Patent Application
20220148690
Diagnostic tests are currently available and performed on a regular basis to detect the presence or absence of a normal state in an individual. These tests, however, do not provide a clear assessment of the immunorepertoire in an individual or insight into how such individual's immunorepertoire is indicative of the presence or absence of wellness. A need, therefore, exists for systems and methods to provide individuals with a means of assessing and displaying their immunorepertoire in a manner that may assist with an assessment of the health of such individual.
In some embodiments, the present disclosure relates to a method of presenting a user's immunorepertoire profile to the user, comprising the steps of: obtaining a blood sample from the user; determining at least one index for selected from the group consisting of the clonotype index, essential index, and diversity index to produce an immunorepertoire profile for the blood sample for the user; and outputting information to the user pertaining to the user's immunorepertoire profile. In some embodiments, the method further comprises the step of obtaining a set of characteristic data associated with the user, wherein the characteristic data associated with the user comprises the user's age and gender. In some embodiments, the characteristic data further comprises the presence of any disease. In some embodiments, the blood sample comprises whole blood. In some embodiments, the blood sample comprises a dried blood spot. In some embodiments, the, method comprises the additional steps of: providing the user with a kit comprising a blood collection card, wherein the blood collection card comprises at least one blood collection area and a QR code; and scanning the QR code by the user to associate the blood sample with the user's account on a software application. In some embodiments, the step of outputting information to the user is performed using a software application.
In some embodiments, the present disclosure relates to a method of presenting a user's immunorepertoire profile to the user, comprising the steps of: providing the user with a kit comprising a blood collection card, wherein the blood collection card comprises at least one blood collection area and a QR code; scanning the QR code by the user to associate the blood sample with the user's account on a software application; obtaining a set of characteristic data associated with the user, wherein the characteristic data associated with the user comprises the user's age, gender and the presence or absence of any disease; obtaining a blood sample from the user; determining at least one index for selected from the group consisting of the clonotype index, essential index, and diversity index to produce an immunorepertoire profile for the blood sample for the user; and outputting information to the user pertaining to the user's immunorepertoire profile using a software application.
This disclose relates to systems and methods for assessing the immunorepertoire and wellness of an individual. As depicted in
In some embodiments, the blood sample may be collected by a user by using a kit comprising a lancet and a sterile blood collection card. The blood collection card may comprise materials suitable for absorbing blood, including but not limited to paper and card stock. A user may use the lancet to draw blood, for example from one of the user's fingertips. The blood collection card comprises one or more blood collection areas on which the user may place a sample of blood and where such blood may dry. The blood collection card may further comprise a QR code, which the user may scan using a smartphone or other device to associate the QR code and the blood sample with the user's account on a software application. The user may then send the blood collection card for rehydration, processing and determination of the user's clonotype index, essential index, and/or diversity index to generate a user report which is stored on a database. The user may then access his or her user report stored on the database using the software application via an internet connected device.
The first index disclosed herein is referred to as the clonotype index. The clonotype index for an individual is obtained by measuring the total number of unique clonotypes in an individual's sample containing lymphocytes, such as a blood sample, and dividing the number of unique clonotypes by the number of unit reads for such sample. As used herein, “blood sample” means peripheral blood, a dried blood spot, cord blood, or other sample containing blood.
The second index disclosed herein is referred to as the essential index. In one embodiment, the essential index is the number of the top 1000 public CDR3s (pCDR3s) in 100,000 of an individual's reads. pCDR3s are CDR3s present in more than one individual. For purposes of determining the top 1000 pCDR3s, the pCDR3s of a cohort of individuals (index pool) is determined and ranked. In other embodiments, fewer than the top 1000 pCDRs are assessed. In other embodiments, more than the top 1000 pCDR3s are assessed. In other embodiments, fewer than the 100,000 reads are taken for an individual. In other embodiments, more than the 100,000 reads are taken for an individual.
In one aspect of the present disclosure, the immunorepertoire of an individual is considered normal if the individual's essential index meets or exceeds a minimum percentage, whereas the immunorepertoire of the individual is considered abnormal of the individual's normality index is below such minimum percentage. In one embodiment, the minimum percentage is 35%.
