WO2023196884A1

WO2023196884A1 - Detection assay for human papillomavirus (hpv) type 16 (hpv-16)

Info

Publication number: WO2023196884A1
Application number: PCT/US2023/065411
Authority: WO
Inventors: Lizhi YU; Yixin Wang
Original assignee: Juno Therapeutics, Inc.
Priority date: 2022-04-06
Filing date: 2023-04-05
Publication date: 2023-10-12

Abstract

Provided herein is a method of detecting, diagnosing, and treating human papillomavirus (HPV) in a subject. Further provided herein is a method of detecting, diagnosing, and treating HPV comprising the generation of a first pre-amplification reaction product.

Description

DETECTION ASSAY FOR HUMAN PAPILLOMAVIRUS (HPV) TYPE 16 (HPV-16)

Cross-Reference to Related Applications

[0001] This application claims priority from U.S. provisional application No. 63/328,198, filed April 6, 2022, entitled “DETECTION ASSAY FOR HUMAN PAPILLOMAVIRUS (HPV) TYPE 16 (HPV-16),” the contents of which are incorporated by reference in their entirety.

Field

[0002] The present disclosure relates in some aspects to a method of detecting human papillomavirus in a subject. The present disclosure also provides a method for diagnosing and treating human papillomavirus.

Incorporation By Reference of Sequence Listing

[0003] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 735042025240SeqList.xml, created April 3, 2023, which is 80,500 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

Background

[0004] Various strategies are available for the detection of human papillomavirus (HPV) in a subject. New assays are needed to improve the specificity and sensitivity of detection of the virus. Provided is a method that meet such needs.

Summary

[0005] Provided herein is a method of detecting human papillomavirus (HPV) in a subject, the method comprising (a) incubating a template DNA obtained from a biological sample in a first incubation with (i) an oligonucleotide set comprising a forward oligonucleotide primer, a reverse oligonucleotide primer, and an oligonucleotide probe and (ii) a DNA polymerase to produce a first pre- amplification reaction product; (b) incubating the first pre-amplification reaction product or sample thereof in a second incubation with the (i) the oligonucleotide set comprising the forward oligonucleotide primer, the reverse oligonucleotide primer, and the oligonucleotide probe and (ii) a DNA polymerase to produce a secondary reaction product, wherein the incubating in (a) and the incubating in (b) is under conditions sufficient for amplification of a HPV16 amplicon if present in the sample; and (c) detecting the generated HPV16 amplicon.

[0006] Provided herein is a method of amplifying a HPV16 amplicon, the method comprising: (a) incubating a template DNA obtained from a biological sample in a first incubation with (i) an oligonucleotide set comprising a forward oligonucleotide primer, a reverse oligonucleotide primer, and an oligonucleotide probe, and (ii) a DNA polymerase to produce a first pre-amplification reaction product; (b) incubating the first pre-amplification reaction product or sample thereof in a second incubation with (i) the oligonucleotide set comprising the forward oligonucleotide primer, the reverse oligonucleotide primer, and the oligonucleotide probe, and (ii) a DNA polymerase to produce a secondary reaction product, wherein the incubating in (a) and the incubating in (b) is under conditions sufficient for amplification of a HPV16 amplicon if present in the sample.

[0007] In some of any embodiments, the HPV16 amplicon is an HPV16 E7 amplicon. In some of any embodiments, the first incubation and the second incubation individually is performed by polymerase chain reaction (PCR). In some embodiment, the PCR is real-time PCR (RT-PCR). In some of any embodiments, the template DNA is complementary DNA (cDNA). In some of any embodiments, prior to the incubating in (a), the method comprises synthesizing the template DNA by reverse-transcribing an RNA template from the biological sample into cDNA. In some of any embodiments, the DNA polymerase is a Taq DNA polymerase.

[0008] In some of any embodiments, the HPV16 amplicon is less than 120 base pairs. In some of any embodiments, the HPV16 amplicon is between 60 and 119 base pairs, inclusive. In some of any embodiments, the HPV16 amplicon is between 70 and 119 base pairs, inclusive. In some of any embodiments, the HPV16 amplicon is between 80 and 119 base pairs, inclusive. In some of any embodiments, the HPV16 amplicon is between 80 and 117 base pairs, inclusive. In some of any embodiments, the (i) the forward oligonucleotide primer comprises 15-30 nucleotides that are complementary to a plus strand of HPV16 E7 template DNA, and (ii) the reverse oligonucleotide primer comprises 15-30 nucleotides that are complementary to a minus strand of HPV16 E7 template DNA. In some of any embodiments, the (i) the forward oligonucleotide primer comprises 18-25 nucleotides that are complementary to a plus strand of HPV16 E7 template DNA, and (ii) the reverse oligonucleotide primer comprises 18-25 nucleotides that are complementary to minus strand of HPV16 E7 template DNA.

[0009] In some of any embodiments, the forward oligonucleotide primer comprises a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to any one of SEQ ID NOs: 3, 6, 9, 12 or 15. In some of any embodiments, the forward oligonucleotide primer comprises the sequence set forth in any of SEQ ID NOs: 3, 6, 9, 12 or 15. In some of any embodiments, the reverse oligonucleotide primer comprises a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to any one of SEQ ID NOs: 4, 7, 10, 13 or 16. In some of any embodiments, the reverse oligonucleotide primer comprises the sequence set forth in any of SEQ ID NOs: 4, 7, 10, 13 or 16. In some of any embodiments, the oligonucleotide probe comprises a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to any one of SEQ ID NOs: 5, 8, 11, 14 or 17. In some of any embodiments, the oligonucleotide probe comprises one of any of the sequences as set forth in SEQ ID NOs: 5, 8, 11, 14 or 17.

[0010] In some of any embodiments, the HPV16 amplicon detected is at or about 75-90 base pairs, optionally at or about 80 base pairs. In some of any embodiments, the forward oligonucleotide primer is set forth in SEQ ID NO: 9 and the reverse oligonucleotide primer is set forth in SEQ ID NO: 10. In some of any embodiments, the forward oligonucleotide primer is set forth in SEQ ID NO: 9, the reverse oligonucleotide primer is set forth in SEQ ID NO: 10 and the oligonucleotide probe is set forth in SEQ ID NO: 11. In some of any embodiments, the forward oligonucleotide primer is set forth in SEQ ID NO: 12 and the reverse oligonucleotide primer is set forth in SEQ ID NO: 13.

[0011] In some of any embodiments, the forward oligonucleotide primer is set forth in SEQ ID NO: 12, the reverse oligonucleotide primer is set forth in SEQ ID NO: 13 and the oligonucleotide probe is set forth in SEQ ID NO: 14. In some of any embodiments, the forward oligonucleotide primer is set forth in SEQ ID NO: 15 and the reverse oligonucleotide primer is set forth in SEQ ID NO: 16. In some of any embodiments, the forward oligonucleotide primer is set forth in SEQ ID NO: 15, the reverse oligonucleotide primer is set forth in SEQ ID NO: 16 and the oligonucleotide probe is set forth in SEQ ID NO: 17. [0012] In some of any embodiments, the HPV16 amplicon is 95 to 105 base pairs, optionally at or about 100 base pairs. In some of any embodiments, the forward oligonucleotide primer is set forth in SEQ ID NO: 6 and the reverse oligonucleotide primer is set forth in SEQ ID NO: 7. In some of any embodiments, the forward oligonucleotide primer is set forth in SEQ ID NO: 6, the reverse oligonucleotide primer is set forth in SEQ ID NO: 7 and the oligonucleotide probe is set forth in SEQ ID NO:8. In some of any embodiments, the HPV16 the amplicon is at or about 110 to 140 base pairs, optionally at or about 117 base pairs. In some of any embodiments, the forward oligonucleotide primer is set forth in SEQ ID NO: 3 and the reverse oligonucleotide primer is set forth in SEQ ID NO: 4. In some of any embodiments, the forward oligonucleotide primer is set forth in SEQ ID NO: 3, the reverse oligonucleotide primer is set forth in SEQ ID NO: 4 and the oligonucleotide probe is set forth in SEQ ID NO:5.

[0013] In some of any embodiments, the oligonucleotide probe comprises a detectable moiety. In some embodiment, the detectable moiety is a fluorescent moiety. In some of any embodiments, the HPV16 amplicon is detected by measuring a detectable signal emitted from the detectable moiety of the second reaction product. In some embodiment, the detectable signal is a fluorescent signal. In some of any embodiment, detecting the HPV16 amplicon comprises determining the quantification cycle (Cq) at which the detectable signal, optionally the fluorescent signal, exceeds background fluorescence.

[0014] In some of any embodiments, the concentration of the oligonucleotide set in the first incubation step is less than the concentration in the second incubation step. In some embodiment, the concentration of the oligonucleotide set in the first incubation is step is between 0.05-fold and 0.001-fold of the concentration in the second incubation step. In some of any embodiments, the concentration of the oligonucleotide set in the first incubation is step is between 0.01-fold and 0.002-fold of the concentration in the second incubation step. In some of any embodiments, the concentration of the oligonucleotide set in the first incubation is step is at or about 0.0025-fold the concentration in the second incubation step.

[0015] In some of any embodiments, the first incubation step comprises thermal profile conditions set forth as: 10 seconds at 95 °C followed by 10 cycles of amplification (95 °C for 15 seconds and 60°C for 4 minutes). In some of any embodiments, the second incubation step comprises thermal profile conditions set forth as: 50°C for 2 minutes, 95°C for 20 seconds, then 40 cycles of amplification comprising 95 °C for 1 second and 60°C for 20 seconds. In some of any embodiments, the subject has, or is suspected of having head and neck cancer. In some of any embodiments, the head and neck cancer is squamous cell carcinoma (HNSCC). In some of any embodiments, the biological sample is a tissue, a buccal sample, a saliva sample, or a blood sample. In some embodiment, the tissue sample is a tumor tissue. In some of any embodiments, the sample is a tissue sample that has been formalin fixed and paraffin embedded.

