthetic amino acids, N-substituted glycines, and N-methyl amino acids.

[0039] Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, substitutions can be made in which an aliphatic amino acid (e.g., G, A, I, L, or V) is replaced with another member of its group. Similarly, an aliphatic polar, uncharged group such as C, S, T, M, N, or Q can be replaced with another member of its group, and basic residues such as K, R, or H can be substituted for each other. In some embodiments, an amino acid with an acidic side chain, such as E or D, can be substituted with its uncharged counterpart, such as Q or N, respectively, and vice versa. Each of the following eight groups contains other exemplary amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) Cysteine ​​(C), Methionine (M) (See, e.g., Creighton, Proteins, 1993).

[0040] The term "amino acid modification" or "amino acid alteration" refers to the substitution, deletion, or insertion of one or more amino acids.

[0041] "Percent sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, where the portion of the sequence within the comparison window (e.g., a peptide described herein) may contain additions or deletions (i.e., gaps) compared to a reference sequence that does not contain additions or deletions due to optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where the identical amino acid residue occurs in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percent sequence identity.

[0042] The term "identical" or percent "identity" in the context of two or more polypeptide or peptide sequences refers to two or more sequences or subsequences that are the same. Two sequences are "substantially identical" if they have a specified percentage of amino acid residues that are the same (i.e., 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity over a specified region, or, if not specified, over the entire sequence of the reference sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. With respect to amino acid sequences, identity or substantial identity can exist over a region that is at least 5, 10, 15, or 20 amino acids in length, optionally at least about 25, 30, 35, 40, 50, 75, or 100 amino acids in length, optionally at least about 150, 200, or 250 amino acids in length, or over the entire length of the reference sequence. For shorter amino acid sequences, for example, amino acid sequences of 20 amino acids or less, substantial identity exists if one or two amino acid residues are conservatively substituted in accordance with conservative substitutions as defined herein.

[0043] For sequence comparison, typically, one sequence serves as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters. Two examples of algorithms suitable for determining percent sequence identity and percent sequence similarity are BLAST and BLAST 2.0, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information.

[0044] An indication that two polypeptide or peptide sequences are substantially identical occurs when a first polypeptide or peptide is immunologically cross-reactive with antibodies raised against the second polypeptide or peptide. Thus, a first polypeptide or peptide is typically substantially identical to a second polypeptide or peptide, e.g., the two sequences differ only by conservative substitutions.

[0045] The term "antigenic fragment" refers to a contiguous subsequence of a polypeptide that binds to an antibody. An antigenic fragment may or may not be immunogenic, i.e., it may or may not induce an immune response.

[0046] The term "conformational antigenic fragment" refers to a spatially contiguous region of a polypeptide or tetramer that may or may not be formed by a contiguous subsequence. A conformational antigenic fragment may or may not be immunogenic.

[0047] The term "epitope" or "antigenic determinant" refers to a site on a peptide or polypeptide to which B cells and / or T cells respond. B cell epitopes can be formed from both contiguous or noncontiguous amino acids juxtaposed by tertiary or quaternary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary or quaternary folding (i.e., conformationally determined) are typically lost upon treatment with denaturing solvents. Epitopes typically contain at least three, more usually at least five or 8-10 amino acids in a unique spatial conformation. Methods for determining the spatial conformation of epitopes include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, "Epitope Mapping Protocols in Methods in Molecular Biology," Vol. 66, Glenn E. Morris, Ed. (1996). Antibodies that recognize the same epitope can be identified in a simple immunoassay demonstrating the ability of one antibody to block the binding of another antibody to its target antigen (e.g., electrochemiluminescence assay, competitive ELISA, solid-phase radioimmunoassay (SPRIA) or blocking Western blot). T cells recognize contiguous epitopes of approximately 9 amino acids for CD8 cells or approximately 13-15 amino acids for CD4 cells. T cells that recognize an epitope are identified by T cells primed in response to the epitope. 3 Identification can be achieved by in vitro assays measuring 3H-thymidine incorporation (Burke et al., J. Inf. Dis. 170, 1110-19 (1994)), antigen-dependent killing (cytotoxic T lymphocyte assay, Tigges et al., J. Immunol. (1996) 156:3901-3910), or antigen-dependent proliferation as determined by cytokine secretion.

[0048] The terms "specifically bind" or "specifically directed" refer to preferential association between a T cell receptor and / or antibody and a target peptide / polypeptide or its antigenic fragment, in whole or in part, compared to other peptides / polypeptides. It is recognized, of course, that some degree of non-specific interaction may occur between an antibody or T cell receptor and a non-target peptide / polypeptide. Nevertheless, specific binding can be distinguished as being mediated through specific recognition of the target peptide / polypeptide or its antigenic fragment. Typically, specific binding or a specific immune response results in a much stronger association between a target peptide / polypeptide and an antibody or T cell receptor against the target peptide / polypeptide than between an antibody or T cell receptor against the target peptide / polypeptide and a non-target peptide / polypeptide. Specific binding typically results in an increase of more than about 10-fold (e.g., more than 100-fold) in the amount of bound antibody against the target peptide / polypeptide (per unit time) to cells or tissues bearing the target peptide / polypeptide compared to cells or tissues lacking the epitope of the target peptide / polypeptide. Specific binding between a target peptide / polypeptide and an antibody against the target peptide / polypeptide generally results in a binding intensity of at least 10 6 M -1 This means affinity. 10 8 M -1 Affinities of greater than 0.01 are preferred. Specific binding can be determined using any assay for antibody binding known in the art, including, but not limited to, Western blot, dot blot, ELISA, flow cytometry, electrochemiluminescence, multiplex bead assays (e.g., using Luminex or fluorescent microbeads), and immunohistochemistry. T cells specifically directed against an epitope of a target peptide / polypeptide typically exhibit about 2-fold greater (e.g., about 5-fold greater or 10-fold greater) antigen-induced proliferation in response to the target peptide / polypeptide than in response to a non-target peptide / polypeptide. T cell proliferation assays are known in the art and include, 3 It can be measured by 3H-thymidine incorporation.

[0049] The term "sample" refers to any biological specimen obtained from a subject, e.g., a human subject. Samples include, but are not limited to, whole blood, plasma, serum, red blood cells, white blood cells, saliva, urine, stool, sputum, bronchial lavage fluid, tears, nipple aspirate, breast milk, any other bodily fluid, tissue samples such as placental biopsies, and cellular extracts thereof. In some embodiments, the sample is whole blood or a fractional component thereof, e.g., plasma, serum, or a cell pellet.

[0050] The terms "subject," "individual," or "patient" typically include humans, but can also include other animals, e.g., other primates, rodents, dogs, cats, horses, sheep, pigs, etc. In certain embodiments, the subject is a human subject.

[0051] The term "increased risk of developing ASD" refers to an increased likelihood or probability that a fetus or child exposed to antibodies that bind to one or more antigens (e.g., NSE) described herein or levels of antibodies to one or more antigens above a predetermined threshold level will develop symptoms of ASD, compared to the risk, likelihood or probability of a fetus or child not exposed to antibodies to one or more antigens or levels of antibodies to one or more antigens below a predetermined threshold level.

[0052] The term "reduced risk of developing ASD" refers to a lower likelihood or probability that a fetus or child exposed to antibodies to one or more antigens (e.g., NSEs) described herein or levels of antibodies to one or more antigens above a predetermined threshold level and whose mother has undergone a therapeutic intervention, such as blocking, inactivating, or removing antibodies that bind to the antigens, will develop symptoms of ASD, compared to the likelihood or probability that a fetus or child exposed to antibodies to the antigens or levels of antibodies to one or more antigens above a predetermined threshold level and whose mother has not undergone the therapeutic intervention will develop symptoms of ASD.

[0053] The term "peptide epitope" or "antigenic peptide" refers to a peptide or fragment of one or more antigens (e.g., NSE) described herein that mimics an epitope (e.g., bound by an antibody against the antigen), although there may not be clear homology between the structure or sequence of such a peptide epitope and the epitope of the native antigen. Instead, mimicry of a peptide epitope relies on similar physicochemical properties and similar spatial organization. Screening and construction of peptide epitopes is known in the art. For example, peptide epitopes can be derived from known epitopes by sequence modification or can be developed de novo using, for example, combinatorial peptide libraries for peptides that bind to antibodies against one or more antigens. See, e.g., Yip and Ward, Comb Chem High Throughput Screen (1999) 2(3):125-128; Sharav, et al., Vaccine (2007) 25(16):3032-37; and Knittelfelder, et al., Expert Opin Biol Then (2009) 9(4):493-506.

[0054] The term "family history" refers to the presence of a disease condition (e.g., ASD or an autoimmune disease) in a family member. Family members can be direct descendants, e.g., parents, children, or grandparents, or close relatives, e.g., siblings, aunts or uncles, cousins. Typically, family members are blood relatives who share a common genetic heritage.

[0055] The term "therapeutically effective amount" refers to an amount of a peptide described herein that can achieve a therapeutic effect or desired result (i.e., a sufficient amount of peptide to block the binding of an antibody to an antigen to a target antigen), preferably with minimal or no side effects. In some embodiments, a therapeutically acceptable amount does not induce or cause undesirable side effects. A therapeutically effective amount can be determined by administering an initial low dose and then incrementally increasing the dose until the desired effect is achieved. A "prophylactically effective amount" and a "therapeutically effective amount" of an antibody blocking agent described herein can prevent the onset of ASD or result in a reduction in the severity of ASD. A "prophylactically effective amount" and a "therapeutically effective amount" can also prevent or ameliorate, respectively, disorders resulting from the activity of maternal antibodies and functional or physical disabilities resulting from disease.

[0056] The term "pharmaceutically acceptable carrier" refers to compounds, chemicals, or molecules useful in preparing pharmaceutical compositions that are generally safe, non-toxic, and not biologically or otherwise undesirable, including those that are acceptable for pharmaceutical use in a subject. Suitable pharmaceutical carriers are described herein and in "Remington's Pharmaceutical Sciences" by E.W. Martin.

[0057] As used herein, the term "administering" includes oral administration, topical contact, administration as a suppository, intravenous, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal, or subcutaneous administration to a subject, or implantation of a sustained-release device, such as a mini-osmotic pump. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarteriolar, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, and the like. Additional methods for administering a therapeutically effective amount of a peptide described herein to prevent or alleviate one or more symptoms associated with the presence or activity of maternal antibodies will be known to those skilled in the art. "Co-administering" means that the peptide described herein is administered simultaneously with, immediately before, or immediately after the administration of a second drug.

[0058] As used herein, the term "treating" refers to any indicia of success in treating or ameliorating a disease state or condition, including any objective or subjective parameter, such as relief, remission, attenuation of symptoms, or making the disease state or condition more tolerable to the patient, slowing the rate of degeneration or decline, making the end point of degeneration less debilitating, or improving the patient's physical or mental well-being. Treatment or amelioration of symptoms can be based on objective or subjective parameters, including the results of a physical examination, a histopathological examination (e.g., analysis of biopsy tissue), clinical laboratory analysis of urine, saliva, tissue samples, serum, plasma, or blood, or imaging.