The CDR3 expressed by individuals exhibits tremendous diversity, with up to 1015 unique CDR3 possible. As such, CDR3 may be used as a basis for immune system diversity. Based on a sampling of 75 million CDR3, the inventor has determined that approximately 81% of randomly-selected CDR3 are unique to a given individual and are not shared among multiple individuals.
The method of the present disclosure may be performed using the following steps to identify a normal immune status or an abnormal immune status in an individual, the method comprising the steps of: (a) amplifying polynucleotides from a population of white blood cells from an individual in a reaction mix comprising target-specific nested primers to produce a set of first amplicons, at least a portion of the target-specific nested primers comprising additional nucleotides which, during amplification, serve as a template for incorporating into the first amplicons a binding site for at least one common primer; (b) transferring a portion of the first reaction mix containing the first amplicons to a second reaction mix comprising at least one common primer; (c) amplifying, using the at least one common primer, the first amplicons to produce a set of second amplicons; (d) sequencing the second amplicons to identify CDR3 sequences in the subpopulation of white blood cells, and (e) identifying CDR3 sequences which constitute pCDR3s; (f) calculating the essential index based on the individual's pCDR3s; and (g) identifying whether the essential index is normal or abnormal, wherein a normal state is characterized by the presence of a minimum percentage of pCDR3 and an abnormal state is characterized by the absence of a minimum percentage of pCDR3.
In certain embodiments, the sequencing includes about 100,000 reads taken per sample. In certain embodiments, the reads are performed multiple times, for example about 10 to 100 times, using random selection. The number of an individual's pCDR3 in the top 1000 pCDR3s of the reference pool provide a percentage, referred to as the “essential index,” which is a number between 0% and 100%. For example, if an individual's sample contains 200 of the top 1000 pCDR3 sequences, then the individual's essential index is 0.20 or 20%. In other embodiments at least 10,000 reads are taken. In other embodiments, more than 100,000 reads are taken. In other embodiments, the reads are performed less than 10 times. In other embodiments, the reads are performed more than 100 times.
In certain embodiments, the index pool is composed of about 1000 individuals. In other embodiments, the index pool contains between 100 and 1000 individuals. In other embodiments, the index pool contains fewer than 100 individuals. In other embodiments, the index pool contains more than 1000 individuals. Relative to the individual, the individuals may be age-matched, gender-matched, healthy, disease-matched, and/or other criteria commonly known in the art when controlling for variables. In certain embodiments, the index pool is composed of healthy controls. In other embodiments, the index pool is composed of a mix of healthy controls and individuals with one or more disease states. In other embodiments, the index pool is composed of individuals with one or more particular disease states.
In certain embodiments, the CDR3 sequences shared by the index pool (i.e., the pCDR3) are determined by comparing each sample from the index pool and identifying those CDR3s that are shared by at least 50% of the individuals tested in such reference pool. In certain embodiments, the pCDR3 includes about the top 1000 shared CDR3 sequences. In other embodiments, the pCDR3 include at least 100 CDR3 sequences. In other embodiments, the pCDR3 includes more than 1000 CDR3 sequences.
It has previously been difficult to assess the immune system in a broad manner, because the number and variety of cells in a human or animal immune system is so large that sequencing of more than a small subset of cells has been almost impossible. The inventor developed a semi-quantitative PCR method (arm-PCR, described in more detail in U.S. Patent Application Publication Number 20090253183), which provides increased sensitivity and specificity over previously-available methods, while producing semi-quantitative results. It is this ability to increase specificity and sensitivity, and thereby increase the number of targets detectable within a single sample that makes the method ideal for detecting relative numbers of clonotypes of the immunorepertoire. The inventor has more recently discovered that using this sequencing method allows comparison of an individual's CDR3 sequences to those commonly shared by an index group, which has led to the development of the present method. The method may be used to evaluate the diversity of the immunorepertoire of subjects relative to an index pool of individuals. For example, the inventor has demonstrated that the presence of disease correlates with decreased immunorepertoire diversity, for example a decrease in the diversity of CDR3 sequences, which can be readily detected using the method of the present disclosure. This method may therefore be useful as an initial diagnostic indicator, much as cell counts and biochemical tests are currently used in clinical practice, of normal versus abnormal immunorepertoire diversity.