[0016] In some of any embodiments, a method of diagnosing human papillomavirus (HPV) in a subject comprises (i) detecting a generated HPV16 amplicon; and (ii) diagnosing a subject with HPV if a detectable amount of HPV is present in the sample. In some embodiment, the detectable amount of HPV is determined by the quantification cycle (Cq).

[0017] Provided herein is an oligonucleotide set comprising a primer pair, comprising (i) a forward oligonucleotide primer comprising a nucleic acid sequence set forth in any of SEQ ID NOs: 6, 9, or 12, and (ii) a reverse oligonucleotide primer comprising a nucleic acid sequence set forth in any of SEQ ID NOs: 7, 10, or 13. In some embodiment, the oligonucleotide set further comprises an oligonucleotide probe set forth in any one of SEQ ID NOs: 5, 8, 11, 14 or 17. In some of any embodiments, (i) the forward oligonucleotide primer comprises a nucleic acid sequence set forth in SEQ ID NO: 6, and (ii) the reverse oligonucleotide primer comprises a nucleic acid sequence set forth in SEQ ID NO: 7. In some embodiment, the oligonucleotide set comprises the oligonucleotide probe is set forth in SEQ ID NO:8.

[0018] In some of any embodiments, (i) the forward oligonucleotide primer comprises a nucleic acid sequence set forth in SEQ ID NO: 9, and (ii) the reverse oligonucleotide primer comprises a nucleic acid sequence set forth in SEQ ID NO: 10. In some embodiment, the oligonucleotide set comprises the oligonucleotide probe is set forth in SEQ ID NO: 11. In some of any embodiments, (i) the forward oligonucleotide primer comprises a nucleic acid sequence set forth in SEQ ID NO: 12, and (ii) the reverse oligonucleotide primer comprises a nucleic acid sequence set forth in SEQ ID NO: 13. In some embodiment, the oligonucleotide set comprises the oligonucleotide probe is set forth in SEQ ID NO: 14. In some of any embodiments, (i) the forward oligonucleotide primer comprises a nucleic acid sequence set forth in SEQ ID NO: 15, and (ii) the reverse oligonucleotide primer comprises a nucleic acid sequence set forth in SEQ ID NO: 16. In some embodiment, the oligonucleotide set comprises the oligonucleotide probe is set forth in SEQ ID NO: 17. In some of any embodiments, (i) the forward oligonucleotide primer comprises a nucleic acid sequence set forth in SEQ ID NO: 3, and (ii) the reverse oligonucleotide primer comprises a nucleic acid sequence set forth in SEQ ID NO: 4. In some embodiment, the oligonucleotide set comprises the oligonucleotide probe is set forth in SEQ ID NO:5.

[0019] In some of any embodiments, the oligonucleotide probe comprises a detectable moiety. In some embodiment, the detectable moiety is a fluorescent moiety. In some of any embodiments, the detectable moiety is at the 5’ end of the oligonucleotide probe. In some of any embodiments, the detectable moiety is selected from FAM, HEX, FITC, Texas Red, TET, JOE, VIC, NED, TAMRA, ROX, ABY, PET, JUN, LIZ, Cy3, or Cy5. In some of any embodiments, the oligonucleotide probe comprises a minor groove binder (MGB) moiety, optionally wherein the MGB is at the 3’ end. In some of any embodiments, the oligonucleotide probe comprises a nonfluorescent quencher (NFQ), optionally wherein the NFQ is at the 3’ end. In some of any embodiments, the detectable moiety is FAM-MGB.

[0020] In some of any embodiments, a kit comprises the oligonucleotide set and one or more reagents for carrying out a polymerase chain reaction. In some embodiment, the one or more reagents comprises a DNA polymerase. In some embodiment, the DNA polymerase is a Taq polymerase. In some of any embodiments, a method of treatment comprises administering a therapeutic to a subject in need thereof for treating an human papillomavirus (HPV) infection, wherein: the subject is selected for treatment if an HPV16 amplicon is detected in a sample obtained from said subject. In some of any embodiments, a method of treatment comprises: selecting a subject in which an HPV16 amplicon is detected; and administering to the selected subject a therapeutic agent for treating an human papillomavirus (HPV) infection. In some embodiment, a method of treatment comprises administering a therapeutic to a subject in need thereof for treating an human papillomavirus (HPV) infection, wherein the subject is diagnosed with an HPV infection.

In some of any embodiments, the therapeutic for treating an HPV infection is selected from the group consisting of vaccines that induce or boost HPV T cell adaptive immunity, adoptive cell therapy, therapeutic antibodies, antiviral therapeutics, immune response modifier compounds, proteasome inhibitors, HD AC inhibitors, and drugs targeting HPV genes. In some of any embodiments, the therapeutic is a T cell therapy comprising a T cells expressing a recombinant antigen receptor specific to an HPV16 epitope. In some embodiment, the HPV 16 epitope is an HVP16 E7 epitope. In some embodiment, the HPV 16 epitope is HPV E7 (11-19). In some of any embodiments, the subject has or is suspected of having a cancer. In some of any embodiments, the subject has, or is suspected of having head and neck cancer. In some embodiment, the head and neck cancer is squamous cell carcinoma (HNSCC).

Brief Description of the Drawings

[0021] FIG. 1 depicts a workflow diagram illustrating the head and neck squamous cell carcinoma (HNSCC) FFPE patient sample processing and analysis workflow described in the Examples.

[0022] FIGs. 2A-C show pl6 immunohistochemistry (IHC) staining of head and neck squamous cell carcinoma (HNSCC) tumor tissue samples. FIG. 2A shows an exemplary tumor tissue sample demonstrating approximately 100% positive staining for pl6. FIG. 2B shows an exemplary tumor tissue sample showing approximately 75-80% positive staining for pl6, indicative of a borderline positive score. FIG. 2C depicts an exemplary tumor tissue sample showing approximately 0% positive staining (i.e., negative staining) for pl 6, indicative of a negative pl6 score.

[0023] FIG. 3 shows a correlation of Cq values of the reference RNA-PCR assay compared to the Real-Time RNA-PCR Assay for each of 25 positive samples are plotted for correlation. Pearson correlation coefficient is 0.96, p<0.0001

[0024] FIG. 4 depicts a pair- wise comparison of Cq values from the reference RNA-PCR assay and the Real-Time RNA-PCR Assay for each of 25 positive samples. Cq values of the Real-Time RNA-PCR Assay are significantly lower than that of the reference RNA-PCR assay, p<0.0001.

Detailed Description

[0025] Provided herein is a method of detecting, diagnosing, and treating human papillomavirus (HPV) in a subject. In certain embodiments, the method comprises incubating DNA from a sample in a first incubation step to produce a first pre-amplification reaction product. In certain embodiments, the method comprises incubating the first preamplification reaction product in a second incubation step to produce a secondary reaction product. In certain embodiments, the incubating in the first and second incubation steps is under conditions sufficient for amplification of a HPV 16 amplicon if present in the sample. In certain embodiments, the method disclosed herein comprises detecting the generated HPV16 amplicon.

[0026] HPV is a causative organism in most cases of cervical cancer and has been implicated in anal, vaginal, vulvar, penile, and oropharyngeal cancers, and other cancers. Generally, the HPV genome contains an early region containing six open reading frames (El, E2, E4, E5, E6 and E7), which encode proteins involved in cell transformation and replication, and a late region containing two open reading frames (LI and L2), which encode proteins of the viral capsid. In general, E6 and E7 are oncogenes that can affect cell cycle regulation and contribute to the formation of cancers. For instance, the E6 gene product can cause p53 degradation and the E7 gene product can cause retinoblastoma (Rb) inactivation.

[0027] High risk human papillomavirus (HPV), particularly type 16 (HPV-16), is established as one of the drivers for head and neck squamous cell carcinomas (HNSCC) (Gillison ML et al., Evidence for a causal association between human papillomavirus and a subset of head and neck cancers. J Natl Cancer Inst. 2000;92(9):709-20). Numerous studies have identified that HPV positivity in HNSCC is correlated with favorable prognosis (Posner MR et al., Survival and human papillomavirus in oropharynx cancer in TAX 324: a subset analysis from an international phase III trial. Ann Oncol. 2011; 22(5): 1071-1077; Ang KK et al., Human Papilloma virus and Survival of Patients with Oropharyngeal Cancer. N Engl J Med. 2010; 363(1): 24-35), which indicates that HPV-related HNSCC is a biological and clinical variant of the disease. Clinical trials are evaluating possible de-escalation of therapy in this patient population due to improved treatment-sensitivity and overall survival rate of the patients (Mirghani H et al. Treatment de-escalation in HPV-positive oropharyngeal carcinoma: Ongoing trials, critical issues and perspectives. Int J Cancer. 2015;136(7):1494- 1503).