[0059] The term "specifically inhibit" refers to the ability of an agent (e.g., a peptide described herein) to inhibit antibody binding to one or more antigens (e.g., NSEs). Specific inhibition typically results in at least about a two-fold inhibition above background, e.g., about a 10-fold, 20-fold, or 50-fold inhibition of antibody binding to the target antigen by comparing antibody binding in the absence of the agent. In some embodiments, antibody binding to the target antigen is completely inhibited or prevented by the agent (e.g., a peptide described herein). Typically, specific inhibition is a statistically significant reduction in antibody binding to the target antigen (e.g., p<0.05) using an appropriate statistical test.

[0060] The term "agent" includes peptides (eg, peptide epitopes), mimotopes, polypeptides (eg, ligands, antibodies), nucleic acids, small organic compounds, and the like.

[0061] The term "solid support" refers to any material suitable for carrying out the methods described herein, such as plastic or glass tubes, beads, slides, microtiter plates, porous filters or membranes, non-porous filters or membranes, non-magnetic beads, microbeads, slides, microarrays, etc.

[0062] The term "neutralizing complex" refers to a complex comprising a maternal antibody bound to a specific peptide described herein that prevents / inhibits / blocks the maternal antibody from binding to its antigen (e.g., NSE). For example, a maternal autoantibody that specifically recognizes the NSE antigen can form a neutralizing complex with an NSE peptide or a mimotope thereof described herein, such that the maternal autoantibody does not bind to the NSE antigen.

[0063] The term "affinity plasmapheresis" refers to an extracorporeal blood purification procedure for removing harmful agents (eg, disease-causing agents) from a subject's plasma.

[0064] III. Detailed Description of the Embodiments The present disclosure provides peptides (e.g., peptide epitopes and mimotopes thereof) that specifically bind to maternal autoantibodies against the endogenous autoantigen NSE protein. The present disclosure also provides compositions and kits comprising the peptides described herein. In addition, the present disclosure provides methods for determining the risk of a child or future offspring (e.g., in the mother or potential mother during or before pregnancy) developing autism spectrum disorder (ASD) by detecting the presence of maternal autoantibodies in a biological sample from the mother or potential mother using the peptides described herein. The present disclosure further provides methods for preventing or reducing the risk of an offspring developing ASD by administering a therapeutically effective amount of a peptide described herein to the mother or potential mother of the offspring to block binding between maternal autoantibodies and their antigens.

[0065] A. Neuron-specific enolase (NSE) NSE is one of the most abundant proteins in the brain, accounting for 0.4–2.2% of total soluble protein depending on the brain region. It has been associated with various roles, including in glycolysis and gluconeogenesis, neuronal differentiation, activation, and proliferation via the PI3K / Akt and MAPK / ERK signaling pathways. Furthermore, NSE plays a role in activating the RhoA kinase pathway, which can result in neurodegeneration or neuroprotection depending on the strength of the signal. Furthermore, NSE has been shown to be involved in CNS inflammatory processes, as its expression is upregulated in M1 microglia and reactive astrocytes. Thus, NSE plays several important roles during neurodevelopment but is also involved in neurodegeneration

[18] .

[0066] Measurement of plasma NSE levels has been used as a biomarker for various applications

[17] . For example, it is a useful indicator of neural maturation and is currently the most widely used biomarker for small cell lung cancer (SCLC), having been shown to have direct effects on cell proliferation and migration in vitro in different SCLC cell lines [28, 29]. Furthermore, it is also used for the diagnosis and prognosis of other types of cancer, such as non-small cell lung cancer (NSCLC), neuroendocrine tumors (NETs), neuroblastoma, brain cancer, and traumatic brain injury (TBI)

[30] . As described in the Examples herein, we addressed the value of autoantibodies against NSE as a potential biomarker or risk factor for MAR ASD, based on the concept that antibody binding to NSE during neurogenesis may affect protein functionality and brain metabolism, potentially resulting in lasting effects on neural tissue function and development.

[0067] As described in the Examples herein, we found that autoantibody reactivity to NSE was present in similar proportions in both experimental groups (ASD and TD). This indicates that intact NSE protein alone is not a biomarker, similar to previous studies demonstrating the need for autoantibody reactivity to multiple antigens rather than a single antigen to confer ASD specificity [8, 12, 13, 31, 32]. When we first discovered seven autoantigens, we found that reactivity to specific antigen combinations was highly significant as a biomarker of ASD risk, including LDH, STIP1, and CRMP1 (13% ASD vs. 0% TD), as well as several other combinations of three or more autoantigens with >98% specificity [6, 8, 9]. Therefore, we tested NSE using a larger dataset and found that it increased the specificity and sensitivity of the MAR ASD assay.

[0068] In a recent study, we performed microarray-based epitope mapping of CRMP1, CRMP2, GDA, LDHA / B, STIP1, and YBX1 and further documented differential reactivity to several epitopes recognized exclusively by autoantibodies from mothers of children with ASD

[15] . Furthermore, we used epitopes from the original set of autoantigens to create an endogenous antigen-driven mouse model of autism in which mice were immunized with peptide epitopes of LDHA, LDHB, CRMP1, and STIP1. This methodology allowed us to continuously expose embryos to autoantibodies against MAR ASD-specific peptides throughout gestation. Thus, we generated a mouse model exhibiting ASD-related behaviors and demonstrated that exposure to this combination of autoantibodies resulted in altered neurogenesis

[11] .

[0069] As described in the Examples herein, we identified NSE as an additional MAR ASD autoantigen and found 16 epitope sequences recognized by maternal autoantibodies present exclusively in the ASD group. Four of these sequences demonstrated statistical significance when compared with the control group using conventional t-test and SAM score t-test analyses. The epitope sequences (ES 408 and 409) SERLAKYNQLMRIEE (SEQ ID NO: 6) and ERLAKYNQLMRIEEE (SEQ ID NO: 3) had the highest OR values ​​(10.1 and 12.6, respectively), indicating a strong association between having autoantibodies against these sequences and the risk of having a child with ASD. These ASD-specific epitope peptides can be used to generate MAR ASD animal models that allow evaluation of the effects of NSE ASD-specific peptides individually and in combination with pathogenic epitopes from other autoantigens, thus providing a better understanding of the role of anti-NSE in autism pathology.

[0070] As a mechanism of action, we hypothesize that the presence of autoantibodies against ASD-specific NSE epitopes could potentially inhibit proper protein function in two different ways: 1) by directly interfering with proper protein folding (tertiary and quaternary structure) or 2) by binding to critical functional sites (catalytic or substrate sites) [33-36]. Anti-NSE antibodies in the developing brain may be able to elicit responses against cells targeted by these autoantibodies, but we lack evidence of tissue destruction based on our previous rodent models. Instead, the presence of MAR ASD autoantibodies against CRMP1, LDHA / B, and STIP1 appears to affect progenitor cell maturation and alterations in dendritic spines and structure in the adult brain [10, 13, 37]. However, the mechanisms of autoantibody-mediated immunopathology in the brain remain poorly understood.

[0071] A final area of ​​interest was the investigation of the relationship between ASD- and non-ASD-specific peptide sequences and the epitope repertoire reported in the Immune Epitope Database (IEDB)

[38] . This interest stems from the possibility of identifying peptidomimetics, providing some understanding of how autoantibodies to these self-proteins are generated. We found that the sequences DYPVVSIEDPFDQDD (SEQ ID NO: 7), YPVVSIEDPFDQDDW (SEQ ID NO: 8), PVVSIEDPFDQDDWA (SEQ ID NO: 9), VVSIEDPFDQDDWAA (SEQ ID NO: 10), and VSIEDPFDQDDWAAW (SEQ ID NO: 11) were recognized by antibodies from both experimental groups and represent immunodominant epitopes recognized by the general population. As expected, these sequences share a high degree of homology with alpha and gamma enolase (NNE and NSE) at 90% stringency and, interestingly, 80% homology with other proteins, including protein ORF73 from human γ-herpesvirus 8 (the causative agent of mononucleosis), protein X from hepatitis B virus, and serpin H1 from humans, indicating the potential for molecular mimicry to directly expose these factors.

[0072] B. Peptide Epitopes In certain aspects, the present disclosure provides isolated peptides that specifically bind to maternal antibodies raised maternally or potentially maternally against the neuron-specific enolase (NSE) protein. NSE is a catalytic enzyme expressed in neurons and neuroendocrine tissues that mediates the conversion of 2-phosphoglycerate (2PG) to 2-phosphoenolpyruvate (2PEP) and the reverse reaction (2PEP to 2PG) in the glycolytic and gluconeogenic pathways, respectively.

[16] In eukaryotic cells, three enolase isoforms exist, encoded by distinct genes and with tissue-specific expression: alpha-enolase (ENO1) is ubiquitously expressed, gamma-enolase (ENO2) is found only in neurons, and beta-enolase (ENO3) is found only in muscle. Enolases exist as dimers, and their function depends on the natural cofactor Mg, which regulates the enzyme's conformation and catalytic activity.

[17] In the brain, NSE is expressed as gamma on neurons and alpha on microglia, astrocytes, and oligodendrocytes. Non-neuronal enolase (NNE, alpha-alpha dimer) is observed on neural tissue during early development, but changes to gamma and alpha-gamma isoforms (NSE) as neuronal and glial differentiation and maturation occur. NSE is involved in cellular metabolism, immune response regulation, neuroinflammation, neurogenesis, and brain homeostasis by regulating cell survival / death signals

[18] . Therefore, the potential of NSE as a target of maternal autoantibodies in the context of ASD is well established due to its distinct role in neurodevelopmental biology.

[0073] In a first aspect, the peptide has at least about 50%, e.g., about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1-6 (DVAASEFYRDGKYDL (SEQ ID NO: 1); IEDPFDQDDWAAWSK (SEQ ID NO: 2); ERLAKYNQLMRIEEE (SEQ ID NO: 3); RLAKYNQLMRIEEEL (SEQ ID NO: 4); DYPVVSIEDPFDQDDWAAW (SEQ ID NO: 5); and SERLAKYNQLMRIEE (SEQ ID NO: 6)). In some embodiments, the peptide comprises at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive amino acids of the amino acid sequence of any one of SEQ ID NOs: 1-6. In other embodiments, the peptide (e.g., an antigenic fragment thereof) has at least about 50%, e.g., about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the length of the amino acid sequence of any one of SEQ ID NOs: 1-6. In some embodiments, the peptide comprises an amino acid sequence that comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 1-6. In other embodiments, the peptide comprises one or more additional amino acid residues at the amino and / or carboxyl termini that correspond to the amino acid residues at those positions in the NSE polypeptide sequence. In certain embodiments, the peptide binds to a parent antibody that binds to the NSE polypeptide.

[0074] In some embodiments, the peptide is about 5 to about 45 amino acids in length, about 8 to about 45 amino acids in length, about 8 to about 25 amino acids in length, about 12 to about 45 amino acids in length, about 5 to about 40 amino acids in length, about 10 to about 40 amino acids in length, about 15 to about 30 amino acids in length, about 15 to about 25 amino acids in length, about 15 to about 22 amino acids in length, about 15 to about 20 amino acids in length, about 17 to about 25 amino acids in length, about 19 to about 25 amino acids in length, or about 45, 40, 35, 30, 25, 20, 15, 10, or 5 amino acids in length. For example, peptides can be about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or more amino acids in length. Typically, peptides should not exceed a length that allows for the formation of tertiary structures, such as more than 45 amino acids, when present as an isolated molecule. However, peptides can exceed 45 amino acids when fused to a larger molecule, such as an antibody or another protein or macromolecule, which may prevent the formation of tertiary structures within the peptide. Peptides can also exceed 45 amino acids when they are bivalent peptides, having first and second peptide fragments that bind to different parent antibodies. In certain embodiments, the peptide is up to about 15, 20, 25, 30, 35, 40, or 45 amino acids in length.