Clonotypes (i.e., clonal types) of an immunorepertoire are determined by the rearrangement of Variable(V), Diverse(D) and Joining(J) gene segments through somatic recombination in the early stages of immunoglobulin (Ig) and T cell receptor (TCR) production of the immune system. The V(D)J rearrangement can be amplified and detected from T cell receptor alpha, beta, gamma, and delta chains, as well as from immunoglobulin heavy chain (IgH) and light chains (IgK, IgL). Cells may be obtained from an individual by obtaining peripheral blood, lymphoid tissue, cancer tissue, or tissue or fluids from other organs and/or organ systems, for example. Techniques for obtaining these samples, such as blood samples, are known to those of skill in the art. Cell counts may be extrapolated from the number of sequences detected by PCR amplification and sequencing.
The CDR3 region, comprising about 30-90 nucleotides, encompasses the junction of the recombined variable (V), diversity (D) and joining (J) segments of the gene. It encodes the binding specificity of the receptor and is useful as a sequence tag to identify unique V(D)J rearrangements.
Wang et al. disclosed that PCR may be used to obtain quantitative or semi-quantitative assessments of the numbers of target molecules in a specimen (Wang, M. et al, “Quantitation of mRNA by the polymerase chain reaction,” (1989) Proc. Nat'l. Acad. Sci. 86: 9717-9721). Particularly effective methods for achieving quantitative amplification have been described previously by the inventor. One such method is known as arm-PCR, which is described in United States Patent Application Publication Number 20090253183A1.
Aspects of the present disclosure include arm-PCR amplification of CDR3 from T cells, B cells, and/or subsets of T or B cells. Such cell types may be sorted and isolated using techniques known in the art including, but not limited to, FACS sorting and magnetic bead sorting. The term “population” of cells, as used herein, therefore encompasses what are generally referred to as either “populations” or “sub-populations” of cells. Large numbers of amplified products may then be efficiently sequenced using next-generation sequencing using platforms such as 454 or Illumina, for example.
The arm-PCR method provides highly sensitive, semi-quantitative amplification of multiple polynucleotides in one reaction. The arm-PCR method may also be performed by automated methods in a closed cassette system (iCubate®, Huntsville, Ala.), which is beneficial in the present method because the repertoires of various T and B cells, for example, are so large. In the arm-PCR method, target numbers are increased in a reaction driven by DNA polymerase, which is the result of target-specific primers being introduced into the reaction. An additional result of this amplification reaction is the introduction of binding sites for common primers which will be used in a subsequent amplification by transferring a portion of the first reaction mix containing the first set of amplicons to a second reaction mix comprising common primers. “At least one common primer,” as used herein, refers to at least one primer that will bind to such a binding site, and includes pairs of primers, such as forward and reverse primers. This transfer may be performed either by recovering a portion of the reaction mix from the first amplification reaction and introducing that sample into a second reaction tube or chamber, or by removing a portion of the liquid from the completed first amplification, leaving behind a portion, and adding fresh reagents into the tube in which the first amplification was performed. In either case, additional buffers, polymerase, etc., may then be added in conjunction with the common primers to produce amplified products for detection. The amplification of target molecules using common primers gives a semi-quantitative result wherein the quantitative numbers of targets amplified in the first amplification are amplified using common, rather than target-specific primers—making it possible to produce significantly higher numbers of targets for detection and to determine the relative amounts of the cells comprising various rearrangements within an individual blood sample. Also, combining the second reaction mix with a portion of the first reaction mix allows for higher concentrations of target-specific primers to be added to the first reaction mix, resulting in greater sensitivity in the first amplification reaction. It is the combination of specificity and sensitivity, along with the ability to achieve quantitative results by use of a method such as the arm-PCR method, which allows a sufficiently sensitive and quantitative assessment of the CDR3 expressed in a population of cells to produce a normality index that is of diagnostic use.
Clonal expansion due to recognition of antigen results in a larger population of cells that recognize that antigen, potentially including antibody-producing B cells or receptor-bearing T cells. This may cause the reads taken pursuant to the method disclosed herein to be biased in favor of the antigen-specific expansion, thereby reducing the percentage of pCDR3 sequences detected. Therefore, a relatively low normality index, for example one below the minimum percentage, may be indicative of the expansion of a particular population of cells that is prevalent in individuals who have been diagnosed with a particular disease or in individuals recently-vaccinated against a particular antigen.