[0028] The difference in prognosis and possible influence in patient management makes accurate diagnosis of HPV status in HNSCC critically important. This need becomes more compelling with emergence of genetically engineered T-cell therapy for advanced HNSCC because this patient population is generally incurable by and resistant to chemotherapy (O’Sullivan B et al. Development and validation of a staging system for HPV-related oropharyngeal cancer by the International Collaboration on Oropharyngeal cancer Network for Staging (ICON-S): A multicentre cohort study. Lancet Oncol. 2016;17(4):440-451). HPV-positive tumors express HPV E6 and E7 oncoproteins which are absent in healthy tissues (Trimble CL, Frazer IH. Development of therapeutic HPV vaccines. Lancet Oncol. 2009; 10(10):975-980), making them attractive targets for genetically engineered T-cells (Doran SL et al. T-Cell Receptor Gene Therapy for Human Papillomavirus-Associated Epithelial Cancers: A First-in-Human, Phase Eli Study. J Clin Oncol. 2019;37(30):2759- 2768). The growing importance of diagnosis of HPV-positive cancer accelerates inclusion of HPV testing in clinical practice. The College of American Pathologists has recently recommended routine HPV testing as part of standard pathologic evaluation of resected oropharyngeal squamous cell carcinomas, a subset of HNSCC (Lewis JS et al. HPV Testing Head & Neck Carcinomas: CAP Guideline. Arch Pathol Lab Med. 2018;142:559-597).

[0029] Despite the need for reliable determination of HPV status, there is not yet a standard method for HPV diagnosis in HNSCC. Methods of HPV testing across laboratories vary considerably not only in assay technologies, but also in the detection targets. Since active transcription of E6 and E7 is required for carcinogenesis, detection of E6 or E7 transcripts by RNA PCR is considered as the gold standard approach (Boscolo-Rizzo P, Pawlita M, Holzinger D. From HPV-positive towards HPV-driven oropharyngeal squamous cell carcinomas. Cancer Treat Rev. 2016;42(l):24-29). Conversely, detection of a surrogate marker, pl6 protein by immunohistochemistry (IHC) is a more common method in clinical laboratories for HPV diagnosis. Although the method is better adopted, it was reported that a significant proportion of patients that exhibited pl6 positivity by IHC was negative for HPV DNA by in-situ hybridization (ISH) or PCR assays (Rietbergen MM et al. Molecular characterization of pl6-immunopositive but HPV DNA-negative oropharyngeal carcinomas. Int J Cancer. 2014; 134(10): 2366-2372; Holzinger D et al., Viral RNA patterns and high viral load reliably define oropharynx carcinomas with active HPV 16 involvement. Cancer Res. 2012;72(19):4993-5003). Meanwhile, HPV DNA genotyping methods such as ISH or PCR are more sensitive than pl6 IHC, but the analytical specificity is relatively poor (Smeets SJ et al., A novel algorithm for reliable detection of human papillomavirus in paraffin embedded head and neck cancer specimen. Int J Cancer. 2007; 121( 11): 2465-2472).

[0030] One major challenge in HPV RNA-PCR assay development is the highly fragmented RNA from Formalin Fixed Paraffin Embedded (FFPE) tissue samples. The challenge is further exacerbated by insufficient tumor content that is frequently encountered in clinical samples. An assay is needed that increases the detection sensitivity for FFPE- derived RNA with low amount and/or low quality. The embodiments provided herein provided improved methods for detecting and diagnosing HPV.

[0031] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0032] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

I. METHODS OF DETECTION AND DIAGNOSIS OF HPV

[0033] Provided herein is a method of detecting human papillomavirus (HPV) in a subject. Also provided herein is a method of diagnosing HPV in a subject. Further provided herein is a method of treating HPV in a subject in which an HPV 16 amplicon has been detected.

[0034] In some embodiments, the methods include amplifying a HPV 16 amplicon if present in a sample, by a method comprising: (a) incubating a template DNA obtained from a biological sample in a first incubation with (i) an oligonucleotide set comprising a forward oligonucleotide primer, a reverse oligonucleotide primer, and an oligonucleotide probe, and (ii) a DNA polymerase to produce a first pre-amplification reaction product; (b) incubating the first pre-amplification reaction product or sample thereof in a second incubation with (i) the oligonucleotide set comprising the forward oligonucleotide primer, the reverse oligonucleotide primer, and the oligonucleotide probe, and (ii) a DNA polymerase to produce a secondary reaction product. In som embodiments, the incubating in (a) and the incubating in (b) is under conditions sufficient for amplification of a HPV 16 amplicon if present in the sample . In some embodiments, the methods can further include detecting the HPV16 amplicon amplified by the provide method.

[0035] In particular embodiments, the provided methods include a first incubation that is a pre-amplification. Pre-amplification is a technique designed to amplify (e.g., by PCR) target nucleic acids prior to downstream analysis. Downstream analysis techniques include quantitative assays such as qPCR (e.g., digital PCR or real time PCR). For example, in the provided methods, a sample comprising DNA template nucleic acids are mixed with (i) DNA primers designed to amplify a target nucleic acid segment of HPV16, such as HPV16 E7, (ii) a DNA polymerase such as Taq or another amplification enzyme, and (iii) appropriate reagents (e.g., dNTPs, buffer, etc.). The amplified region by the preamplification includes sequences containing the amplicon. In some embodiments, the reaction product of the preamplification may consist essentially of the amplicon or may alternatively include a larger product that flanks the amplicon on either or both of the 5' and 3' end. After preamplification, the product (optionally diluted or processed to separate the amplification product) is added to the downstream analysis reaction in a second incubation. In some embodiments, the pre-amplification step increases the amount of the targeted nucleic acid available for further analysis, and is thus useful for detection, quantification, and analysis of rare sequences.

[0036] In some embodiment, the provided methods include a first incubation as a preamplification reaction that includes: (i) an HPV16 DNA template from a sample suspected of containing the target nucleic acid; (ii) an oligonucleotide set containing amplification primers and probe specific for the DNA template; and (ii) appropriate reagents (e.g. DNA polymerase, nucleotides, buffer, etc.). The pre-amplification reaction is then carried out to produce a product. Assuming the pre-amplification worked, the product would include amplification products of the target nucleic acids from the sample. The product of the first amplification can be added to a single reaction mixture for the second nucleic acid analysis in a second amplification incubation. For example, the second incubation may be carried out under conditions for amplification of the pre-amplification product by qPCR, in which the reaction mixture can include primers and probes specific for the amplification product of the first nucleic acid template and appropriate reagents (e.g. DNA polymerase, nucleotides, buffer, etc..).

[0037] Thus, in provided embodiments, the method comprises incubating DNA from a sample in a first incubation step with an oligonucleotide set comprising a forward primer, a reverse primer and an oligonucleotide probe under conditions to produce a first preamplification reaction product. Then, the method includes incubating the first preamplification reaction product in a second incubation step with the oligonucleotide set comprising the forward primer, the reverse primer and the oligonucleotide probe to produce a secondary reaction product. In certain embodiments, the incubating in the first and second incubation steps is under conditions sufficient for amplification of a HPV16 amplicon if present in the sample. In certain embodiments, the method disclosed herein comprises detecting the generated HPV16 amplicon. In particular embodiments, the HPV16 amplicon is an amplicon from HPV16 E7.

[0038] Primers for the pre-amplification of step in the first incubation and amplification in the second incubation (e.g. qPCR reaction) can be the same, i.e. primers which are specific for the amplicon of interest. However, in some embodiments, the approach of “nested PCR” can also be used, wherein the amplified DNA stretch in the pre-amplification step is longer than the amplicon targeted for amplification in the second incubation (e.g. qPCR).

[0039] In some embodiments, the methods more specifically are carried out by polymerase chain reaction (PCR), such as real-time PCR (RT-PCR). In some embodiments, the RT-PCR is quantitative PCR (qPCR) . In some embodiments, the first incubation amplifies by PCR a pre-amplification product of the HPV16 gene, such as a pre-amplification product of HPV16 E7. In some embodiments, the second incubation amplifies by PCR an HPV16 amplicon derived from an HPV16 gene, such as an HPV16 E7 amplicon.

[0040] Provided methods also include methods of diagnosing a subject with an HPV infection. For instance, a subject is diagnosed as having an HPV infection if the HPV16 amplicon, such as HPV 16 E7 amplicon, is detected. In particular embodiments, the provided methods provide for greater assay sensitivity as compared to other methods, which is advantageous in screening clinical samples where the copy number of HPV may be low. Because the physical manifestations of HPV infection are often covert and the latency period prolonged, infection with HPV may not be detected until the patient has been diagnosed with cervical intraepithelial neoplasia (CIN), which, if allowed to go untreated, can progress to carcinoma. Typically, higher grade lesions (CIN2, CIN3 and carcinoma) are associated with high HPV copy number, which may be detectable by traditional methods known in the art. However, many assays currently in use are not sensitive or specific enough to detect low copy number HPV. Thus, the provided methods offer advantages for early detection of HPV when HPV copy numbers are low and therapeutic intervention is more likely to be effective.

A. Sample

[0041] The method disclosed herein comprises detecting HPV amplicon in a biological sample obtained from a subject. In certain embodiments, a biological sample is obtained from a subject who has or who is suspected of having HPV. [0042] In some embodiments, a biological sample includes, but is not limited to, sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histologic purposes, blood and blood fractions or products (e.g., serum, plasma, platelets, red blood cells, and the like), sputum or saliva, lymph and tongue tissue, cultured cells, e.g., primary cultures, explants, and transformed cells, stool, urine, etc. A biological sample is typically obtained from a eukaryotic organism, most preferably a mammal such as a primate e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish. In particular embodiments, the biological sample is from a human subject.

[0043] In certain embodiments, the biological sample is a sample of tissue, serum, or plasma. In some embodiments, the sample can be a whole blood sample, a partially purified blood sample, a peripheral blood sample, a serum sample, a cell sample or a lymph node sample. In some embodiments, the sample can be peripheral blood mononuclear cells (PBMC).