[0075] In some embodiments, the peptide further comprises a label, such as a detectable label. In certain instances, the label is selected from the group consisting of biotin, a fluorescent label, a chemiluminescent label, and a radioactive label. In certain other instances, the label is covalently attached to the peptide.

[0076] In other embodiments, the peptides include variants that have been further modified to improve resistance to proteolysis, optimize solubility properties, or make them more suitable as therapeutic agents. For example, the peptides further include analogs containing residues other than naturally occurring L-amino acids, such as D-amino acids or non-naturally occurring synthetic amino acids. D-amino acids can be substituted for some or all of the amino acid residues.

[0077] In certain embodiments, the peptides comprise naturally occurring amino acids and / or unnatural amino acids, examples of unnatural amino acids include D-amino acids, ornithine, ornithine diaminobutyrate, ornithine norleucine, pyriylalanine, thienylalanine, naphthylalanine, phenylglycine, alpha and alpha-disubstituted amino acids, N-alkylamino acids, lactic acid, halide derivatives of naturally occurring amino acids (e.g., trifluorotyrosine, p-Cl-phenylalanine, p-Br-phenylalanine, pI-phenylalanine, etc.), L-allyl-glycine, β-alanine, La-aminobutyric acid, Lg-aminobutyric acid, La-aminoisobutyric acid, Le ...aminoisobutyric acid, and the like. Examples of suitable peptides include, but are not limited to, aminocaproic acid, 7-aminoheptanoic acid, L-methionine sulfone, L-norleucine, L-norvaline, p-nitro-L-phenylalanine, L-hydroxyproline, L-thioproline, and methyl derivatives of phenylalanine (e.g., 1-methyl-Phe, pentamethyl-Phe, L-Phe(4-amino), L-Tyr(methyl), L-Phe(4-isopropyl), L-Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), L-diaminopropionic acid, and L-Phe(4-benzyl)). The peptides may be further modified. For example, one or more amide bonds may be replaced with ester or alkyl backbone bonds. Constraints such as N- or C-alkyl substituents, side chain modifications, or disulfide bridges or side chain amide or ester bonds may be present.

[0078] In some embodiments, peptides include both modified peptides and synthetic peptide analogs. Peptides can be modified to improve formulation and storage properties or to protect labile peptide bonds by incorporating non-peptidic structures.

[0079] In other embodiments, the peptide may be cyclized. Methods for introducing cyclic structures into peptides to select and provide conformational constraints on the structure that result in improved stability are well known in the art. For example, a C- or N-terminal cysteine ​​can be added to a peptide, so that when oxidized, the peptide contains a disulfide bond, generating a cyclic peptide. Other methods for cyclizing peptides include the formation of thioethers and carboxyl- and amino-terminal amides and esters. Several synthetic techniques have been developed to generate synthetic cyclic peptides (see, e.g., Tarn et al., Protein Sci., 7:1583-1592 (1998); Romanovskis et al., J. Pept. Res., 52:356-374 (1998); Camarero et al., J. Amer. Chem. Soc., 121:5597-5598 (1999); Valero et al., J. Pept. Res., 53(1):56-67 (1999)). In general, cyclized peptides serve a dual purpose: (1) to reduce hydrolysis in vivo and (2) to thermodynamically destabilize the unfolded state and promote secondary structure formation.

[0080] In some embodiments, the present disclosure provides a plurality of peptides comprising at least two of the same or different peptides covalently or non-covalently linked, e.g., in some embodiments, at least two, three, four, five, or six of the same or different peptides are covalently linked, e.g., such that they have appropriate size and / or binding properties but avoid undesirable aggregation.

[0081] The peptides described herein may be produced by any suitable means known in the art or later discovered, for example, synthesized in vitro, purified or substantially purified from natural sources, or recombinantly produced from eukaryotic or prokaryotic cells.

[0082] Peptides can be prepared by in vitro synthesis using conventional methods known in the art. For example, peptides can be produced by chemical synthesis, for example, using solid-phase techniques and / or automated peptide synthesizers. In a specific example, peptides can be synthesized using a solid-phase strategy on an automated multiple peptide synthesizer (Abimed AMS 422) using 9-fluorenylmethyloxycarbonyl (Fmoc) chemistry. The peptides can then be purified by reverse-phase HPLC and lyophilized. Synthesizers can be used to substitute unnatural amino acids for naturally occurring amino acids. The specific sequence and preparation method are determined by convenience, economy, required purity, etc. Alternatively, peptides can be prepared by cleavage of longer peptides or full-length protein sequences.

[0083] Peptides can also be isolated and purified according to conventional methods of recombinant synthesis. A lysate of the expression host can be prepared and the lysate purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification techniques. Methods well known to those skilled in the art can be used to construct expression vectors containing coding sequences and appropriate transcriptional / translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination / genetic recombination. Alternatively, RNA capable of encoding the desired peptide can be chemically synthesized. Those skilled in the art can readily utilize well-known codon usage tables and synthetic methods to provide a suitable coding sequence for any of the peptides described herein. See, for example, Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd Ed., 2001, Cold Spring Harbor Laboratory Press; and Ausubel, et al., Current Protocols in Molecular Biology, 1987-2009, John Wiley Interscience.

[0084] In other aspects, the present disclosure provides compositions comprising any one or more of the peptides described herein. As a non-limiting example, the composition comprises multiple peptides that bind to maternal antibodies against NSE. As a further non-limiting example, the composition comprises one or more peptides selected from the group consisting of SEQ ID NOS: 1-6. As a further non-limiting example, the composition comprises peptides corresponding to SEQ ID NOS: 1-6.

[0085] C. Mimotope In certain aspects, the present disclosure provides mimotopes that immunologically mimic the peptide epitopes described herein (e.g., peptides that bind to parent antibodies that bind to NSE proteins). In some embodiments, a mimotope is a peptide sequence that immunologically mimics a peptide epitope and has sequence homology with an antigenic site. In other embodiments, a mimotope is a peptide sequence that immunologically mimics a peptide epitope and has a three-dimensional conformation similar to the antigenic site, but does not have sequence homology.

[0086] In some embodiments, mimotopes induce antibody responses similar to those elicited by peptide epitopes. In certain instances, the antibody response of a mimotope corresponds to binding to the same antigenic site on the parent antibody to which the peptide epitope binds. The ability of a mimotope to act as a molecular mimic that binds to the parent antibody can be used to block the antibody from binding to its original target antigen (e.g., NSE protein).

[0087] In some embodiments, mimotopes are obtained by biopanning from phage display libraries. Phage display libraries suitable for screening and identifying candidate mimotopes are typically large numbers of phage expressing random amino acid sequences at positions to which antibodies can bind, typically less than 100 amino acids in length, less than 75 amino acids, less than 50 amino acids, less than 25 amino acids, particularly in the range of about 3 to about 25 amino acids.

[0088] In other embodiments, mimotopes are obtained by screening a peptide library. In some examples, the peptide library is an overlapping peptide library. In other examples, the peptide library is a truncated peptide library that can be used to identify the shortest amino acid sequence required for activity. In yet other examples, mimotopes are obtained by alanine scanning, in which alanine is used to sequentially replace each residue to identify the specific amino acid residue responsible for the peptide's activity. In a further example, mimotopes are obtained by positional scanning, in which an amino acid of interest at a single position is identified and substituted with all other natural amino acids one at a time to identify a preferred amino acid residue at that position for increasing the peptide's activity. In related cases, positional scanning can include two-position combinatorial scanning or three-position combinatorial scanning. Further methods for designing, screening, and determining mimotopes are described, for example, in U.S. Pat. No. 4,833,092, the disclosure of which is incorporated by reference in its entirety for all purposes.

[0089] In further embodiments, mimotopes may comprise peptide sequences that are more structurally constrained than the linear form of the sequence. Unsubstituted linear peptides, as they exist free in solution, can typically adopt a number of different conformations. In contrast, peptides that are structurally constrained by having one, or usually two or more, substituents that presumably reduce the number of possible conformations that can be assumed, are also within the scope of the present disclosure.

[0090] Substituents, such as covalent or intramolecular bonds to additional peptide chains, structurally constrain the peptide. For example, a peptide can form part of the primary structure of a larger polypeptide that includes the amino acid sequence of the peptide. In certain instances, the peptide comprises a cyclic peptide.

[0091] Other substituents include covalent attachment to other moieties, such as macromolecular structures, including biological and non-biological structures. Examples of biological structures include, but are not limited to, carrier proteins. Examples of non-biological structures include lipid vesicles, such as liposomes, micelles, and lipid nanoparticles.

[0092] In some embodiments, a carrier protein is conjugated to the mimotope. Many carriers are known for this purpose, including various protein-based carriers such as albumin (e.g., bovine serum albumin (BSA)), keyhole limpet hemocyanin (KLH), ovalbumin (OVA), tetanus toxoid (TT), high molecular weight protein (HMP) from nontypeable Haemophilus influenzae, diphtheria toxoid, or bacterial outer membrane proteins, all of which can be obtained from biochemical or pharmaceutical supply companies or prepared by standard methodologies.

[0093] In other embodiments, the mimotope is a component of a vaccine. The vaccine may incorporate one or more mimotopes, each capable of binding to the same or different parent antibodies, to block the binding of the antibody to its original target antigen (e.g., NSE protein). Multiple mimotopes may be conjugated to each other, for example, using polylysine to which each mimotope is conjugated.

[0094] In certain embodiments, peptide mimotopes are designed using single amino acid substitutions, followed by affinity testing of each peptide construct to determine which peptide mimics have the ability to block autism-specific maternal autoantibodies. In certain examples, D-amino acids are used to synthesize peptide mimotopes because peptides synthesized from D-amino acids are more resistant to proteolytic digestion and have a longer half-life in vivo. In other examples, peptide mimotopes for each autoantigen are fused to a polyethylene glycol (PEG) backbone, resulting in the creation of heteromultimers that can neutralize autism-specific maternal autoantibodies. See, for example, Kessel et al., Chem Med Chem. 4(8):1364-70, 2009.

[0095] Mimotope peptides linked to a PEG backbone are useful as antibody blocking agents because peptides on a PEG backbone are less immunogenic than individual peptides. Peptide mimotopes can be synthesized by 9-fluorenyl-methoxy-carbonyl-protected amino acid chemistry on a suitable polyethylene glycol (PEG)-PS resin (GenScript Corporation; Piscataway, New Jersey) using an automated peptide synthesizer (Pioneer; Applied Biosystems; Foster City, California). Cleavage of the peptide from the resin and removal of the protecting groups from the side chains can be achieved by using trifluoroacetic acid with a scavenger. Crude peptides are purified by preparative C fractionation with a gradient of solvent A [95% / 5% HO (0.1% trifluoroacetic acid) / acetonitrile] and solvent B (100% acetonitrile). 18 The peptide can be purified by reversed-phase high performance liquid chromatography using a column. The purity of the peptide can then be confirmed by analytical C 18The peptides are analyzed by high-performance liquid chromatography using a column. The identity of the synthesized peptides can also be confirmed by matrix-assisted laser desorption / ionization / time-of-flight mass spectrometry. In certain instances, peptide mimotopes can be PEGylated using a strategy involving reversible protection of specific residues on the peptide. This procedure is possible only because peptides generally contain only a few nucleophilic groups and are more stable than full-length proteins against the harsh chemical treatments involved in this process. This method involves three steps: (1) protection with appropriate reagents of residues known to be important for activity and ultimately for purification of the desired isomer; (2) PEGylation at the level of the single unprotected reactive target residue; and (3) removal of all protecting groups.