Primers for amplifying and sequencing variable regions of immune system cells are available commercially, and have been described in publication such as the inventor's published patent applications WO2009137255 and US201000021896A1.
There are several commercially available high-throughput sequencing technologies, such as Hoffman-LaRoche, Inc.'s 454® sequencing system. In the 454® sequencing method, for example, the A and B adaptor are linked onto PCR products either during PCR or ligated on after the PCR reaction. The adaptors are used for amplification and sequencing steps. When done in conjunction with the arm-PCR technique, A and B adaptors may be used as common primers (which are sometimes referred to as “communal primers” or “superprimers”) in the amplification reactions. After A and B adaptors have been physically attached to a sample library (such as PCR amplicons), a single-stranded DNA library is prepared using techniques known to those of skill in the art. The single-stranded DNA library is immobilized onto specifically-designed DNA capture beads. Each bead carries a unique singled-stranded DNA library fragment. The bead-bound library is emulsified with amplification reagents in a water-in-oil mixture, producing microreactors, each containing just one bead with one unique sample-library fragment. Each unique sample library fragment is amplified within its own microreactor, excluding competing or contaminating sequences. Amplification of the entire fragment collection is done in parallel. For each fragment, this results in copy numbers of several million per bead. Subsequently, the emulsion PCR is broken while the amplified fragments remain bound to their specific beads. The clonally amplified fragments are enriched and loaded onto a PicoTiterPlate® device for sequencing. The diameter of the PicoTiterPlate® wells allows for only one bead per well. After addition of sequencing enzymes, the fluidics subsystem of the sequencing instrument flows individual nucleotides in a fixed order across the hundreds of thousands of wells each containing a single bead. Addition of one (or more) nucleotide(s) complementary to the template strand results in a chemilluminescent signal recorded by a CCD camera within the instrument. The combination of signal intensity and positional information generated across the PicoTiterPlate® device allows the software to determine the sequence of more than 1,000,000 individual reads, each is up to about 450 base pairs, with the GS FLX system.
Having obtained the sequences using a quantitative and/or semi-quantitative method, it is then possible to calculate the normality index, for example, by determining the percentage of pCDR3 represented by a predetermined number of reads of an individual sample. Each individual's normality index may be compared to a predetermined threshold to determine whether the individual's normality index falls within the normal range, and therefore is normal, or below the threshold, and thereby is abnormal.
The method of the present disclosure provides a physician with an additional clinical test for diagnostic purposes to determine whether an individual's immunorepertoire is abnormal. Further, the method of the present disclosure, particularly if used in an automated system such as that described by the inventor in U.S. Patent Application Publication Number 201000291668A1, may be used to analyze samples from multiple individuals, with detection of the amplified targets sequences being accomplished by the use of one or more microarrays.
Whole blood samples (40 ml) collected in sodium heparin or peripheral blood mononuclear cells (PBMCs) were obtained from 1100 individuals, representing a mixed population of both healthy individuals and those with disease. The 1100 individuals were placed randomly into 11 different groups with 100 samples per group.
RNA extraction was performed using the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. For each target, a set of nested sequence-specific primers (Forward-out, Fo; Forward-in, Fi; Reverse-out, Ro; and Reverse-in, Ri) was designed using primer software available at www.irepertoire.com. A pair of common sequence tags was linked to all internal primers (Fi and Ri). Once these tag sequences were incorporated into the PCR products in the first few amplification cycles, the exponential phase of the amplification was carried out with a pair of communal primers. In the first round of amplification, only sequence-specific nested primers were used. The nested primers were then removed by exonuclease digestion and the first-round PCR products were used as templates for a second round of amplification by adding communal primers and a mixture of fresh enzyme and dNTP. Each distinct barcode tag was introduced into amplicon from the same sample through PCR primer.
Barcode tagged amplicon products from different samples were pooled together and loaded into a 2% agarose gel. Following electrophoresis, DNA fragments were purified from DNA band corresponding to 250-500 bp fragments extracted from agarose gel. DNA was sequenced using the 454 GS FLX system with titanium kits (SeqWright, Inc.).