[0044] In a specific embodiment, the sample is a tissue biopsy. The biopsy can be from any organ or tissue, for example, skin, liver, lung, heart, colon, kidney, bone marrow, teeth, lymph node, hair, spleen, brain, breast, or other organs. Any biopsy technique known by those skilled in the art can be used for isolating a sample from a subject, for instance, open biopsy, close biopsy, core biopsy, incisional biopsy, excisional biopsy, or fine needle aspiration biopsy. In some embodiments, the sample is a lymph node biopsy. In some embodiments, the sample can be a frozen tissue sample. In some embodiments, the sample can be a formalin-fixed paraffin-embedded (“FFPE”) tissue sample. In some embodiments, the sample can be a deparaffinised tissue section.

[0045] In certain embodiments, the tissue biopsy can be tissue derived from healthy tissue. In certain embodiments, the sample is obtained from the head or neck of the subject. In certain embodiments, the sample is obtained from the throat, larynx, nasal cavity, sinuses, mouth, tongue, or salivary gland of a subject. In certain embodiments, the sample is a tumor biopsy. In certain embodiments, the tumor is a sarcoma or a carcinoma. In certain embodiments, the tumor biopsy is obtained from a solid tumor.

[0046] In some embodiments, mRNA can be isolated from the sample. In some embodiments, the sample can be a tissue sample. In a specific embodiment, the tissue sample can be a tumor biopsy. General methods for mRNA extraction are well known in the art and are disclosed in standard textbooks of molecular biology, including Ausubel et al.. Current Protocols of Molecular Biology. John Wiley and Sons (1997). In particular, RNA isolation can be performed using a purification kit, buffer set, and protease from commercial manufacturers, such as Roche or Qiagen, according to the manufacturer's instructions. For example, total RNA from cells in culture can be isolated using High Pure FFPE RNA Isolation kit (Roche, Indianapolis, IN). Other commercially available RNA isolation kits include M ASTERPURE® Complete DN.A and RNA Purification Kit (EPICENTRE®, Madison. Wis.), and Paraffin Block RNA Isolation Kit (Ambion. Inc.). Total RNA from tissue samples can be isolated using RNA Stat-60 (Tel-Test). RNA prepared from tumors can be isolated, for example, by cesium chloride density gradient centrifugation. In certain embodiments, the RNA is extracted from a tissue section of 2 μm.3 μm.4 μm.5 μm. 6 μm.7 μm.8 μm. 9 μm. or 10 pm thickness. In a specific embodiment the RNA is extracted from a tissue sample of 5 pm thick.

[0047] In some embodiments, reverse transcription of the extracted RNA is performed to produce cDNA. In certain embodiments, a combined reverse-transcription-polymerase chain reaction (RT-PCR) reaction may be used. For example, extracted RNA can be reverse- transcribed using a High Capacity RNA to cDNA Reverse Transcription Kit (Thermo Fisher, Foster City, CA), following the manufacturer's instructions. In certain embodiments, the amount of RNA used in the RT-PCR reaction can be 10 ng, 15 ng, 20 ng, 25 ng, 30 ng, 35 ng, 40 ng, 45 ng, or 50 ng. In certain embodiments, the amount of RNA used in the RT-PCR reaction is between 10 ng and 20 ng, between 15 ng and 25 ng, between 20 ng and 30 ng, between 25 ng and 35 ng, between 30 ng and 40 ng, between 35 ng and 45 ng, or 40 ng and 50 ng. In a specific embodiment, the amount of RNA used in the RT-PCR reaction is between 25 ng and 35 ng. In a specific embodiment, the amount of RNA used in the RT- PCR reaction is 30 ng.

[0048] In certain embodiments, the volume of the RT-PCR reaction is between 10 pL and 30 pL. In some embodiments, the volume of the RT-PCR reaction is 10 μL, 11 μL, 12 μL, 13 μL, 14 μL, 15 μL, 16 μL, 17 μL, 18 μL, 19 μL, 20 μL, 21 μL, 22 μL, 23 μL, 24 μL, 25 μL, 26 μL, 27 μL, 28 μL, 29 μL, or 30 pL. In some embodiments, the volume of the RT- PCR reaction is between 10 pL and 12 μL, 11 pL and 13 μL, 12 pL and 14 μL, 13 pL and 15 μL, 14 pL and 16 μL, 15 pL and 17 μL, 16 pL and 18 μL, 17 pL and 19 μL, 18 pL and 20 μL, 19 pL and 21 μL, 20 pL and 22 μL, 21 pL and 23 μL, 22 pL and 24 μL, 23 pL and 25 μL, 24 pL and 26 μL, 25 pL and 27 |aL, 26 |iL and 28 |iL, 27 |aL and 29 |iL, or 28 |iL and 30 μL,. In a specific embodiment, the volume of the RT-PCR reaction is 20 pL.

[0049] In certain embodiments, the derived cDNA can be used as a template for a subsequent PCR reaction. In certain embodiments, the derived cDNA is incubated in a first incubation step with a forward oligonucleotide primer, a reverse oligonucleotide primer, and a Taq DNA polymerase to produce a first pre-amp lification reaction product.

7 ffPV Virus

[0050] Provided herein is a method of detecting human papillomavirus (HPV) in a subject. Also provided herein is a method of diagnosing HPV in a subject. Also provided herein is a method of treating HPV in a subject. In certain embodiments, the HPV detected, diagnosed, or treated by the method described herein is an alpha-HPV, beta-HPV, gamma- HPV, mu-HPV, or nu-HPV. In a specific embodiment, the HPV detected, diagnosed, or treated by the method described herein is an alpha-HPV.

[0051] Of the 65 HPV types belonging to Alphapapillomavirus (alpha-HPV types), a subset of viruses (e.g., HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59) are considered oncogenic and associated with the development of cervical cancer and its precursor lesions (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2012, Schiffman et al., 2016). These HPV types are the etiological agents of several cancers, such as those of the cervix, vagina, vulva, anus, penis, and a subset of head and neck cancers (HNCs). In a certain embodiment, the HPV detected, diagnosed, or treated by the method described herein is a HPV type 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59. In a specific embodiment, the HPV detected, diagnosed, or treated by the method described herein is a HPV 16.

[0052] The key drivers of HPV-mediated transformation are the oncoproteins E5, E6 and E7. In certain embodiments, the method described herein detects or diagnoses HPV in a subject by way of the detection of contiguous region of nucleotides of HPV 16. In certain embodiments, the method described herein detects a contiguous region of nucleotides of HPV16 E5. In certain embodiments, the method described herein detects a contiguous region of nucleotides of HPV16 E6. In certain embodiments, the method described herein detects a contiguous region of nucleotides of HPV 16 E7.

[0053] Provided herein is a method of detecting, diagnosing, or treating human papillomavirus (HPV) in a subject comprising incubating the HPV DNA, if present, obtained from a sample. In certain embodiments, the DNA is incubated in a first incubation step. In certain embodiments, the first incubation step comprises incubation of DNA, a forward oligonucleotide primer, a reverse oligonucleotide primer, and a DNA polymerase to produce a first pre-amplification reaction product.

B. Amplification

[0054] In some embodiments, the provided methods include amplifying, such as by PCR, nucleic acid from a DNA template in the presence of a DNA polymerase and an oligonucleotide set in a first incubation to produce a pre-amplification product and in a second incubation to produce an HPV16 amplicon. In some embodiments, in the first and second incubations the oligonucleotide set is the same. In some embodiments, each oligonucleotide set is composed of (a) a forward primer able to hybridize to a first location (first region) of a nucleic acid sequence of an HPV16 gene, (b) a reverse primer able to hybridize to a second location (second region) of the nucleic acid sequence of the HPV16 gene downstream of the first location, and (c) a probe containing a detectable moiety that is able to emit or produce a detectable signal, in which the probe is able to hybridize to a location of the nucleic acid sequence of the HPV gene between the first and the second locations. In some embodiments the detectable moiety is a fluorescent moiety or fluorophore that emits energy at a unique emission maxima. In some embodiments, the probe is labeled with a quencher molecule.

[0055] In some embodiments of the method provided herein, the oligonucleotide sets specifically hybridize to the E7 genes of HPV. In some embodiments, the oligonucleotide primers and probe of the oligonucleotide set are able to hybridize to the E7 gene. Thus, in aspects of the provided methods, a pre-amplification product of an HPV 16 E7 gene is produced by the first incubation, and the second incubation amplifies an HPV 16 E7 amplicon.

[0056] In some embodiments, the sample is positive for HPV if the HPV 16 amplicion (e.g. HPV16 E7) amplicon is detected. The generation of the HPV16 amplicon can be detected when a change of detectable signal of the generated amplicon is detected. In some embodiments, the detectable signal is generated from the detectable moiety on the probe present in the oligonucleotide sets during the incubation. In some embodiments, the change of the detectable signal (e.g. fluorescence) corresponds to the occurrence of nucleic acid amplification. In some embodiments, a sample is positive for the HPV if a detectable change of the detectable signal is measured, such as a change greater than the background signal or a pre-determined threshold.

[0057] In some embodiments, the DNA polymerase is characterized by the degradation of double-stranded DNA encountered during extension by the PCR primer pairs. The probe carrying a detectable moiety (e.g. fluorescent moiety) also is annealed to the amplicon during the reaction and will be degraded in a similar manner, thus releasing the detectable moiety (e.g. fluorophore) from the oligonucleotide. In some embodiments, the probe also contains a quencher. When the probe is intact and the detectable moiety (e.g. fluorophore) is in close proximity to the quencher dye, little to no detectable signal (e.g. fluorescence) will result because of suppression of the detectable signal due to an energy transfer between the two dyes. During polymerization and extension, strand synthesis will begin to displace the probe that have hybridized to the target sequence. Upon release of the probe, the detectable moiety (e.g. flurophore) and the quencher become dissociated so that the detectable signal (e.g. fluorescence emitted by the fluorophore) is no longer quenched, which results in a detectable change in the detectable signal.