[0096] ELISA assays can be used to determine whether multimerized peptide mimotopes bind to anti-brain autoantibodies in patient serum. ELISA assays can also be used to determine whether multimerized peptide mimotopes inhibit antigen-antibody interactions with native antigen proteins. This can be achieved by preincubating maternal antibody-positive plasma with the heteromultimers before performing the ELISA.

[0097] Animal models can be used to examine the efficacy, safety, and / or pharmacokinetic properties of peptide mimotopes in vivo. As a non-limiting example, a mouse model of maternal autoantibody-associated (MAR) autism can be used. See, for example, Example 5 of WO 2016 / 210137. MAR autism and control dams (i.e., pregnant female mice) can be randomly assigned to one of two treatment conditions: pregnancy mimotope treatment or saline control. Once tolerance is broken in the MAR autism dams, dams assigned to the treatment group can be administered mimotope peptides via intravenous injection. In vivo mimotope efficacy can be determined by administering 200 μg of mimotope to dams every 24 hours for a total of four injections. The reduction in mouse autoantibody titers by the peptide mimotope after treatment can be determined using an ELISA assay against the whole target antigen protein. A series of treatment trials can be performed to determine the number of treatments required to reduce / block mouse maternal antibodies during pregnancy. Once the ideal treatment regimen is determined, dams can be bred to produce offspring for subsequent behavioral analysis.

[0098] In certain embodiments, the peptide is a mimotope of the amino acid sequence of any one of SEQ ID NOs: 1-6, comprising D-amino acids at some (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) or all positions in the amino acid sequence, and / or comprising amino acid modifications (e.g., substitutions) at one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) positions in the amino acid sequence.

[0099] D. Kit The present disclosure also provides kits for diagnosing or prognosing whether an offspring, such as a fetus or a child, is at increased risk of developing an autism spectrum disorder (ASD). Relatedly, the kits are also used for diagnosing or prognosing whether a mother or potential mother is at increased risk of giving birth to a child that will develop an ASD.

[0100] Materials and reagents for carrying out these various methods can be provided in kits to facilitate carrying out the methods. As used herein, the term "kit" includes a combination of items that facilitate a process, assay, analysis, or procedure. In particular, kits containing the peptides or compositions described herein find utility in a wide range of applications, including, for example, diagnosis, prognosis, immunotherapy, etc.

[0101] In certain embodiments, the kit comprises any one or more peptides described herein (e.g., peptide epitopes and mimotopes thereof) that specifically bind to parent antibodies against the NSE antigen, and a solid support. In some examples, the kit comprises a peptide (e.g., peptide epitopes and / or mimotopes thereof) corresponding to any one of SEQ ID NOs: 1-6, or a combination thereof.

[0102] In some embodiments, the solid support comprises at least one peptide, e.g., at least 1, 2, 3, 4, 5, or 6 peptides. In particular examples, the solid support comprises an NSE peptide epitope. In some embodiments, the solid support comprises one or more peptides having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a peptide described herein, e.g., to the amino acid sequence of any one of SEQ ID NOs: 1-6. In other embodiments, the solid support comprises one or more peptides having the amino acid sequence set forth in any one of SEQ ID NOs: 1-6, or fragments thereof.

[0103] In some embodiments, the peptide or peptides can be immobilized on a solid support. In other embodiments, the solid support is a multiwell plate, an ELISA plate, a microarray, a chip, beads, a porous strip, or a nitrocellulose filter. Immobilization can be achieved via covalent or non-covalent binding. In some embodiments, immobilization is via a capture antibody that specifically binds to one or more peptides. In certain examples, the solid support in the kit is provided prepared with one or more immobilized peptides.

[0104] In certain embodiments, the plurality of peptides in the kit comprises at least 2, 3, 4, 5, or 6 different peptides, e.g., selected from SEQ ID NOs: 1-6 and mimotopes thereof. In particular examples, each of the different peptides in the kit binds to the same maternal antibody, e.g., all of the different peptides bind to a maternal antibody against the NSE antigen.

[0105] In certain embodiments, the plurality of peptides in the kit comprises one or more panels, each panel comprising a combination of peptides that bind to maternal antibodies against the NSE antigen. As a non-limiting example, each panel comprises a combination of peptides that bind to maternal antibodies against the NSE antigen. As a further non-limiting example, the kit comprises one or more peptide combinations selected from the group consisting of SEQ ID NOs: 1-6, each combination having 2, 3, 4, 5, or 6 peptides selected from the peptides of SEQ ID NOs: 1-6.

[0106] The kits may include chemical reagents and other components. Additionally, the kits described herein may include, but are not limited to, instructions for the user of the kit, equipment and reagents for sample collection and / or purification, equipment and reagents for product collection and / or purification, reagents for bacterial cell transformation, reagents for eukaryotic cell transfection, previously transformed or transfected host cells, sample tubes, holders, trays, racks, dishes, plates, solutions, buffers or other chemical reagents, and appropriate samples used for standardization, normalization, and / or control samples. The kits described herein can also be packaged, for example, in a box with a lid, for convenient storage and safe transport.

[0107] In some embodiments, the kit also includes a labeled secondary antibody used to detect the presence of maternal autoantibodies that bind to one or more peptides. The secondary antibody binds to the constant or "C" region of different classes or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE. Typically, a secondary antibody against the IgG constant region, such as a secondary antibody against one of the IgG subclasses (e.g., IgG1, IgG2, IgG3, and IgG4), is included in the kit. The secondary antibody may be a fluorophore (e.g., fluorescein, phycoerythrin, quantum dots, Luminex beads, fluorescent beads), an enzyme (e.g., peroxidase, alkaline phosphatase), a radioisotope (e.g., 3 H, 32 P, 125 The antibody can be labeled with any directly or indirectly detectable moiety, including a fluorescent antibody (I) or a chemiluminescent moiety. The label signal can be amplified using a conjugate of biotin and a biotin-binding moiety (e.g., avidin, streptavidin, neutravidin). Fluorescently labeled anti-human IgG antibodies are commercially available from Molecular Probes, Eugene, OR. Enzyme-labeled anti-human IgG antibodies are commercially available from Sigma-Aldrich, St. Louis, MO, and Chemicon, Temecula, CA.

[0108] The kit may further include instructions for contacting the solid support with a biological sample from the mother or potential mother, and instructions for correlating the presence of maternal antibodies or a level of maternal antibodies above a threshold level with an increased likelihood that the mother's or potential mother's fetus or child will develop an ASD.

[0109] In some embodiments, the kit also contains a negative control sample and a positive control sample for detecting maternal antibodies. In some instances, the negative control sample is obtained from a mother of a TD child. In other instances, the negative and / or positive control sample is reactive to the NSE antigen. In yet other instances, the negative and / or positive control sample is non-reactive to the NSE antigen. In some embodiments, the kit includes samples for preparing a titration curve of maternal antibodies in a sample to aid in assessing the quantified level of antibodies in a test biological sample. In certain embodiments, the kit includes one or more peptides set forth in SEQ ID NOS: 1-6, e.g., 1, 2, 3, 4, 5, or 6 of the peptides set forth in SEQ ID NOS: 1-6.

[0110] The kit is used to provide a diagnosis or prognosis to a woman of childbearing age. The diagnosis or prognosis can be determined before, during, or after pregnancy. Detection of maternal antibodies can be performed in one or more of the first, second, and / or third trimesters of pregnancy. In some embodiments, detection of maternal antibodies is performed on a biological sample obtained from a woman whose fetus has begun to develop its brain (e.g., after about 12 weeks of pregnancy). In some embodiments, the presence or absence of maternal antibodies or the quantified level of maternal antibodies is assessed one or more times postpartum, for example, during the first four weeks after birth and / or while the mother is breastfeeding her child. In some embodiments, the presence or quantified level of maternal antibodies is assessed one or more times before pregnancy or in any woman who is not pregnant.

[0111] E. Patients who are candidates for diagnosis or treatment The methods described herein can be performed on any mammal, e.g., a human, a non-human primate, a laboratory mammal (e.g., a mouse, a rat, a rabbit, a hamster), a domestic mammal (e.g., a cat, a dog), or an agricultural mammal (e.g., a cow, a sheep, a pig, a horse). In some embodiments, the patient is female and human.

[0112] Any woman capable of bearing a child can benefit from the methods described herein. The child may or may not be pregnant, i.e., the woman may be pregnant, but need not be. In some embodiments, the woman has a child that is a newborn. In some embodiments, the woman is of childbearing age, i.e., the woman has begun menstruating and has not reached menopause.

[0113] In some embodiments, the diagnostic and preventative and / or treatment methods described herein are performed on a woman carrying a fetus (i.e., who is pregnant). The methods can be performed at any time during pregnancy. In some embodiments, the methods are performed on a woman carrying a fetus whose brain has begun to develop. For example, the fetus may be about 12 weeks pregnant or later. In some embodiments, the woman being treated or diagnosed is in the second or third trimester of pregnancy. In some embodiments, the woman being treated or diagnosed is in the first trimester of pregnancy. In some embodiments, the woman is postpartum, e.g., within six months of giving birth. In some embodiments, the woman is postpartum and breastfeeding.

[0114] Women who benefit from the methods may, but need not, have a family history of ASD or autoimmune disease. For example, the woman may have ASD or have family members (e.g., parents, children, grandparents) who have ASD. In some embodiments, the woman has an autoimmune disease or has family members (e.g., parents, children, grandparents) who have an autoimmune disease.

[0115] In some embodiments, the methods described herein include determining that a diagnosis or treatment is appropriate for a patient, for example, based on previous medical or familial medical history, or pregnancy status, or any other relevant criteria.

[0116] F. Methods for assessing risk of developing autism spectrum disorder In certain aspects, the present disclosure provides methods for determining the likelihood or risk that a fetus or child will develop an autism spectrum disorder (ASD), comprising identifying the presence of maternal autoantibodies that bind to an NSE antigen in a biological sample from the mother or potential mother of the fetus or child, the method comprising detecting the presence or absence in the biological sample of maternal autoantibodies that bind to any one of the peptides described herein, wherein the presence of maternal autoantibodies that bind to a peptide or peptides indicates an increased likelihood or risk that the fetus or child will develop an ASD.

[0117] Regarding the biological sample taken from the mother or potential mother, any fluid sample containing antibodies can be used. For example, the biological sample can be blood, serum, plasma, amniotic fluid, urine, breast milk, or saliva. Of course, one or more different bodily fluids can be evaluated for antibodies that specifically bind to one or more peptides.

[0118] In certain embodiments, a biological sample is assessed for the presence of maternal antibodies that specifically bind to at least one or more of the peptides described herein (e.g., SEQ ID NOS: 1-6), e.g., at least one, two, three, four, five, or six of the peptides set forth in SEQ ID NOS: 1-6. In some embodiments, the presence of maternal antibodies that specifically bind to an NSE antigen is detected in a sample using one or more of the peptides described herein (e.g., SEQ ID NOS: 1-6). As a non-limiting example, one or more peptides, e.g., one, two, three, four, five, or six different peptides set forth in SEQ ID NOS: 1-6, can be used to detect the presence or absence of maternal antibodies in a sample.