Sequences for each sample were sorted out according to barcode tag. Following sequence separation, sequence analysis was performed in a manner similar to the approach reported by Wang et al. (Wang C, et al. High throughput sequencing reveals a complex pattern of dynamic interrelationships among human T cell subsets. Proc Natl Acad Sci USA 107(4): 1518-1523). Briefly, germline V and J reference sequences, which were downloaded from the IMGT server (http://www.imgt.org), were mapped onto sequence reads using the program IRmap. The boundaries defining CDR3 region in reference sequences were mirrored onto sequencing reads through mapping information. The enclosed CDR3 regions in sequencing reads were extracted and translated into amino acid sequence.
Table 1 below lists exemplary pCDR3 from cord blood. Table 2 below lists exemplary pCDR3 from adult blood.
The third index disclosed herein is referred to as the diversity index. This method uses the difference between the level of immune cell diversity generally seen in a normal, healthy individual and the generally lower level of diversity seen in an individual who has one or more disease conditions as a diagnostic indicator of the presence of a normal or abnormal immune status. In one aspect of the invention, the diversity level is referred to as the D50, with D50 being defined as the minimum percentage of distinct CDR3s accounting for at least half of the total CDR3s in a population or subpopulation of immune system cells. The third complementarity-determining region (CDR3) being a region whose nucleotide sequence is unique to each T or B cell clone, the higher the number, the greater the level of diversity. D50 may be described as follows. Where the “significant percentage” of the total number cells is fifty percent (50%), the diversity index (D50) may also be defined as a measure of the diversity of an immune repertoire of J individual cells (the total number of CDR3s) composed of S distinct CDR3s in a ranked dominance configuration where ri is the abundance of the ith most abundant CDR3, r1 is the abundance of the most abundant CDR3, r2 is the abundance of the second most abundant CDR3, and so on. C is the minimum number of distinct CDR3s, amounting to 50% of the total sequencing reads. D50 therefore is given by C/S×100.
The method of the invention may be performed using the following steps for assessing the level of diversity of an immunorepertoire: (a) amplifying polynucleotides from a population of white blood cells from a human or animal subject in a reaction mix comprising target-specific nested primers to produce a set of first amplicons, at least a portion of the target-specific nested primers comprising additional nucleotides which, during amplification, serve as a template for incorporating into the first amplicons a binding site for at least one common primer; (b) transferring a portion of the first reaction mix containing the first amplicons to a second reaction mix comprising at least one common primer; (c) amplifying, using the at least one common primer, the first amplicons to produce a set of second amplicons; (d) sequencing the second amplicons to identify V(D)J rearrangement sequences in the subpopulation of white blood cells, (e) using the identified V(D)J rearrangement sequences to quantify both the total number of cells in a population of immune system cells and the total numbers of cells within each of the clonotypes identified within the population; and (f) identifying the number of clonotypes that comprise a significant percentage of a total number of cells counted within that population, wherein a normal state is characterized by the presence of a greater variety of clonotypes represented within the significant percentage of the total number of cells and an abnormal state is characterized by the presence of a lesser number of clonotypes represented within a significant percentage of the total number of cells.
It has previously been difficult to assess the immune system in a broad manner, because the number and variety of cells in a human or animal immune system is so large that sequencing of more than a small subset of cells has been almost impossible. The inventor developed a semi-quantitative PCR method (arm-PCR, described in more detail in U.S. Patent Application Publication Number 20090253183), which provides increased sensitivity and specificity over previously-available methods, while producing semi-quantitative results. It is this ability to increase specificity and sensitivity, and thereby increase the number of targets detectable within a single sample that makes the method ideal for detecting relative numbers of clonotypes of the immunorepertoire. The inventor has more recently discovered that using this sequencing method allows him to compare immunorepertoires of individual subjects, which has led to the development of the present method. The method has been used to evaluate subjects who appear normal, healthy, and asymptomatic, as well as subjects who have been diagnosed with various forms of cancer, for example, and the inventor has demonstrated that the presence of disease correlates with decreased immunorepertoire diversity, which can be readily detected using the method of the invention. This method may therefore be useful as a diagnostic indicator, much as cell counts and biochemical tests are currently used in clinical practice.