[0058] In embodiments of the provided methods, the generated HPV16 amplicon can be detected by detection of the detectable signal. For instance, during exponential growth of the PCR product, the amplicon- specific detectable signal (e.g. fluorescence) increases to a point at which the sequence detection can distinguish it from the background fluorescence of nonamplifying samples. In some cases, a detection of a positive product can be determined by the quantification cycle (Cq) for the samples, which is the cycle at which this detetable signal (e.g. fluorescence) increases above a pre-determined threshold or background.

[0059] The method disclosed herein comprises a first and second incubation step. In certain embodiments, the first incubation step comprises the incubation of a first reaction mixture to produce a first pre-amplification reaction product. In certain embodiments, the second incubation step comprises the incubation of a second reaction mixture. In certain embodiments, the second reaction mixture comprises the first pre-amplification reaction product. In certain embodiments, the incubation in the first incubation step and the second incubation step is under conditions sufficient for amplification of a HPV16 amplicon if present in the sample. [0060] The method described herein comprises a first and second incubation step. In certain embodiments, one or more of the incubation steps comprises a polymerase chain reaction.

/. Oligonucleotide Sets, Primer and Prole Oligonucleotides

[0061] In some embodiments, the provided embodiments utilize a set of primers (primer pair) containing a forward oligonucleotide primer and a reverse oligonucleotide primer that are capable of participating in PCR amplification of a segment of nucleic acid of an HPV16 gene in the presence of a DNA polymerase to produce a PCR amplicon. In some embodiments, the primers that comprise the primer pair are specific to HPV16 E7 gene, resulting in an HPV16 E7 amplicon.

[0062] In some embodiments, the primers are able to amplify an HPV16 amplicon composed of a contiguous region of nucleotides of an HPV16 gene, such as HPV16 E7. For instance, the forward primer is able to hybridize to a plus strand of a first region of the DNA template and the second primer is able to hybridize to a minus strand of a second region of the DNA template that is downstream of the first region. In some embodiments, the locations of the first and second primers at which hybridization occurs is such that extension of the primers produces an amplicon that is greater than 60 base pairs in length and less than 120 base pairs in length.

[0063] In certain embodiments, the amplicon is less than 120 base pairs. In certain embodiments, the size of the amplicon is between 60 and 119 base pairs inclusive, 70 and 119 base pairs inclusive, 80 and 119 base pairs inclusive, 80 and 119 base pairs inclusive, 80 and 118 base pairs inclusive, or 80 to 117 base pairs inclusive. In a specific embodiment, the size of the amplicon is between 80 and 117 base pairs, inclusive. In certain embodiments, the size of the amplicon is at or about 80 base pairs. In certain embodiments, the size of the amplicon is at or about 90 base pairs. In certain embodiments, the size of the amplicon is at or about 100 base pairs. In certain embodiments, the size of the amplicon is at or about 110 base pairs. In certain embodiments, the size of the amplicon is at or about 115 base pairs. In certain embodiments, the size of the amplicon is at or about 117 base pairs. In certain embodiments, the size of the amplicon is at or about 120 base pairs.

[0064] In some embodiments, the primers uses for the preamplification step amplifies sequences containing the amplicon. These amplicons may be used for quantitative analysis such as qPCR. In some embodiments the preamplification product amplifies the amplicon sequence. For instance, in some embodiments, the preamplification product consists essentially of the amplicon. In other embodiments, the preamplification product also includes sequences 5' of the amplicon, 3' of the amplicon, or both. In some embodiments, the preamplification product may be about 20 to about 500 nucleotides longer, more generally about 20 to about 200 nucleotides longer, than the corresponding amplicon. In certain embodiments, the preamplification product may be about 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500, or more nucleotides longer than the corresponding amplicon. In other embodiments, the length of the preamplification product is the same as the amplicon.

[0065] In some embodiments, primers used in the preamplification step may be the same as those used to amplify or quantify the amplicon. Alternatively, the 5' forward primer, the 3' reverse primer, or both primers used in the preamplification step may flank, bracket, or nest the corresponding amplicon primers. In one embodiment, the preamplificaion utilizes two primers that flank the corresponding qPCR primers for the corresponding amplicon. In particular embodiments, the preamplification PCR uses the same forward and reverse primer as the primers used in the qPCR.

[0066] In some embodiments, the forward oligonucleotide primer is complementary to, and able to hybridize to, a plus strand of a a first region of the HPV16 DNA template, such as HPV16 E7 DNA template. In some embodiments, the forward oligonucleotide primer is 5-15, 10-20, 15-25, 16-25, 17-25, 18-25, 19-25, 20-30, 25-35, 5-20, 10-25, 15-30, or 20-35 nucleotides in length. In a specific embodiment, the forward oligonucleotide primer is 18-25 nucleotides in length.

[0067] In some embodiments, the reverse oligonucleotide primer is complementary to, and able to hybridize to, a minus strand of a second region of the HPV16 DNA template, such as HPV16 E7 DNA template. In some embodiments, the reverse oligonucleotide primer is 5-15, 10-20, 15-25, 16-25, 17-25, 18-25, 19-25, 20-30, 25-35, 5-20, 10-25, 15-30, or 20-35 nucleotides in length. In a specific embodiments, the reverse oligonucleotide primer is 18-25 nucleotides in length.

[0068] In certain embodiments, the forward and reverse oligonucleotide primers have a melting temperature (Tm) of 45 °C, 50 °C, 55 °C, 56 °C, 57 °C, 58 °C, 59 °C, 60 °C, 61 °C, 62 °C, 63 °C, 64 °C, 65 °C, 70 °C, or 75 °C. In certain embodiments, the forward and reverse oligonucleotide primers have a melting temperature between 45 °C to 60 °C, 50 °C to 65 °C, 55 °C to 70 °C, or 60 °C to 75 °C inclusive. In certain embodiments, the forward and reverse oligonucleotide primers have a melting temperature between 45 °C to 55 °C, 50 °C to 60 °C, 55 °C to 65 °C, 60 °C to 70 °C, or 65 °C to 75 °C inclusive. In certain embodiments, the forward and reverse oligonucleotide primers have a melting temperature between 55 °C to 60 °C, 56 °C to 61 °C, 57 °C to 62 °C, 58 °C to 63 °C, or 59 °C to 64 °C inclusive.

[0069] In some embodiments, the primers and probes specifically hybridize to sequences of an HPV16 E7 DNA template. In some embodiments, HPV16 E7 primers and probes of the provided embodiments do not share full homology with other HPV types.

[0070] In certain embodiments, the forward oligonucleotide primer can comprise a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to any one of SEQ ID NOs: 3, 6, 9, 12, or 15. In certain embodiments, the forward oligonucleotide primer can comprise a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 3. In certain embodiments, the forward oligonucleotide primer can comprise a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 6. In certain embodiments, the forward oligonucleotide primer can comprise a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 9. In certain embodiments, the forward oligonucleotide primer can comprise a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 12. In certain embodiments, the forward oligonucleotide primer can comprise a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 15.

[0071] In certain embodiments, the forward oligonucleotide primer comprises the sequence set forth in any of SEQ ID NOs: 3, 6, 9, 12, or 15. In certain embodiments, the forward oligonucleotide primer comprises the sequence set forth in SEQ ID NO: 3. In certain embodiments, the forward oligonucleotide primer comprises the sequence set forth in SEQ ID NO: 6. In certain embodiments, the forward oligonucleotide primer comprises the sequence set forth in SEQ ID NO: 9. In certain embodiments, the forward oligonucleotide primer comprises the sequence set forth in SEQ ID NO: 12. In certain embodiments, the forward oligonucleotide primer comprises the sequence set forth in SEQ ID NO: 15.

[0072] In certain embodiments, the reverse oligonucleotide primer comprises a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to any one of SEQ ID NOs: 4, 7, 10, 13, or 16. In certain embodiments, the reverse oligonucleotide primer comprises a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 4. In certain embodiments, the reverse oligonucleotide primer comprises a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 7. In certain embodiments, the reverse oligonucleotide primer comprises a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 10. In certain embodiments, the reverse oligonucleotide primer comprises a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 13. In certain embodiments, the reverse oligonucleotide primer comprises a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 16.

[0073] In certain embodiments, the reverse oligonucleotide primer comprises the sequence set forth in any of SEQ ID NOs: 4, 7, 10, 13, or 16. In certain embodiments, the reverse oligonucleotide primer comprises the sequence set forth in SEQ ID NO: 4. In certain embodiments, the reverse oligonucleotide primer comprises the sequence set forth in SEQ ID NO: 7. In certain embodiments, the reverse oligonucleotide primer comprises the sequence set forth in SEQ ID NO: 10. In certain embodiments, the reverse oligonucleotide primer comprises the sequence set forth in SEQ ID NO: 13. In certain embodiments, the reverse oligonucleotide primer comprises the sequence set forth in SEQ ID NO: 16.

[0074] In certain embodiments, the primer pair comprises a forward oligonucleotide primer comprising a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to any one of SEQ ID NOs: 3, 6, 9, 12, or 15, and a reverse nucleotide primer comprising a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to any one of SEQ ID NOs: 4, 7, 10, 13, or 16. In certain embodiments, the primer pair comprises a forward oligonucleotide primer comprising the sequence set forth in any of SEQ ID NOs: 3, 6, 9, 12, or 15, and a reverse oligonucleotide primer comprising the sequence set forth in any one of SEQ ID NOs: 4, 7, 10, 13, or 16.