[0119] In certain cases, the presence of maternal antibodies against NSE can be detected using one, two, three, four, five or six peptides set forth in SEQ ID NOs: 1-6 or antigenic fragments thereof.

[0120] In some embodiments, detecting the presence of maternal antibodies (versus the absence of detection of maternal antibodies) indicates that the fetus or child is likely to have or develop an ASD.

[0121] In some embodiments, the level or titer of maternal antibodies in a biological sample is compared to a threshold level or titer. A level or titer of antibodies in a biological sample greater than the threshold level or titer indicates that the fetus or child has or is likely to develop an ASD. Similarly, a level or titer of antibodies in a biological sample less than the threshold level or titer does not indicate that the fetus or child has or is likely to develop an ASD (i.e., indicates no increased probability). A threshold level or titer of maternal antibodies in a particular biological fluid can be determined by assessing the level of maternal antibodies in a population of pregnant women and comparing the antibody level or titer in the biological fluid of mothers when their children develop an ASD with the antibody level or titer in the biological fluid of mothers when their children do not develop an ASD. The threshold level or titer can also be determined at different times during pregnancy, for example, every four weeks, every two weeks, or every week during the fetus's pregnancy. The threshold antibody level or titer can also be measured after the child is born, for example, during the first four weeks after birth and / or while the mother is breastfeeding her child.

[0122] The presence of maternal antibodies to NSE antigens or quantified levels of maternal antibodies to NSE antigens can be determined before, during, or after pregnancy. If determined during pregnancy, maternal antibody detection can be performed once, twice, three times, four times, or more times, as needed, at any time during the course of pregnancy. For example, maternal antibody detection can be performed in one or more of the first, second, and / or third trimesters of pregnancy. In some embodiments, maternal antibody detection is performed on a biological sample obtained from a woman whose fetus is beginning to develop its brain (e.g., after about 12 weeks of pregnancy). In some embodiments, the presence or absence of maternal antibodies or quantified levels of maternal antibodies is assessed one or more times postpartum, for example, during the first four weeks after birth and / or while the mother is breastfeeding her child. In some embodiments, the presence or absence of maternal antibodies or quantified levels of maternal antibodies is assessed one or more times before pregnancy or in any non-pregnant woman.

[0123] The presence of maternal antibodies can be determined one or more times, as needed or desired, hi some embodiments, the presence or absence of maternal antibodies or quantified levels of maternal antibodies are assessed every four weeks, every two weeks or every week throughout pregnancy, or more or less frequently, as needed.

[0124] In some embodiments, the presence of maternal antibodies is determined without comparing the test sample (i.e., a biological sample from the mother or potential mother) to a control sample. In other embodiments, the test sample is compared to a control. The control can be from the same individual at a different time point. For example, the test sample can be collected during pregnancy, and the control sample can be collected from the same individual before pregnancy. In some examples, the test sample is collected relatively late in pregnancy, and the control sample is collected from the same individual earlier in pregnancy. In this case, if the level of maternal antibodies is greater in the test sample than in the control sample, the fetus or child is at increased risk of developing ASD. If several samples are evaluated over the course of pregnancy, an increase in the level or titer of maternal antibodies over the course of pregnancy indicates an increased risk that the fetus or child will develop ASD. Similarly, an absence or decrease in the level or titer of maternal antibodies over the course of pregnancy indicates a low or decreased risk that the fetus or child will develop ASD.

[0125] The control can also be derived from a different individual with a known status for the presence of maternal antibodies. The control can also be a calculated value from a population of individuals with a known status for the presence of maternal antibodies. The control can be a positive control or a negative control. In some instances, the negative control is obtained from a mother with a TD child. In other instances, the negative and / or positive control sample reacts with the NSE antigen. In yet other instances, the negative and / or positive control sample does not react with the NSE antigen.

[0126] In some embodiments, the control is a negative control from another individual or population of individuals. If the known status of the control sample is negative for the antibody, a higher level of maternal antibody in the test sample than in the negative control sample indicates an increased risk of the fetus or child developing ASD. A similar level of maternal antibody in the test sample to the negative control sample indicates a non-increased, i.e., low or reduced, risk of the fetus or child developing ASD.

[0127] In some embodiments, the control is a positive control from another individual or population of individuals, or the control reflects a predetermined threshold level of the antibody. If the known status of the control sample is positive for the antibody, a similar or higher level of maternal antibodies in the test sample compared to the positive control sample indicates an increased risk of the fetus or child developing an ASD. A lower level of maternal antibodies in the test sample relative to the control sample indicates that the fetus or child is not at elevated risk or is at low or reduced risk of developing an ASD.

[0128] The difference between the control sample or value and the test sample need only be sufficient to detect, in some embodiments, an increase in the level of maternal antibodies in the test sample, and thus an increased risk of ASD, is determined when the antibody level is at least, e.g., 10%, 25%, 50%, 1-fold, 2-fold, 3-fold, 4-fold or more, compared to a negative control or a previously determined control.

[0129] The presence of maternal antibodies to any subtype, isoform or isoenzyme of the NSE antigen can be determined for the purpose of diagnosing an increased likelihood that the fetus or child will develop ASD.

[0130] Maternal antibodies can be detected using any method known in the art, including, but not limited to, Western blot, dot blot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), electrochemiluminescence, and multiplex bead assays (e.g., using Luminex or fluorescent microbeads).

[0131] The peptide can be an antigenic fragment of the NSE antigen. The peptide can be derived from a known antigenic epitope of the NSE antigen in which one or more amino acids have been substituted, deleted, added, or otherwise modified. The peptide can be purified or substantially purified from a natural source, or can be produced recombinantly or synthetically.

[0132] In some embodiments, the peptide used to detect the parent antibody can be immobilized on a solid support. The solid support can be, for example, a multiwell plate, a microarray, a chip, beads, a porous strip, or a nitrocellulose filter. Immobilization can be via covalent or non-covalent attachment. In some embodiments, immobilization is by a capture antibody that specifically binds to the target epitope.

[0133] For detection of parent antibodies, a sample can be incubated with one or more of the peptides described herein under conditions (e.g., time, temperature, concentration of sample) sufficient to allow specific binding of any antibodies that specifically bind to one or more target antigens present in the sample. The one or more peptides can be bound to a solid support. For example, the one or more peptides can be exposed to the sample for about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 hours, or overnight, or for about 8, 10, 12, 14, or 16 hours. However, incubation times can be longer or shorter depending, for example, on the composition of the one or more peptides, the composition of the one or more target antigens, the dilution of the sample, and the temperature for incubation. Incubations using less diluted samples and higher temperatures can be performed for shorter times. Incubations are typically performed at room temperature (about 25°C) or biological temperature (about 37°C), and can be performed in a refrigerator (about 4°C). Washing to remove unbound sample before addition of the secondary antibody is performed according to known immunoassay methods.

[0134] Labeled secondary antibodies are generally used to detect antibodies in a sample that bind to one or more of the peptides described herein. Secondary antibodies bind to the constant or "C" region of different classes or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE. Typically, secondary antibodies directed against the IgG constant region are used in the above methods. Secondary antibodies directed against IgG subclasses, such as IgG1, IgG2, IgG3, and IgG4, also find use in the methods of the present invention. Secondary antibodies can be labeled with fluorophores (e.g., fluorescein, phycoerythrin, quantum dots, Luminex beads, fluorescent beads), enzymes (e.g., peroxidase, alkaline phosphatase), radioisotopes (e.g., 3 H, 32 P, 125 The antibody can be labeled with any directly or indirectly detectable moiety, including a fluorescent antibody (I) or a chemiluminescent moiety. The label signal can be amplified using a conjugate of biotin and a biotin-binding moiety (e.g., avidin, streptavidin, neutravidin). Fluorescently labeled anti-human IgG antibodies are commercially available from Molecular Probes, Eugene, OR. Enzyme-labeled anti-human IgG antibodies are commercially available from Sigma-Aldrich, St. Louis, MO, and Chemicon, Temecula, CA.

[0135] The method for detecting the presence or absence or differential presence of autoantibodies in a sample corresponds to the selection of the label of the secondary antibody. For example, when one or more of the peptides described herein are transferred onto a membrane substrate suitable for immunoblotting, the detectable signal (i.e., blot) can be quantified using a digital imager if an enzyme label is used, or using an X-ray film developer if a radioisotope label is used. In another example, when one or more of the peptides described herein are transferred onto a multi-well plate, the detectable signal can be quantified using an automated plate reader that can detect and quantify fluorescent, chemiluminescent, and / or colorimetric signals. Such detection methods are well known in the art.

[0136] General immunoassay techniques are well known in the art.Guidelines for parameter optimization can be found, for example, in Wu, Quantitative Immunoassay: A Practical Guide for Assay Establishment, Troubleshooting, and Clinical Application, 2000, AACC Press; Principles and Practice of Immunoassay, Price and Newman, eds., 1997, Groves Dictionaries, Inc.; The Immunoassay Handbook, Wild, ed., 2005, Elsevier Science Ltd.; Ghindilis, Pavlov and Atanassov, Immunoassay Methods and Protocols, 2003, Humana Press; Harlow and Lane, Using Antibodies: A Laboratory Manual, 1998, Cold Spring Harbor Laboratory Press; and Immunoassay Automation: An Updated Guide to Systems, Chan, ed., 1996, Academic Press.

[0137] In certain embodiments, the presence or increased presence of maternal antibodies is indicated by a detectable signal (e.g., blot, fluorescence, chemiluminescence, color, radioactivity) in an immunoassay in which a biological sample from the mother or potential mother is contacted with one or more of the peptides described herein. This detectable signal can be compared to a signal from a control sample or a threshold value. In some embodiments, increased presence is detected and indicates an increased risk of ASD when the detectable signal of maternal antibodies in the test sample is at least about 10%, 20%, 30%, 50%, 75% greater than the signal of maternal antibodies in the control sample or a predetermined threshold value. In some embodiments, increased presence is detected and indicates an increased risk of ASD when the detectable signal of maternal antibodies in the test sample is at least 1-fold, 2-fold, 3-fold, 4-fold, or more greater than the signal of maternal antibodies in the control sample or a predetermined threshold value.

[0138] In some embodiments, the results of the maternal antibody determination are recorded on a tangible medium. For example, the results of the diagnostic assay (e.g., observing the presence or increased presence of maternal antibodies) and the diagnosis of whether an increased risk of ASD is determined can be recorded on paper or electronic media (e.g., audiotape, computer disk, CD, flash drive, etc.).

[0139] In other embodiments, the method further comprises providing the patient (i.e., the mother or potential mother) with a diagnosis of whether the patient's fetus or child is at increased risk for developing ASD based on the results of the maternal antibody determination.

[0140] G. Methods of Reducing Risk by Administering Peptide Epitopes In certain aspects, the present disclosure provides methods for preventing and / or reducing the risk of developing autism spectrum disorder (ASD) in a fetus or child by administering a blocking agent (e.g., an NSE peptide described herein or a mimotope thereof that specifically binds to a maternal autoantibody associated with ASD) to the mother or potential mother in vivo. The blocking agent can prevent maternal antibodies from specifically binding to endogenous NSE autoantigens present in the fetus or child.