Clonotypes (i.e., clonal types) of an immunorepertoire are determined by the rearrangement of Variable(V), Diverse(D) and Joining(J) gene segments through somatic recombination in the early stages of immunoglobulin(Ig) and T cell receptor (TCR) production of the immune system. The V(D)J rearrangement can be amplified and detected from T cell receptor alpha, beta, gamma, and delta chains, as well as from immunoglobulin heavy chain (IgH) and light chains (IgK, IgL). Cells may be obtained from an individual by obtaining peripheral blood, lymphoid tissue, cancer tissue, or tissue or fluids from other organs and/or organ systems, for example. Techniques for obtaining these samples, such as blood samples, are known to those of skill in the art. “Quantifying clonotypes,” as used herein, means counting, or obtaining a reliable approximation of, the numbers of cells belonging to a particular clonotype. Cell counts may be extrapolated from the number of sequences detected by PCR amplification and sequencing.
The CDR3 region, comprising about 30-90 nucleotides, encompasses the junction of the recombined variable (V), diversity (D) and joining (J) segments of the gene. It encodes the binding specificity of the receptor and is useful as a sequence tag to identify unique V(D)J rearrangements.
Wang et al. disclosed that PCR may be used to obtain quantitative or semi-quantitative assessments of the numbers of target molecules in a specimen (Wang, M. et al., “Quantitation of mRNA by the polymerase chain reaction,” (1989) Proc. Nat'l. Acad. Sci. 86: 9717-9721). Particularly effective methods for achieving quantitative amplification have been described previously by the inventor. One such method is known as arm-PCR, which is described in United States Patent Application Publication Number 20090253183A1.
Aspects of the invention include arm-PCR amplification of CDR3 from T cells, B cells, and/or subsets of T or B cells. The term “population” of cells, as used herein, therefore encompasses what are generally referred to as either “populations” or “sub-populations” of cells. Large numbers of amplified products may then be efficiently sequenced using next-generation sequencing using platforms such as 454 or Illumina, for example. If the significant percentage that is chosen is 50%, the number may be referred to as the “D50.” D50 may then be the percent of dominant and unique T or B cell clones that account for fifty percent (50%) of the total T or B cells counted in that sample. For high-throughput sequencing, for example, the D50 may be the number of the most dominant CDR3s, among all unique CDR3s, that make up 50% of the total effective reads, where total effective reads is defined as the number of sequences with identifiable V and J gene segments which have been successfully screened through a series of error filters.
The arm-PCR method provides highly sensitive, semi-quantitative amplification of multiple polynucleotides in one reaction. The arm-PCR method may also be performed by automated methods in a closed cassette system (iCubate®, Huntsville, Ala.), which is beneficial in the present method because the repertoires of various T and B cells, for example, are so large. In the arm-PCR method, target numbers are increased in a reaction driven by DNA polymerase, which is the result of target-specific primers being introduced into the reaction. An additional result of this amplification reaction is the introduction of binding sites for common primers which will be used in a subsequent amplification by transferring a portion of the first reaction mix containing the first set of amplicons to a second reaction mix comprising common primers. “At least one common primer,” as used herein, refers to at least one primer that will bind to such a binding site, and includes pairs of primers, such as forward and reverse primers. This transfer may be performed either by recovering a portion of the reaction mix from the first amplification reaction and introducing that sample into a second reaction tube or chamber, or by removing a portion of the liquid from the completed first amplification, leaving behind a portion, and adding fresh reagents into the tube in which the first amplification was performed. In either case, additional buffers, polymerase, etc., may then be added in conjunction with the common primers to produce amplified products for detection. The amplification of target molecules using common primers gives a semi-quantitative result wherein the quantitative numbers of targets amplified in the first amplification are amplified using common, rather than target-specific primers—making it possible to produce significantly higher numbers of targets for detection and to determine the relative amounts of the cells comprising various rearrangements within an individual blood sample. Also, combining the second reaction mix with a portion of the first reaction mix allows for higher concentrations of target-specific primers to be added to the first reaction mix, resulting in greater sensitivity in the first amplification reaction. It is the combination of specificity and sensitivity, along with the ability to achieve quantitative results by use of a method such as the arm-PCR method, that allows a sufficiently sensitive and quantitative assessment of the type and number of clonotypes in a population of cells to produce a diversity index that is of diagnostic use.