[0075] In certain embodiments, the primer pair comprises a forward oligonucleotide primer comprising a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 3 and a reverse oligonucleotide primer comprising a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 4. In certain embodiments, the primer pair comprises a forward oligonucleotide primer comprising a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 6 and a reverse oligonucleotide primer comprising a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 7. In certain embodiments, the primer pair comprises a forward oligonucleotide primer comprising a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 9 and a reverse oligonucleotide primer comprising a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 10. In certain embodiments, the primer pair comprises a forward oligonucleotide primer comprising a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 12 and a reverse oligonucleotide primer comprising a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 13. In certain embodiments, the primer pair comprises a forward oligonucleotide primer comprising a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 15 and a reverse oligonucleotide primer comprising a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 16.

[0076] In certain embodiments, the primer pair comprises a forward oligonucleotide primer comprising the sequence set forth in SEQ ID NO: 3, and a reverse oligonucleotide primer comprising the sequence set forth in SEQ ID NO: 4. In certain embodiments, the primer pair comprises a forward oligonucleotide primer comprising the sequence set forth in SEQ ID NO: 6, and a reverse oligonucleotide primer comprising the sequence set forth in SEQ ID NO: 7. In certain embodiments, the primer pair comprises a forward oligonucleotide primer comprising the sequence set forth in SEQ ID NO: 9, and a reverse oligonucleotide primer comprising the sequence set forth in SEQ ID NO: 10. In certain embodiments, the primer pair comprises a forward oligonucleotide primer comprising the sequence set forth in SEQ ID NO: 12, and a reverse oligonucleotide primer comprising the sequence set forth in SEQ ID NO: 13. In certain embodiments, the primer pair comprises a forward oligonucleotide primer comprising the sequence set forth in SEQ ID NO: 15, and a reverse oligonucleotide primer comprising the sequence set forth in SEQ ID NO: 16.

[0077] In some embodiments, the oligonucleotide sets further comprises a probe. In some embodiments, the probe sequence is a sequence that is able to hybridize specifically in the DNA target region of interest between the two PCR primers. [0078] In some embodiments, the probe is designed to have a slightly higher annealing temperature compared to the PCR primers, such that the probe will hybridize after the extension (polymerization) of the primers begin. In certain embodiments, the probe has a melting temperature (Tm) of 50 °C, 55 °C, 56 °C, 57 °C, 58 °C, 59 °C, 60 °C, 61 °C, 62 °C, 63 °C, 64 °C, 65 °C, 70 °C, 75 °C, or 80 °C. In certain embodiments, the probe has a melting temperature between 50 °C to 65 °C, 55 °C to 70 °C, 60 °C to 75 °C, or 65 °C to 80 °C inclusive. In certain embodiments, the probe has a melting temperature between 50 °C to 60 °C, 55 °C to 65 °C, 60 °C to 70 °C, 65 °C to 75 °C, or 70 °C to 80 °C inclusive. In certain embodiments, the probe has a melting temperature between 65 °C to 69 °C, 66 °C to 70 °C, 67 °C to 71 °C, 68 °C to 72 °C, or 69 °C to 73 °C inclusive. In certain embodiments, the probe has a melting temperature of 65 °C, 66 °C, 67 °C, 68 °C, 69 °C, 70 °C, 71 °C, 72 °C, or 73 °C.

[0079] In certain embodiments, the probe comprises a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to any one of SEQ ID NOs: 5, 8, 11, 14, or 17. In certain embodiments, the probe comprises a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 5. In certain embodiments, the probe comprises a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 8. In certain embodiments, the probe comprises a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 11. In certain embodiments, the probe comprises a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 14. In certain embodiments, the probe comprises a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 17.

[0080] In certain embodiments, the probe comprises the sequence set forth in any of SEQ ID NOs: 5, 8, 11, 14, or 17. In certain embodiments, the probe comprises the sequence set forth in SEQ ID NO: 5. In certain embodiments, the probe comprises the sequence set forth in SEQ ID NO: 8. In certain embodiments, the probe comprises the sequence set forth in SEQ ID NO: 11. In certain embodiments, the probe comprises the sequence set forth in SEQ ID NO: 14. In certain embodiments, the probe comprises the sequence set forth in SEQ ID NO: 17.

[0081] In some embodiments, the reaction mixtures of the first incubation and second incubation described herein independently comprises an oligonucleotide set composed of a forward primer, a reverse primer and an oliogonucleotide probe as described herein. In certain embodiments, the forward primer comprises a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to any one of SEQ ID NOs: 3, 6, 9, 12, or

15, the reverse primer comprises a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to any one of SEQ ID NOs: 4, 7, 10, 13, or 16, and the probe comprises a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to any one of SEQ ID NOs: 5, 8, 11, 14, or 17. In certain embodiments, the forward primer comprises the sequence set forth in any of SEQ ID NOs: 3, 6, 9, 12, or 15, the reverse primer comprises the sequence set forth in any one of SEQ ID NOs: 4, 7, 10, 13, or

16, and the probe comprises the sequence set forth in any one of SEQ ID NOs: 5, 8, 11, 14, or

17,

[0082] In certain embodiments, the forward primer comprises a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 3, the reverse primer comprises a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 4, and the probe comprises a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 5. In certain embodiments, the forward primer comprises a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 6, the reverse primer comprises a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 7, and the probe comprises a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 8. In certain embodiments, the forward primer comprises a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 9, the reverse primer comprises the sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 10, and the probe comprises a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 11. In certain embodiments, the forward primer comprises a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 12, the reverse primer comprises a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 13, and the probe comprises a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 14. In certain embodiments, the forward primer comprises a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 15, the reverse primer comprises a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 16, and the probe comprises a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 17.

[0083] In certain embodiments, the forward primer comprises the sequence set forth in SEQ ID NO: 3, the reverse primer comprises the sequence set forth in SEQ ID NO: 4, and the probe comprises the sequence set forth in SEQ ID NO: 5. In certain embodiments, the forward primer comprises the sequence set forth in SEQ ID NO: 6, the reverse primer comprises the sequence set forth in SEQ ID NO: 7, and the probe comprises the sequence set forth in SEQ ID NO: 8. In certain embodiments, the forward primer comprises the sequence set forth in SEQ ID NO: 9, the reverse primer comprises the sequence set forth in SEQ ID NO: 10, and the probe comprises the sequence set forth in SEQ ID NO: 11. In certain embodiments, the forward primer comprises the sequence set forth in SEQ ID NO: 12, the reverse primer comprises the sequence set forth in SEQ ID NO: 13, and the probe comprises the sequence set forth in SEQ ID NO: 14. In certain embodiments, the forward primer comprises the sequence set forth in SEQ ID NO: 15, the reverse primer comprises the sequence set forth in SEQ ID NO: 16, and the probe comprising the sequence set forth in SEQ ID NO: 17.

[0084] In certain embodiments, the probe further comprises a detectable moiety. In certain embodiments, the detectable moiety can be a fluorescent moiety (also termed a fluorophore). In certain embodiments, the fluorescent moiety can be selected from the group consisting of fluorescein-family dyes, polyhalofluorescein-family dyes, hexachloro fluorescein-family dyes, coumarin-family dyes, rhodamine-family dyes, cyanine- family dyes, oxazine-family dyes, thiazine-family dyes, squaraine-family dyes, chelated lanthanide-family dyes, and BODIPYO-family dyes. In a certain embodiment, the fluorescent moiety can be, but is not limited to, FAM, HEX, FITC, Texas Red, TET, JOE, VIC, NED, TAMRA, ROX, ABY, PET, JUN, LIZ, Cy3, or Cy5. In particular embodiments, the fluorescent moiety is 6-carboxy-fluorescein (FAM), 4-dichloro-6-carboxyfluorescein (VIC), 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (JOE), or 5-tetrachloro-fluorescein (TET). In a preferred embodiment, the fluorescent moiety is 6-carboxyfluoroscein (FAM). In certain embodiments, the generated HPV16 amplicon is detected by measuring the fluorescence emitted from the detectable moiety of the second reaction product. [0085] In some embodiments, the detectable moiety (e.g. fluorescent moiety) may be attached to the probe at any location, such as at the 5’ end, the 3’ end or internal to either end. For instance, in some embodiments, the detectable moiety (e.g. fluorescent moiety) may be attached to any one of the nucleotides of the probe sequence capable of hybridizing to the specific HPV16 gene region that the probe was designed to detect. In particular embodiments, the detectable moiety is a fluorescent organic dye that is derivatized for attachment to the 3’ carbon or terminal 5’ carbon of the probe via a linking moiety. In some embodiments, the detectable moiety (e.g. fluorescent moiety) is attached to a 5’ terminal nucleotide of the probe sequence.

[0086] In certain embodiments, the probe comprises a quencher moiety. The quencher moiety can be any quencher moiety known to one of skill in the art without limitation. In some embodiments, the quencher molecules are also organic dyes, which may or may not be fluorescent, depending on the embodiment of the invention. In some embodiments, the quencher molecule is non-fluorescent. In some embodiments, the quencher is a fluorescent molecule, Generally, whether the quencher molecule is fluorescent or simply releases the transferred energy from the reporter by non-radiative decay, the absorption band of the quencher should substantially overlap the fluorescent emission band of the reporter molecule. Non-fluorescent quencher molecules that absorb energy from excited reporter molecules,- but which do not release the energy radiatively, are referred to herein as “dark quenchers,” “dark quencher molecules,” “non-fluorescent quenchers” or “non-fluorescent quencher molecules."

[0087] In certain embodiments, the quencher moiety can be selected from the group consisting of fluorescein-family dyes, polyhalofluorescein-family dyes, hexachloro fluorescein-family dyes, coumarin-family dyes, rhodamine-family dyes, cyanine- family dyes, oxazine-family dyes, thiazine-family dyes, squaraine-family dyes, chelated lanthanide-family dyes, BODIPY®-family dyes, and non-fluorescent quencher moieties.