[0141] In some embodiments, the method comprises administering to the mother or potential mother at least one blocking agent comprising one or more peptides described herein (e.g., SEQ ID NOS: 1-6) or mimotopes thereof, e.g., at least 1, 2, 3, 4, 5, or 6 of the peptides set forth in SEQ ID NOS: 1-6 or mimotopes thereof. In particular examples, the blocking agent comprises a peptide corresponding to SEQ ID NOS: 1-6 or a mimotope thereof, and combinations thereof. In some examples, the blocking agent specifically binds to maternal antibodies that recognize the NSE antigen.

[0142] The disclosed prevention and / or treatment methods using a blocking agent or agents can be provided to a woman before, during, or after pregnancy. In some embodiments, the blocking agent can be administered once, twice, three times, four times, or more times as needed at any time during pregnancy. For example, the blocking agent can be administered in one or more of the first, second, and / or third trimesters of pregnancy. In some embodiments, the blocking agent is administered to a woman whose fetus has begun brain development, e.g., about 12 weeks into pregnancy. In some embodiments, the blocking agent is administered one or more times, e.g., during the first four weeks after birth and / or while the mother is breastfeeding her child. In some embodiments, the blocking agent is administered one or more times before conception, e.g., to a woman who has tested positive for maternal antibodies and is trying to conceive.

[0143] In some embodiments, multiple agents are administered, including two or more peptides or mimotopes thereof. The multiple agents can be administered separately or together. The multiple agents can be a pool of individual peptides or mimotopes. In some embodiments, two or more peptides or mimotopes with different epitopes are chemically linked. The multiple antigenic epitopes can be derived from the same or different antigenic polypeptides. The chemical linkage in this case can be by direct peptide bonding or by use of a chemical scaffold or linker. In some embodiments, two or more peptides or mimotopes with different peptide epitopes are fused together. Peptide epitope fusions can be expressed recombinantly or chemically synthesized.

[0144] In some embodiments, the method further comprises administering to the mother or potential mother a therapeutic or prophylactic regimen of one or more blocking agents (e.g., one or more peptides of SEQ ID NOs: 1-6 or mimotopes thereof) to reduce, inhibit, or prevent binding of maternal autoantibodies to the NSE antigen.

[0145] In particular examples, the administered blocking agent or agents that reduce, inhibit or prevent maternal antibody binding to the NSE polypeptide comprise one, two, three, four, five or six of the peptides set forth in SEQ ID NOs: 1-6, or antigenic fragments or mimotopes thereof. In other examples, the administered blocking agent or agents that reduce, inhibit or prevent maternal antibody binding to the NSE polypeptide comprise one, two, three, four, five or six of the peptides set forth in SEQ ID NOs: 1-6, or antigenic fragments or mimotopes thereof.

[0146] The administered blocking agents may contain modifications to reduce or minimize their immunogenicity. Amino acid modifications in peptides or mimotopes include, but are not limited to, amide moieties or pyroglutamyl residues, or the addition of polyethylene glycol chains (PEGylation). These modifications may contribute to a reduced tendency to form R-sheet conformations or to reduced peptide stability, solubility, and immunogenicity. In some instances, more stable, soluble, and less immunogenic peptides are desirable. Many peptides modified at the C-terminus with a CONH2 (amide) group appear to be resistant to attack by carboxypeptidases, while many peptides with a pyroglutamyl residue at the N-terminus are more resistant to attack by broad-specificity aminopeptidases. PEGylated peptides have been shown to have increased plasma half-lives and reduced immunogenicity compared to unmodified peptides. Furthermore, sequence analysis of blocking agents allows for the minimization of known T-cell epitopes through conservative modifications. Cyclic peptides that are resistant to attack by both carboxypeptidases and aminopeptidases are also included as peptides described herein. Additionally, oral administration of blocking agents can help minimize immunogenicity.

[0147] In some embodiments, prevention and / or treatment methods involve first determining the presence or increased presence of maternal antibodies that bind to the NSE antigen in the mother or potential mother using the detection methods described herein. Women who test positive for the presence of maternal antibodies or have levels above a threshold level are candidates for administration of a blocking agent that specifically binds to maternal antibodies. Women who test negative for the presence of maternal antibodies or have levels below the threshold level do not need to receive a blocking agent that specifically binds to maternal antibodies.

[0148] Pharmaceutical compositions suitable for use in the present disclosure include compositions containing a therapeutically effective amount of the active ingredient. The amount of the composition administered will, of course, depend on the subject being treated, the subject's weight, the severity of the affliction, the mode of administration, and the judgment of the prescribing physician. Determining an effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein. Generally, an effective amount or doses of one or more maternal antibody blocking agents are determined by initially administering a low or small dose of the blocking agent, followed by incrementally increasing the dose or doses administered, and / or adding a second blocking agent(s) as needed, until the desired effect, e.g., eliminating or reducing the presence of unbound or free maternal antibodies below a predetermined threshold level with minimal or no toxicity or undesirable side effects, is observed in the treated subject. Applicable methods for determining appropriate doses and administration schedules for administering the pharmaceutical compositions of the present disclosure are described, for example, in Goodman and Gilman's The Pharmacological Basis of Therapeutics, 11th Ed., Brunton, et al., Eds., McGraw-Hill (2006), and Remington: The Science and Practice of Pharmacy, 21st Ed., University of the Sciences in Philadelphia (USIP), 2005, Lippincott, Williams and Wilkins.

[0149] Dosage and interval can be individually adjusted to provide plasma or tissue levels of blocking agent sufficient to maintain therapeutic efficacy. Single or multiple administrations of a composition comprising an effective amount of one or more blocking agents can be administered at dose levels and patterns selected by the treating physician. The dose and administration schedule can be determined and adjusted, for example, based on the level of maternal antibodies in the mother or potential mother, which can be monitored throughout the course of treatment according to methods commonly practiced by clinicians or methods described herein. In some embodiments, therapeutic levels are achieved by administering a single dose per day. In other embodiments, the dosing schedule can include multiple daily dose schedules. In still other embodiments, administration every other day, twice a week, or once a week is encompassed by the present disclosure.

[0150] For example, the blocking agent can be administered monthly, biweekly, weekly, or daily, as needed. In some embodiments, if maternal antibodies are present or present at levels above a predetermined threshold level, the level of maternal antibodies in the mother or potential mother is monitored and the blocking agent is administered. The blocking agent can be administered for about 1, 2, 3, 4, 5, 10, 12, 15, 20, 24, 30, 32, 36 weeks, or more or less, as needed. For example, if the level of maternal antibodies falls below a predetermined threshold level, administration of the blocking agent(s) can be discontinued. The blocking agent(s) can be administered throughout the entire pregnancy or during one or more of the first, second, or third trimesters of pregnancy. Administration can begin before conception and can continue after birth, for example, while the mother is breastfeeding her child.

[0151] In some embodiments, where the blocking agent(s) is a peptide or mimotope thereof, a typical dosage may range from about 0.1 μg / kg body weight to about 1 g / kg body weight or less, for example, from about 1 μg / kg body weight to about 500 mg / kg body weight, hi some embodiments, the dose of peptide or mimotope is about 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg / kg body weight.

[0152] The exact dose will depend on various factors described herein, including the particular inhibitor, the severity of the disease, and the route of administration. The determination of the exact therapeutically effective dose can be determined by the clinician without undue experimentation, and can include any dose that falls within the ranges disclosed above.

[0153] The blocking agent(s) are administered by a route of administration that allows the agent(s) to bind to maternal antibodies, prevent antibody binding to endogenous autoantigens associated with the risk of developing ASD, and minimize immune responses to the agent(s). Typically, the agent(s) are administered systemically. In some embodiments, the agent(s) are administered parenterally, for example, intravenously or intra-amniotically (i.e., directly into the amniotic sac). Additionally, the agent(s) may be administered orally.

[0154] The blocking agent(s) can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. For injection, the blocking agent(s) can be formulated into a preparation by dissolving, suspending, or emulsifying in an aqueous or non-aqueous solvent, such as vegetable oil or other similar oil, synthetic aliphatic acid glyceride, ester of higher fatty acid, or propylene glycol, optionally with conventional additives, such as solubilizers, isotonicity agents, suspending agents, emulsifiers, stabilizers, and preservatives. In some embodiments, the combination of blocking agents can be formulated in an aqueous solution, preferably in a physiologically compatible buffer, such as Hank's solution, Ringer's solution, or physiological saline buffer. Injectable preparations can be provided in unit dosage form, such as ampoules or multi-dose containers, with added preservatives. The compositions can take the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulating agents such as suspending agents, stabilizing agents, and / or dispersing agents.

[0155] Pharmaceutical preparations for parenteral administration include aqueous solutions of the blocking agent(s) in water-soluble form. Furthermore, suspensions of the blocking agent(s) can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension can also contain stabilizers or suitable agents that increase the solubility of the compounds, allowing for the preparation of highly concentrated solutions. Alternatively, the blocking agent(s) can be in powder form for constitution with a suitable vehicle, for example, sterile pyrogen-free water, before use.

[0156] Treatment with blocking agent(s) is considered effective if the level or titer of maternal antibodies that actively bind to the NSE antigen is reduced or eliminated in a biological sample from an individual after one or more administrations of the blocking agent(s) compared to before administration of the blocking agent(s). For example, a reduction in maternal antibodies that actively bind to the NSE antigen in at least about 10%, 25%, 50%, 75%, or 100% of the sample after one or more administrations of one or more blocking agents indicates that administration of the blocking agent(s) was effective. If a threshold level has been established, treatment with blocking agent(s) is considered effective if the level or titer of maternal antibodies that actively bind to the NSE antigen is reduced below the threshold level. Maternal antibodies that actively bind to the NSE antigen can be measured using any method known in the art, including those described herein.

[0157] H. How to reduce risk by removing maternal antibodies In certain aspects, the present disclosure provides methods for preventing or reducing the risk of an offspring, such as a fetus or a child, developing autism spectrum disorder (ASD) by removing maternal antibodies from the biological fluid of a mother or potential mother ex vivo and then returning the biological fluid containing reduced or eliminated levels of maternal antibodies to the mother or potential mother.

[0158] In some embodiments, biological fluids containing maternal antibodies can be removed from the mother or potential mother and contacted with one or more of the peptides described herein. In other embodiments, one or more of the peptides described herein can be administered to the mother or potential mother to block binding between maternal autoantibodies and their autoantigens in the biological fluid, thereby neutralizing the maternal autoantibodies, and the neutralizing complexes present in the biological fluid are removed using extracorporeal treatments such as affinity plasmapheresis.

[0159] In some embodiments, biological fluids from the mother or potential mother are contacted with one or more peptides immobilized on a solid support. The solid support can be, for example, a multiwell plate, an ELISA plate, a microarray, a chip, beads, a column, a porous strip, a membrane, or a nitrocellulose filter. Immobilization can be via covalent or non-covalent attachment. In some embodiments, immobilization is by a capture antibody that specifically binds to the target peptide epitope. The peptide(s) attached to the solid support are the stationary phase that captures the maternal antibodies in the biological fluid, allowing the biological fluid with reduced or eliminated levels of maternal antibodies to be separated from the solid support, i.e., as the mobile phase, and returned to the mother or potential mother.

[0160] In some embodiments, the biological fluid treated ex vivo is plasma, and maternal antibodies are removed by plasmapheresis, a process well known in the art. The plasma is contacted with a solid support containing one or more immobilized peptides. Maternal antibodies in the plasma bind to the immobilized peptides. The plasma, with reduced or eliminated levels of maternal antibodies, is then returned to the mother or potential mother.