Clonal expansion due to recognition of antigen results in a larger population of cells that recognize that antigen, and evaluating cells by their relative numbers provides a method for determining whether an antigen exposure has influenced expansion of antibody-producing B cells or receptor-bearing T cells. This is helpful for evaluating whether there may be a particular population of cells that is prevalent in individuals who have been diagnosed with a particular disease, for example, and may be especially helpful in evaluating whether or not a vaccine has achieved the desired immune response in individuals to whom the vaccine has been given.
Primers for amplifying and sequencing variable regions of immune system cells are available commercially, and have been described in publication such as the inventor's published patent applications WO2009137255 and US201000021896A1.
There are several commercially available high-throughput sequencing technologies, such as Hoffman-LaRoche, Inc.'s 454® sequencing system. In the 454® sequencing method, for example, the A and B adaptor are linked onto PCR products either during PCR or ligated on after the PCR reaction. The adaptors are used for amplification and sequencing steps. When done in conjunction with the arm-PCR technique, A and B adaptors may be used as common primers (which are sometimes referred to as “communal primers” or “superprimers”) in the amplification reactions. After A and B adaptors have been physically attached to a sample library (such as PCR amplicons), a single-stranded DNA library is prepared using techniques known to those of skill in the art. The single-stranded DNA library is immobilized onto specifically-designed DNA capture beads. Each bead carries a unique singled-stranded DNA library fragment. The bead-bound library is emulsified with amplification reagents in a water-in-oil mixture, producing microreactors, each containing just one bead with one unique sample-library fragment. Each unique sample library fragment is amplified within its own microreactor, excluding competing or contaminating sequences. Amplification of the entire fragment collection is done in parallel. For each fragment, this results in copy numbers of several million per bead. Subsequently, the emulsion PCR is broken while the amplified fragments remain bound to their specific beads. The clonally amplified fragments are enriched and loaded onto a PicoTiterPlate® device for sequencing. The diameter of the PicoTiterPlate® wells allows for only one bead per well. After addition of sequencing enzymes, the fluidics subsystem of the sequencing instrument flows individual nucleotides in a fixed order across the hundreds of thousands of wells each containing a single bead. Addition of one (or more) nucleotide(s) complementary to the template strand results in a chemilluminescent signal recorded by a CCD camera within the instrument. The combination of signal intensity and positional information generated across the PicoTiterPlate® device allows the software to determine the sequence of more than 1,000,000 individual reads, each is up to about 450 base pairs, with the GS FLX system.
Having obtained the sequences using a quantitative and/or semi-quantitative method, it is then possible to calculate the D50, for example, by determining the percent of clones that account for at least about 50% of the total clones detected in the individual sample. Normal ranges may be compared to the numbers obtained for an individual individual, and the result may be reported both as a number and as a normal or abnormal result. This provides a physician with an additional clinical test for diagnostic purposes. Results for individual samples from a healthy individual, an individual with colon cancer, and an individual with lung cancer are shown below in Table 1. These results are from T-cell populations, expressed as an average of results from 8 (age matched normal) to 10 (colon cancer, lung cancer) samples.
As each number represents the percent of clones making up about 50 percent of the total number of sequences detected in the population being assessed, it is clear from the numbers above that a lack of immunorepertoire diversity, expressed as a deviation from normal, may be a useful criterion for use in diagnostic test panels. The method of the invention, particularly if used in an automated system such as that described by the inventor in U.S. Patent Application Publication Number 201000291668A1, may be used to analyze samples from multiple individuals, with detection of the amplified targets sequences being accomplished by the use of one or more microarrays.
Hybridization, utilizing at least one microarray, may also be used to determine the D50 of an individual's immunorepertoire. In such a method, the D50 would be calculated as the percentage of the most dominant variable genes (V and/or J genes) which would account for at least 50% of the total signal from all the V and or J genes.