[0088] In some embodiments, the quencher is a fluorescent quencher, providing that said fluorescent quencher does not interfere with detection of the energy emitted by each of the chosen fluorophores. An example of a fluorescent quencher is 6-carboxy-tetramethyl- rhodamine (TAMRA™, Applera Corp., Norwalk, CT). In some embodiments, the fluorescent moiety of the probe is FAM or VIC and the quencher dye is TAMRA.

[0089] In some embodiments, the quencher molecule is non-fluorescent quencher (NFQ, also termed dark quencher). In some cases, dark quenchers have a lower background fluorescence and do not emit light, allowing additional fluorophore options. In certain embodiments, the non-fluorescent quencher moieties can be BHQT™-family dyes, such as Black Hole Quencher™ 1 (BHQ1), BHQ™-2, or BHQ™-3. Other non-fluoresecent quenchers include, for example, Eclipse® Dark Quencher, Deep Dark Quencher™ I and II ((DDQ), Iowa Black™, or Dabcyl.

[0090] In some embodiments, the detectable moiety (e.g. fluorescent moiety) is attached to a 5’ terminal nucleotide of the probe sequence and the quencher is attached to a 3’ terminal nucleotide of the probe sequence.

[0091] In some embodiments, the probe is particularly designed with a minor groove binder (MGB) at the 3’ end which fits into the minor groove of duplex DNA. This additional feature in the probe construction allows enhanced stabilization during probe annealing (Kutyavin et al., 2000). In certain embodiments, the probe comprises a minor groove binder (MGB) moiety at the 3’ end. In some embodiments, the 3’ end of the probe is covalently attached with MGB and a NFQ (MGB-NFQ).

[0092] Optimal quenchers for a probe in embodiments herein are selected based on their ability to quench the fluorescence of a selected fluorescent dye, said dye emitting energy in the form of light with a defined spectrum. One of skill in the art can readily identify a fluorophore-quencher pair for use in the methods of the present invention. In some embodiments, the fluorophore-quencher pairs include: FAM-BHQ1, JOE-BHQ1, TET-BHQ1, Cy3-BHQ2, Cy5-BHQ3, TET-TAMRA, HEX-TAMRA, Texas Red-DDQ I or DL. In some embodiments, the 3’ end of the probe is covalently attached with MGB-NFQ and the 5’ end of the probe is covalently attached with FAM dye. One of skill in the art will realize that the particular quencher chosen must be capable of effectively quenching the fluorescence of the chosen fluorophore at the wavelength said fluorescence is emitted.

[0093] Oligonucleotide probes and primers for use in the methods provided herein can be synthesized by a number of methods. See, e.g., Ozaki et al., Nucleic Acids Research 20: 5205-5214 (1992); Agrawal et al., Nucleic Acids Research 18: 5419-5423 (1990). For example, oligonucleotide probes can be synthesized on an automated DNA synthesizer such as the ABI 3900 DNA Synthesizer (Applied Biosystems, Foster City, CA). Alternative chemistries, e.g. resulting in non-natural backbone groups, such as phosphorothioate, phosphoramidate, and the like, may also be employed provided that the hybridization efficiencies of the resulting oligonucleotides are not adversely affected. In some embodiments, commercially available linking moieties are employed that can be attached to an oligonucleotide during synthesis, e.g. available from Clontech Laboratories (Palo Alto, Calif.).

2. Reactions and Thermocycling Programs

[0094] The provided methods include two reactions involving a first incubation for preamplification (also referred to as first amplification) of a reaction product from an HPV16 DNA template and a second incubation for amplification of the first reaction product (also called second amplification) to generate an HPV16 amplicon if present in the sample. In some embodiments, both incubations involve PCR in the presence of a DNA polymerase for extension of the primers. In some embodiments, the pre-amplification is of an HPV16 DNA template (e.g. HPV16 E7 DNA) from a sample, and employs amplification by PCR. In some embodiments, at least the second incubation is a qPCR reaction of the reaction product from the pre-amplification for detection of the HPV16 amplicon if present in the sample.

[0095] In some embodiments, the PCR incubations are carried out under conditions for extension (polymerization) of the primers. The method described herein include the presence of a DNA polymerase to extend the primers from their 3’ ends. The DNA polymerase for use in the provided methods is one that possesses 5’ - 3’ exonuclease activity. Several suitable polymerases are known in the art, including but not limited to, Taq (Thermus aquaticus), Tbr (Thermus brockianus) and Tth (Thermus thermophilus) polymerases. In some embodiments, the DNA polymerase is a TAQ DNA polymerase.

[0096] In certain embodiments, the concentration of polymerase used in the first or second amplification step is between 0.001-2.0 μM, inclusive. In certain embodiments, the concentration of polymerase used in the first or second amplification step is between 0.001- 1.0 μM, 0.005-1.05 μM, 0.01-1.1 μM, 0.05-1.2 μM , 0.10-1.3 μM, 0.5-1.4 μM, 0.6-1.5 μM, 0.7-1.6 μM, 0.8-1.7 μM, 0.9-1.8 μM, or 1.0-2.0 μM. In a specific embodiment, the concentration of polymerase used in the first or second amplification step is between 0.005-1 μM, inclusive. In a specific embodiment, the concentration of polymerase used in the first or second amplification step is between 0.1-0.5 μM inclusive.

[0097] The PCR amplification steps can be performed by standard techniques well known in the art (See, e.g., Sambrook, E.F. Fritsch, and T. Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989); U.S. Patent No. 4,683,202; and PCR Protocols: A Guide to Methods and Applications, Innis et al., eds., Academic Press, Inc., San Diego (1990) which are hereby incorporated by reference).

[0098] In certain embodiments, the polymerase chain reaction of the method described herein can be selected from the group including, but not limited to, amplified fragment length polymorphism (AFLP) PCR, allele- specific PCR, Alu PCR, Arbitrary primed PCR, assembly PCR, asymmetric PCR, COLD PCR, colony PCR, conventional PCR, digital PCR (dPCR), drop digital PCR (ddPCR), fast-cycling PCR, high-fidelity PCR, High-Resolution Melt (HRM) PCR, hot-start PCR, GC-rich PCR, in situ PCR, inter sequence- specific (ISSR) PCR, inverse PCR, LATE (linear after the exponential) PCR, ligation-mediated PCR, long-range PCR, methylation- specific PCR (MSP), miniprimer PCR, multiplex-PCR, Nanoparticle- Assisted PCR (nanoPCR), nested PCR, overlap extension PCR, Real-Time PCR (quantitative PCR or qPCR), repetitive sequence-based PCR, Reverse-Transcriptase (RT-PCR), Reverse- Transcriptase Real-Time PCR (RT-qPCR), RNase H-dependent PCR (rhPCR), single cell PCR, Single Specific Primer-PCR (SSP-PCR), solid phase PCR, suicide PCR, thermal asymmetric interlaced PCR (TAIL-PCR), touch down (TD) PCR, and Variable Number of Tandem Repeats (VNTR) PCR.

[0099] In a specific embodiment, the method described herein comprises Real-Time PCR (qPCR). In a specific embodiment, the method described herein comprises reversetranscriptase Real-Time PCR (RT-qPCR).

[0100] In certain embodiments, the method described herein comprises Real-Time PCR (qPCR). In certain embodiments, the fluorescence emitted from the detectable moiety of the second reaction product is detected by qPCR. The qPCR chemistry of the method are selected from the group including, but not limited to, TaqMan®, MGB, SYBR® Green, Molecular Beacon, Amplifluor®, Scorpion®, Locked Nucleic Acid (LNA^fe) Probes, Cycling Probe Technology (CPT), Light Upon extension (Lux™) Fluorogenic Primers, and Plexor™ Technology. In a specific embodiment, the method described herein comprises TaqMan® qPCR chemistry.

[0101] In some embodiments, the PCR of the first and second incubation is carried out in a reaction mixture containing the DNA polymerase and also an oligonucleotide set containing the forward primer, reverse primer and the oligonucleotide probe, such as any described above including any particular combination of the forward primer, reverse primer and oligonucleotide probe as described above. In some embodiments, the DNA polymerase is a Taq DNA polymerase. Suitable additional other reagents for a PCR reaction in the provided methods are known to a skilled artisan and can include nucleotides, salts, buffering agents, various additives and PCR enhancers.

[0102] Primers and probes that make up the oligonucleotide set for the pre-amplification reaction of the first incubation and for the RT-PCR (e.g. qPCR) reaction of the second incubation can be the same, i.e. primers and probes which are specific for the amplicon of interest. In certain embodiments, the concentration of the oligonucleotide set in the first incubation step is less than the concentration of the oligonucleotide set in the second incubation. In some embodiments, the concentration of the oligonucleotide set in the first incubation is from 50-fold (50x) to 1000-fold (l000x) less than the concentration of the oligonucleotide set in the second incubation. For example, the concentration of the oligonucleotide set in the first incubation is at or about l00x less, 200x less, 300x less, 400x less, 500x less, or 600x less than the concentration of the oligonucleotide set in the second incubation step. In a specific embodiment, the concentration of the oligonucleotide set in the first incubation step is at or about 400x less than the concentration of the oligonucleotide set in the second incubation step.

[0103] In some embodiments, the concentration of the oligonucleotide set in the first incubation is between 0.05x (0.05-fold) and 0.00lx (0.001-fold) of the concentration of the oligonucleotide set in the second incubation, such as between 0.01 -fold and 0.002-fold of the concentration in the second incubation step. In a specific embodiment, the concentration of the oligonucleotide set in the first incubation step is at or about 0.0025-fold (0.0025x) the concentration of the oligonucleotide set in the second incubation step.