[0161] Ex vivo removal of maternal antibodies can be performed on a woman before, during, or after pregnancy. In some embodiments, maternal antibodies are removed from biological fluids one, two, three, four, or more times, as needed, at any time during pregnancy. For example, maternal antibodies can be removed in one or more of the first, second, and / or third trimesters of pregnancy. In some embodiments, maternal antibodies are removed from a woman whose fetus has begun to develop its brain, e.g., about 12 weeks into pregnancy. In some embodiments, maternal antibodies are removed one or more times postpartum, e.g., during the first four weeks after birth and / or while the mother is breastfeeding her child. In some embodiments, maternal antibodies are removed one or more times before pregnancy, e.g., in a woman who is trying to conceive and has tested positive for maternal antibodies.

[0162] The ex vivo maternal antibody removal process can be performed one, two, three, four, or more times as needed to eliminate or reduce maternal antibodies from the mother or potential mother. Ex vivo maternal antibody removal can be performed daily, weekly, biweekly, monthly, or bimonthly as needed. In some embodiments, the level of maternal antibodies in the mother or potential mother is monitored and ex vivo maternal antibody removal is performed if the presence of maternal antibodies exceeds a predetermined threshold level. Ex vivo maternal antibody removal can be performed for 1, 2, 3, 4, 5, 10, 12, 15, 20, 25, 35, 36 weeks or longer or shorter periods as needed. For example, if the level of maternal antibodies falls below a predetermined threshold level, ex vivo maternal antibody removal can be discontinued. Ex vivo maternal antibody removal can be performed throughout the entire pregnancy or during one or more of the first, second, or third trimesters of pregnancy. Maternal antibody removal can begin before conception and can continue after birth, for example, while the mother is breastfeeding her child.

[0163] The biological fluid containing maternal antibodies is typically blood, serum, plasma, or milk. In some embodiments, the biological fluid is amniotic fluid. [Example]

[0164] The present disclosure will now be described in more detail with reference to specific examples. The following examples are provided for illustrative purposes only and are not intended to limit the present disclosure in any way.

[0165] Example 1. Materials and Methods 1.1 Research Subjects

[0166] This study included mothers enrolled in the CHARGE study (Childhood Autism Risk from Genetics and Environment) at the MIND Institute at UC Davis

[19] . CHARGE participants in this study included mothers with children diagnosed with ASD (n = 246) and mothers with children selected from the general population (neurotypical, TD; n = 149). We used recruitment, eligibility, and psychometric assessment procedures as previously described [7, 19]. ASD diagnoses were verified at the MIND Institute according to the Diagnostic and Statistical Manual of Mental Disorders-5 (DSM-5)

[20] . All procedures were approved by the California Committee for the Protection of Human Subjects and the Institutional Review Boards at UC Davis and UC Los Angeles. Prior to participation, subjects provided written informed consent in either English or Spanish. Demographic information for this sample is shown in Table 1. [Table 1]

[0167] 1.2 Sample collection and preparation

[0168] Blood was collected into acid citrate dextrose (BD Diagnostic) and plasma was separated, coded, aliquoted and stored at −80° C. Prior to use, samples were thawed and centrifuged at 13,000 RPM for 10 minutes.

[0169] 1.3 Preparation of fetal brain antigen

[0170] Tissue processing was performed as previously described [8]. Briefly, we used embryonic 152-day-old fetal rhesus monkey brains (FMBs) provided by the California National Primate Research Center. FMBs were mechanically homogenized in buffer using a Polytron 3000 homogenizer (Brinkman), sonicated for 3 minutes, and centrifuged at 3,000 × g for 10 minutes. The supernatant was then collected, concentrated by ultrafiltration, and its protein content was measured by bicinchoninic acid assay (BCA).

[0171] 1.4 Prep Cell

[0172] Protein fractionation was performed as previously described [8]. Briefly, 40 mg of FMB was electrophoresed on a 10% polyacrylamide gel for 17 hours at 12 watts using a Prep Cell apparatus (Bio-Rad, Hercules, CA) and separated by molecular weight. Protein fractions were collected at 5-minute intervals at a flow rate of 0.75 ml / min. A total of 110 fractions were obtained, concentrated to 5 mg / ml by ultrafiltration, and probed by Western blot (WB) to determine molecular weight and antigen reactivity (Figures 1A-1D). Ponceau staining confirmed protein enrichment and fractionation in the range of approximately 5 kDa / fraction. Fraction 12 contained proteins ranging from 37 to 45 kDa and was therefore selected for antigen identification (Figures 2A-2E).

[0173] 1.5 Western blot

[0174] To test for autoantibody reactivity against FMB fraction 12, which contains proteins between 37 and 45 kDa, the fraction was probed with maternal plasma samples as previously described (Figure 1D) [8]. Briefly, 200 μg of protein was denatured by heating in SDS buffer at 100°C for 10 min and separated on a 12% SDS-PAGE gel at 200 V for 1 h. Proteins were transferred to a 0.2 μm nitrocellulose membrane overnight at 4°C (10 V for 16 h). To confirm the transfer, the membrane was stained with Ponceau dye, cut into 3 mm strips, which were labeled and blocked with 1% casein buffer. Plasma samples were then diluted (1:400), added to the strips, and incubated at room temperature for 1.5 h, followed by five washes and incubation with 1:20,000 goat anti-human IgG-HRP for 30 min. After five washes, detection was performed by adding 800 μl of Super Signal substrate and placing the strips on a glass plate that was imaged using a FluoroChem 8900 imager. Images were scored as 0 for negative and 1 for positive.

[0175] 1.6 Two-dimensional (2-D) gel electrophoresis

[0176] Protein fractions targeted by maternal autoantibodies were separated by 2-D electrophoresis as previously described [8]. Briefly, 300 μg of protein fractions in the 30-40 kDa range were labeled with Cy2 (GE Life Sciences, Pittsburgh, PA, USA) in preparation for 2-D electrophoresis (all gels were run in duplicate). First, 15 μg of each sample was isoelectrically focused using 3-10 isoelectric focusing strips (GE Healthcare, Piscataway, NJ, USA). The strips were then loaded onto two 10.5% polyacrylamide gels (GE Healthcare) for second-dimensional electrophoresis. Images were captured using Quant software (version 6.0, GE Healthcare). One of the gels was transferred to a nitrocellulose membrane and assayed by WB for maternal plasma reactivity to bands around 37-39 kDa, but not to GDA, LDHA / B, or YBX1. The resulting positive spots were mapped back onto Cy2-stained duplicate 2-D gels, excised from the gels, and digested with trypsin (Promega, Madison, Wis., USA) in preparation for mass spectrometry.

[0177] 1.7 Mass spectrometry

[0178] Mass spectrometry analysis was performed as described previously [8]. Digested peptides were desalted (Zip-tip C18, Millipore, Billerica, MA, USA) and spotted onto a MALDI plate (model ABI 01-192-6-AB). MALDI-TOF MS and TOF / TOF tandem MS / MS data were acquired using an ABI 4700 mass spectrometer (Applied Biosystems, Framingham, MA, USA). The resulting peptide masses and associated fragmentation spectra were analyzed using a GPS Explorer workstation with the MASCOT search engine (Matrix Science, Boston, MA, USA) and used to perform BLAST searches on NCBI. Candidates with either a protein score confidence interval (CI%) or ion CI% greater than 95 were considered positive (Table 1).

[0179] The top four commercially available antigens identified by mass spectrometry using 100 CI were selected for further evaluation. To assess antibody reactivity to our top hits, including NSE, NNE, ALDOC, and CKB, 2 μg of recombinant protein (Novus Biologicals, Littleton, CO) was probed with diluted maternal plasma (1:800) by WB as previously described.

[0180] 1.8 Enzyme-linked immunosorbent assay (ELISA)

[0181] Once NSE was identified as a viable antigen candidate by WB, we evaluated a larger sample set for NSE reactivity using ELISA. We tested plasma from 418 mothers enrolled in the CHARGE study, including mothers of at least one child with ASD (n = 232) or control samples from mothers of typically developing children (TD; n = 186). Microtiter plates were coated with 100 μl of 2 μg / ml NSE (Novus Biologicals, Littleton, CO) in carbonate coating buffer, pH 9.6, incubated overnight at 4°C, washed four times with PBST 0.05%, and blocked with 2% Super Block (Thermo Scientific, Rockford, IL) for 1 hour at room temperature (RT). Plasma samples were diluted 1:500 and run in duplicate. After dilution, 100 μl of diluted sample was added to each well, incubated for 1.5 hours, washed four times, and then incubated with 1:10,000 goat anti-human IgG-HRP IgG (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MA) for 1 hour. The plate was then washed, and detection was performed by adding 100 μl of BD optEIA liquid substrate for ELISA (BD Biosciences, San Jose, CA). After 4 minutes, the reaction was stopped with 50 μl of 2N HCl. Absorbance was measured at 490–450 nm using an iMark Microplate Absorbance Reader (Biorad, Hercules, CA).

[0182] 1.9 Receiver Operating Characteristic (ROC) Curve

[0183] For the ELISA assay, the positive cutoff value for reactivity to NSE was determined using an ROC curve. ROC curves were generated by plotting the true positive rate against the false positive rate at various threshold settings. Therefore, we generated our curve using seven positive samples (labeled +) from mothers whose children with ASD tested positive in WB along with the test samples (true positive samples). By using positive samples as the reference event, the cutoff sacrifices some sensitivity (limit of detection) but has higher specificity (fewer false positives). ROC plots sensitivity versus 1-specificity for each value, generating the area under the curve (AUC), which is a representation of the test's accuracy. We calculated the cutoff using Youden's index [21, 22].

[0184] 1.10 Microarray screening

[0185] The complete NSE sequence (NP_001966.1) was obtained from NCBI and translated into a library of contiguous 15-mer peptides with 14 amino acid (aa) overlaps on microarray slides. Discovery peptide microarrays were synthesized by PEPperPRINT as previously described

[23] , whereby targeted 15-mer peptide sequences are directly printed in duplicate onto glass slides using solid-phase Fmoc chemistry (PEPperPRINT, Heidelberg, Germany). Peptides derived from human influenza hemagglutinin (HA) (YPYDVPDYAG) and polio vaccine (KEVPALTAVETGAT) were also included as positive controls.

[0186] To test antibody reactivity to the printed peptides, we probed the arrays with plasma from mothers enrolled in the CHARGE study (ASD = 27 and TD = 22) according to the manufacturer's instructions. Microarray slides were first incubated with standard buffer (PBS containing 0.05% Tween 20, pH 7.4) for 10 minutes and then blocked at room temperature (Rockland Blocking Buffer MB-070; Rockland Immunochemicals Inc.) for 45 minutes. The slides were then incubated overnight at 4°C with shaking, and individual maternal plasma samples were diluted 1:250 in staining buffer, followed by three washes with standard buffer. For signal detection, slides were incubated with goat anti-human IG(H+L)-DyLight 649 (Rockland Immunochemicals Inc.) at a dilution of 1:5000 in staining buffer (standard buffer containing 10% blocking buffer) for 30 minutes at room temperature. After secondary antibody incubation, the microarrays were imaged using a GenePix 4000 B Microarray Scanner (Molecular Devices, Sunnyvale, CA).