Table 2 illustrates the difference in B-cell diversity, as evidenced by the D50, between (8) normal, healthy individual and (20) individuals with chronic lymphocytic leukemia, and (12) Lupus individuals
Recently, researchers in various laboratories have reported that microbial diversity within a human or animal (the “microbiome”) also shifts when the healthy state changes to a more unhealthy state. For example, shifts in microbial populations have been associated with various gastrointestinal disorders, with obesity, and with diabetes, for example. Zaura et al. (Zaura, E. et al. “Defining the healthy ‘core microbiome’ of oral microbial communities.” BMC Microbiology (2009) 9: 259) reported that a major proportion of bacterial sequences of unrelated healthy individuals is identical, and the proportion shifts in individuals who have oral disease. The arm-PCR method, combined with high-throughput sequencing, provides a relatively fast, highly sensitive, specific, and semi-quantitative method for evaluating diversity of microbial populations to establish a microbial D50 value, for example, for various human or animal tissues. Arm-PCR has been shown to be quite effective for identifying bacteria within mixed populations obtained from clinical samples.
Whole blood samples (40 ml) collected in sodium heparin from 10 lung and 10 colon, and 10 breast cancer individuals were purchased from Conversant Healthcare Systems (Huntsville, Ala.). Whole blood samples (40 ml) collected in sodium heparin from 8 normal control samples were purchased from ProMedDx (Norton, Mass.).
T cell isolations were performed using superparamagnetic polystyrene beads (MiltenyiBiotec) coated with monoclonal antibodies specific for each T cell subset. From whole blood, mononuclear cells were obtained by Ficoll prep, and monocytes removed using anti-CD14 microbeads. This monocyte-depleted mononuclear fraction was then used as a source for specific T cell subset fractions.
Cytotoxic CD8+ T cells were isolated by negative selection using anti-CD4 multisort beads (MiltenyiBiotec), followed by positive selection with anti-CD8 beads. CD4+ T cells were isolated by positive selection with anti-CD4 beads. Anti-CD25 beads (MiltenyiBiotec) were used to select CD4+CD25+ regulatory T cells. All isolated cell populations were immediately resuspended in RNAprotect (Qiagen).
RNA extraction was performed using the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. For each target, a set of nested sequence-specific primers (Forward-out, Fo; Forward-in, Fi; Reverse-out, Ro; and Reverse-in, Ri) was designed using primer software available at www.irepertoire.com. A pair of common sequence tags was linked to all internal primers (Fi and Ri). Once these tag sequences were incorporated into the PCR products in the first few amplification cycles, the exponential phase of the amplification was carried out with a pair of communal primers. In the first round of amplification, only sequence-specific nested primers were used. The nested primers were then removed by exonuclease digestion and the first-round PCR products were used as templates for a second round of amplification by adding communal primers and a mixture of fresh enzyme and dNTP. Each distinct barcode tag was introduced into amplicon from the same sample through PCR primer.
Barcode tagged amplicon products from different samples were pooled together and loaded into a 2% agarose gel. Following electrophoresis, DNA fragments were purified from DNA band corresponding to 250-500 bp fragments extracted from agarose gel. DNA was sequenced using the 454 GS FLX system with titanium kits (SeqWright, Inc.).
Sequences for each sample were sorted out according to barcode tag. Following sequence separation, sequence analysis was performed in a manner similar to the approach reported by Wang et al. (Wang C, et al. High throughput sequencing reveals a complex pattern of dynamic interrelationships among human T cell subsets. Proc Natl Acad Sci USA 107(4): 1518-1523). Briefly, germline V and J reference sequences, which were downloaded from the IMGT server (http://www.imgt.org), were mapped onto sequence reads using the program IRmap. The boundaries defining CDR3 region in reference sequences were mirrored onto sequencing reads through mapping information. The enclosed CDR3 regions in sequencing reads were extracted and translated into amino acid sequence.
This application references various publications. The disclosures of these publications, in their entireties, are hereby incorporated by reference into this application to describe more fully the state of the art to which this application pertains. The references disclosed are also individually and specifically incorporated herein by reference for material contained within them that is discussed in the sentence in which the reference is relied on.
The systems, methodologies and the various embodiments thereof described herein are exemplary. Various other embodiments of the systems and methodologies described herein are possible.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/033451 | 5/18/2020 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62849587 | May 2019 | US |