[0187] Fluorescence signal quantification of spot intensity (FI) and peptide annotation was performed using PepSlide Analyser software (PEPperPRINT) based on the manufacturer's recommendations. Data preprocessing methods were performed as reported in previous peptide microarray studies. Briefly, net fluorescence intensity (FI) was calculated using the correction method reported by Zue et al. [24, 25]. A 3 × 2 window was set for each spot, and the median value of six spots was used as the "neighborhood background" for the central spot. To normalize net fluorescence intensity (FI), a 3 × 1 "sliding window" was set for each spot, and the median value of three spots was used as the normalized signal for the central spot [24, 25]. Corrected net intensity was calculated by subtracting the corrected background from the normalized signal. If the background signal was high compared to the spot (negative FI), the signal was set to 1, as reported in similar studies [26, 27].

[0188] Finally, after background correction and signal normalization, the corrected net signal was obtained by calculating the median of the replicates, and the coefficient of variation was calculated. Samples with a CV higher than 50% were flagged and corrected. Values ​​below 200 FI were treated as negative due to nonspecific binding, and only sequences with values ​​above 200 were considered positive for statistical analysis [26, 27].

[0189] statistical analysis

[0190] To thoroughly examine the data for sequences that significantly differed between diagnostic groups and identify epitopes specific to a given group (TD or ASD), we used two different analytical methods: 1) a t-test—a parametric test that compares two independent samples via mean difference and allows for the assumption of normal distribution of the data; and 2) significance analysis of microarrays (SAM)—a permutation-based approach that measures the strength of the relationship between epitope expression and a response variable, in this case, ASD and TD diagnoses. The SAM score is directly proportional to the significance of the relationship in the data (maximum score = 2). XLSTAT 2015.1 software (Addinsoft, Paris, France) was used to conduct t-tests, and the R statistical computing environment was used to perform SAM analysis. Furthermore, we compared the prevalence of epitope reactivity between the ASD and TD groups using Fisher's exact test. Differences were considered significant when p<0.05. For significant sequences, odds ratios (or 95% CI) were also calculated using GraphPad Prism software (GraphPad Software, San Diego, CA).

[0191] Example 2. Antigen Identification Fetal monkey brain (FMB) was separated into 110 fractions by molecular weight, and fraction 12, containing proteins between 37 and 45 kDa (Figures 1B and 1C), was analyzed by a pair of 2-D gels / Western blots (Figures 2A-2E). One gel was transferred to a nitrocellulose membrane and used to verify the reactivity of autoantibodies to proteins between 37 and 45 kDa in a mother of a child with ASD (Figures 1B and 1C), who had previously tested negative for previously described autoantigens in that molecular weight range (GDA, LDHA, LDHB, and YBX1) by WB (Figure 1D). Multiple spots were observed, and all identified spots were collected from a second, matching 2-D gel for mass spectrometry analysis (Figures 2A-2E). Proteins around 37 to 45 kDa with 100% CI were selected for verification, and detailed mass spectrometry results for the verified antigens are listed in Table 2. [Table 2]

[0192] The top four commercially available proteins recognized by maternal autoantibodies with a 100% CI, including neuron-specific enolase (NSE), nonspecific enolase (NNE), fructose bisphosphate aldolase C (ALDOC), and creatine kinase B (CKB), were selected for further evaluation. Each of these proteins was tested, and maternal autoantibody reactivity to each individual antigen was assessed using recombinant proteins. NSE was subsequently identified as corresponding to a band between 37 and 45 kDa by maternal samples, recognized with the greatest specificity in the samples tested, and therefore selected as the most likely candidate for further MAR ASD target autoantigens.

[0193] Example 3. Antigen validation NSE was identified by mass spectrometry as a potential target of maternal autoantibodies, and based on its important role in neurodevelopment, we chose to further evaluate NSE as a potential MAR ASD biomarker. Using recombinant NSE, we first verified maternal autoantibody reactivity by WB followed by ELISA. We observed reactivity in 26 of 232 mothers with children with ASD (6.2%) and 21 of 186 mothers with typically developing children (TD, 5%), suggesting that NSE alone is not a MAR ASD biomarker. Therefore, we utilized a similar approach to that used for the seven previously described MAR autoantigens to probe samples for differential epitope recognition between ASD and TD groups.

[0194] Example 4. Epitope mapping The entire NSE sequence (NP_001966.1) was translated into 434 distinct 15-mer peptides with 14 aa overlap, printed in duplicate on glass microarrays, and then probed with diluted plasma from mothers from the ASD and control groups. After data preprocessing steps, we divided the samples into two categories based on their reactivity by ELISA (positive: samples with antibodies against NSE; negative: samples that are negative for NSE in their native form but may have reactivity against cryptic epitopes) for statistical analysis. For ELISA (+) samples, we found 16 sequences that were ASD-specific (0% TD) and five sequences (FI > 200) recognized by antibodies from both groups. From the 16 ASD-specific sequences, four sequences were statistically significant using both t-tests and SAM t-tests (Table 3). DVAASEFYRDGKYDL (SEQ ID NO: 1) (p=0.047; SAM score 1.49), IEDPFDQDDWAAWSK (SEQ ID NO: 2) (p=0.049; SAM score 1.49), ERLAKYNQLMRIEEE (SEQ ID NO: 3) (p=0.045; SAM score 1.57), and RLAKYNQLMRIEEEL (SEQ ID NO: 4) (p=0.017; SAM score 1.82). [Table 3]

[0195] Additionally, to assess the association of epitope sequences with a given group, we used Fisher's exact test and found no significant differences, likely due to our small sample size. Instead, we calculated odds ratios (ORs) with 95% confidence intervals (95% CI) for each individual peptide. We found that all ASD-specific sequences had ORs greater than 3, with SERLAKYNQLMRIEE (SEQ ID NO: 6) (OR 10.1, CI 95% 0.5094-200.7) and ERLAKYNQLMRIEEE (SEQ ID NO: 3) (OR 12.6, CI 95% 0.6408-247.7) being the two epitopes with the highest ORs (Figure 3). As described above, we found five consecutive epitope sequences recognized by plasma from both sample groups, suggesting a large immunodominant epitope containing the printed sequences DYPVVSIEDPFDQDD (SEQ ID NO: 7), YPVVSIEDPFDQDDW (SEQ ID NO: 8), PVVSIEDPFDQDDWA (SEQ ID NO: 9), VVSIEDPFDQDDWAA (SEQ ID NO: 10), and VSIEDPFDQDDWAAW (SEQ ID NO: 11) (Table 3). As shown in Figure 3, the sequences highlighted in red indicate the conserved amino acids recognized by antibodies in each of the five distinct peptide epitopes. Reactivity against the large, major immunodominant epitope was also observed in the ELISA(-) sample, suggesting it is a mimotope primarily recognized by the general population (Table 4). Interestingly, we also found one ASD-specific epitope sequence, QDFVRDYPVVSIEDP (p=0.054, SAM score 1.97, OR 12.6, CI 95% 0.6408-247.7; SEQ ID NO: 23), recognized by the ELISA(-) sample, suggesting that this is likely non-reactive with the native structure of NSE and likely binds to a cryptic determinant (Table 4). [Table 4]

[0196] Example 5. Bioinformatics To better understand the potential origin of reactivity to recently identified epitopes, we analyzed the epitope homology with all epitopes reported in the IEDB database using the Immune Epitope Database tool (IEDB). We performed BLAST searches at 90% and 80% sequence homology settings and found that each of the identified sequences shared 90% homology with other isoforms of enolase, primarily alpha-enolase (Table 5). The DYPVVSIEDPFDQDD (SEQ ID NO: 7) and DFVRDYPVVSIEDPF (SEQ ID NO: 16) epitopes each shared 90% homology with protein ORF73 from human gamma-herpesvirus 8 (the causative agent of mononucleosis), and DVAASEFYRDGKYDL (SEQ ID NO: 1) shared 90% homology with outer surface protein A from Borrelia burgdorferi (the causative agent of Lyme disease). Other sequences shared 80% homology with peptides from different organisms, including the genomic polyprotein from hepatitis C virus, the virion packaging protein UL25 from human betaherpesvirus 6B, Alt a 6 from Alternaria alternata, the ATP-dependent RNA helicase RhlB from Vibrio cholerae, and protein X from hepatitis B virus (Table 5). 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[0197] It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light thereof will be suggested to those skilled in the art and are intended to be included within the spirit and scope of this application and the appended claims. All publications, patents, patent applications, and sequence accession numbers cited herein are incorporated by reference in their entirety for all purposes.

Claims

1. An isolated peptide consisting of the amino acid sequence of SEQ ID NO: 3 or 4.

2. The peptide of claim 1 , wherein the peptide binds to a parent antibody that binds to the neuron-specific enolase (NSE) protein.

3. The peptide of claim 1 or 2, wherein the peptide is a mimotope.

4. The peptide of claim 3, wherein the mimotope comprises D-amino acids.

5. The peptide of any one of claims 1 to 4, wherein the peptide further comprises a label.

6. 6. The peptide of claim 5, wherein the label is selected from the group consisting of biotin, a fluorescent label, a chemiluminescent label, and a radioactive label.

7. A composition comprising a peptide or a plurality thereof according to any one of claims 1 to 6.

8. The composition of claim 7 further comprising a pharmaceutically acceptable carrier.

9. The composition of claim 7 or 8, wherein the plurality of peptides comprises at least two different peptides.

10. The composition of claim 9 , wherein the different peptides bind to the same parent antibody.

11. A kit comprising a peptide or a plurality of peptides according to any one of claims 1 to 6 and a solid support.

12. 12. The kit of claim 11, wherein the solid support is a multiwell plate, an ELISA plate, a microarray, a chip, a bead, a porous strip, or a nitrocellulose filter.

13. 13. The kit of claim 11 or 12, wherein the peptide or peptides are immobilized on the solid support.

14. The kit of any one of claims 11 to 13, wherein the plurality of peptides comprises at least two different peptides.

15. The kit of claim 14 , wherein the different peptides bind to the same parent antibody.

16. The kit of any one of claims 11 to 15, further comprising instructions for use.

17. 1. A composition for use in determining the risk of an offspring to develop an autism spectrum disorder (ASD), comprising: the presence of maternal antibodies that bind to the peptide or a plurality thereof in a biological sample from the mother or potential mother indicates an increased risk that the offspring will develop ASD; A composition comprising a peptide or a plurality thereof according to any one of claims 1 to 6.

18. 18. The composition of claim 17, wherein the sample consists of blood, serum, plasma, amniotic fluid, breast milk, or saliva.

19. 19. The composition of claim 17 or 18, wherein the plurality of peptides comprises at least two different peptides.

20. The composition of claim 19 , wherein the different peptides bind to the same parent antibody.

21. The composition of any one of claims 17 to 20, wherein the peptide or peptides are attached to a solid support.

22. 22. The composition of claim 21, wherein the solid support is a multiwell plate, an ELISA plate, a microarray, a chip, a bead, a porous strip, or a nitrocellulose filter.

23. 23. The composition of any one of claims 17 to 22, wherein the maternal antibodies are detected by Western blot, dot blot, ELISA, radioimmunoassay, immunoprecipitation, electrochemiluminescence, immunofluorescence, FACS analysis or multiplex bead assay.

24. 24. The composition of any one of claims 17 to 23, wherein the mother or potential mother has a child with ASD.

25. 25. The composition of any one of claims 17 to 24, wherein the mother or potential mother has a family history of ASD or autoimmune disease.

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