[0104] In some embodiments, the forward and reverse oligonucleotide primers are used in the first incubation (first amplification step or pre-amplification step) at a concentration of at or about 0.000lμM, 0.0005(lM, 0.00lμM, 0.005μM, 0.0lμM, 0.02μM, 0.03μM, 0.04μM, 0.05μM, 0.06μM, 0.07μM, 0.08μM, 0.09μM, 0.lμM, 0.2μM, 0.3μM, 0.4μM, 0.5μM, 0.6μM, 0.7μM, 0.8μM, 0.9μM, l.0μM, l.lμM, 1.2μM, 1.3μM, 1.4μM, 1.5μM, 1.6μM, 1.7μM, 1.8μM, 1.9μM, or 2.0μM, or any value between any of the foregoing. In certain embodiments, the forward and reverse oligonucleotide primers are used in the first incubation (first amplification step or pre-amplification step) at a concentration between 0.0001 μM and 0.00lμM, between 0.0005μM and 0.005μM, between 0.00lμM and 0.0lμM, between 0.005μM and 0.02μM, between 0.03μM and 0.05μM, between 0.04μM and 0.06μM, between 0.05μM and 0.07μM, between 0.06μM and 0.08μM, between 0.07μM and 0.09μM, between 0.08μM and 0.1μM, between 0.09μM and 0.2μM, between 0.1μM and 0.3μM, between 0.2μM and 0.4μM , between 0.3μM and 0.5μM, between 0.4μM and 0.6μM, between 0.5μM and 0.7μM , between 0.6μM and 0.8μM, between 0.7μM and 0.9μM, between 0.8μM and 1.0μM , between 0.9μM and l.lμM, between 1 .0μM and 1.2μM, between l.lμM and 1.3μM , between 1.2μM and 1.4μM, between 1.3μM and 1.5μM, between 1.4μM and 1.6μM , between 1.5μM and 1.7μM, between 1.6μM and 1.8μM, between 1.7μM and 1.9μM , or between 1.8μM and 2.0μM .

[0105] In certain embodiments, the first incubation step (first amplification step or preamplification step) comprises a probe at a concentration of at or about 0.lnM, 0.2nM, 0.3nM, 0.4nM, 0.5nM, 0.6nM, 0.7nM, 0.8nM, 0.9nM, l.0nM, 2.0nM, 3.0nM, 4.0nM, 5.0nM, 6.0nM, 7.0nM, 8.0nM, 9.0nM, 10nM, 20nM, 30nM, 40nM, 50nM, 60nM, 70nM, 80nM, 90nM, 100nM, 110nM, 120nM, 130nM, 140nM, 150nM, 160nM, 170nM, 180nM, 190nM, 200nM, 210nM, 220nM, 230nM, 240nM, 250nM, 260nM 270nM, 280nM, 290nM, 300nM, 310nM, 320nM, 330nM, 340nM, 350nM, 360nM, 370nM, 380nM, 390nM or 400nM, or any value between any of the foregoing. In certain embodiments, the first incubation step (first amplification step or pre-amplification step) comprises a probe at a concentration between 0.1nM and 5nM, l.0nM and 10nM, 10nM and 30nM, 20nM and 40nM, 30nM and 50nM, 40nM and 60nM, 50nM and 70nM, 60nM and 80nM, 70nM and 90nM, 80nM and 100nM, 90nM and 110nM, 100nM and 120nM, 110nM and 130nM, 120nM and 140nM, 130nM and 150nM, 140nM and 160nM, 150nM and 170nM, 160nM and 180nM, 170nM and 190nM, 180nM and 200nM, 190nM and 210nM, 200nM and 220nM, 210nM and 230nM, 220nM and 240nM, 230nM and 250nM, 240nM and 260nM, 250nM and 270nM, 260nM and 280nM, 270nM and 290nM, 280nM and 300nM, 290nM and 310nM, 300nM and 320nM, 310nM and 330nM, 320nM and 340nM, 330nM and 350nM, 340nM and 360nM, 350nM and 370nM, 360nM and 380nM, 370nM and 390nM, or 380nM and 400nM.

[0106] The duration and temperature of each phase in the pre-amplification first incubation step, i.e. denaturation, annealing and extension, may vary based on the DNA polymerase and sample material used. However, a skilled person of the art can choose appropriate PCR reaction conditions and thus the present method is not limited to any certain cycle pattern. The pre-amplification in the first incubation can include an initial denaturation step, which can be performed by heating the PCR reaction mixture to a temperature ranging from about 90°-98°C for a duration of 1-30 second. The pre-amplification also can include a thermal cycling reaction for denaturation, annealing and extension, in a limited number of cycles. In some embodiments, the thermal cycling reaction includes heating the PCR reaction mixture to a temperature ranging from about 90°-98°C for a duration of 1-30 second followed by annealing and extension. The annealing and extension can occur as separate steps (three-step PCR) or in a combined step (two-step PCR). Typically, the preamplification includes an extended annealing time (3 min or more) in a limited number of cycles (usually 20 cycles or less). In some embodiments, the annealing time is at a temperature of 45-60°C and is for 3 min to 5 min, such as for at or about 4 min. In some embodiments, annealing and extension may be combined into a one phase (i.e. 2-step-PCR). In some embodiments, the annealing and extension can be carried out at 60-72°C, such as at or about 60 °C, and is for 3 min to 5 min, such as for at or about 4 min.

[0107] In some embodiments, the initial denaturation step in the first pre-amplification step is for a duration of 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds, 10 seconds, 11 seconds, 12 seconds, 13 seconds, 14 seconds, 15 seconds, 16 seconds, 17 seconds, 18 seconds, 19 seconds, or 20 seconds. In certain embodiments, the the initial denaturation step in the first pre-amplification step is between 1 second and 5 seconds, 5 seconds and 10 seconds, 10 seconds and 15 seconds, or 15 seconds and 20 seconds. In a specific embodiment, the initial denaturation step in the first preamplification step is for a duration of 10 seconds. In some embodiments, the temperature of the initial denaturation step in the first pre-amplification step is at or about 85 °C, 86 °C, 87 °C, 88 °C, 89 °C, 90 °C, 91 °C, 92 °C, 93 °C, 94 °C, 95 °C. 96 °C. 97 °C, 98 °C, 99 °C, or 100 °C. In certain embodiments, the temperature held during the initial denaturation step in the first pre-amplification step is between 85 °C to 91 °C, 90 °C to 96 °C, or 95 °C to 100 °C. In a specific embodiment, the temperature during the initial denaturation step in the first preamplification step is 95 °C.

[0108] In some embodiments, the thermal cycling of the pre-amplification incubation includes heating the reaction mixture to a temperature of between 85°C and 100°C for a period of time (e.g. 5 seconds to 20 seconds) followed by annealing/extension at a temperature between 50°C and 70°C for a period of time (e.g. 3 minutes to 5 minutes). In some embodiments, the thermal cycling is repeated for at least 4 cycles, such as 4-24 cycles, of denaturation, annealing and extension. The number of cycles can be 10-20, 10-24, 5-15 cycles, depending on the expected concentration of the target nucleic acid, and the amount of first and second template in the reaction. Typically the number of cycles is less than 20 cycles, such as 5-12 cycles. In some embodiments, the number of cycles is at or about 10 cycles. The aim of this step is to pre-amplify the DNA template so that the amount of target DNA is increases, such as increased at least 10 times, such as increased 50 to 10000 times or more.

[0109] An exemplary thermal profile for preamplification includes: 95 °C for 10 seconds followed by 10 cycles of amplification at 95 °C for 15 seconds and 60°C for 4 minutes.

[0110] In some embodiments, the methods include a step for removing or degrading excess deoxynucleoside triphosphates after the preamplification step. This step may be performed concurrently with the step of removing or degrading excess primers. Many commercial products can be used to remove both excess primer and deoxynucleoside triphosphates, such as ExoSAP-IT (USB), PCR cleanup kit (Qiagen), GenElute (Sigma- Aldrich), and the like. In other embodiments, such as where the primers are the same, the step of removal after the preamplification step may not be necessary.

[0111] In some embodiments, the forward and reverse oligonucleotide primers are used in the second incubation (second amplification step) at a concentration of at or about 0.000lμM, 0.0005μM, 0.001μM, 0.005μM, 0.01μM, 0.02μM, 0.03μM, 0.04μM, 0.05μM, 0.06μM, 0.07μM, 0.08μM, 0.09μM, 0.lμM, 0.2μM, 0.3μM, 0.4μM, 0.5μM, 0.6μM, 0.7μM, 0.8μM, 0.9μM, l.0μM, l.lμM, 1.2μM, 1.3μM, 1.4μM, 1.5μM, 1.6μM, 1.7μM, 1.8μM, 1.9μM, or 2.0μM, or any value between any of the foregoing. In certain embodiments, the forward and reverse oligonucleotide primers are used in the second incubation (second amplification step) at a concentration between 0.0001 μM and 0.001 μM, between 0.0005μM and 0.005μM, between 0.00lμM and 0.0lμM, between 0.005μM and 0.02μM, between 0.03μM and 0.05μM, between 0.04μM and 0.06μM, between 0.05μM and 0.07 μM, between 0.06μM and 0.08μM, between 0.07μM and 0.09μM, between 0.08μM and 0.1 μM, between 0.09μM and 0.2μM, between 0.lμM and 0.3μM, between 0.2μM and 0.4μM, between 0.3μM and 0.5μM, between 0.4μM and 0.6μM, between 0.5μM and 0.7μM, between 0.6μM and 0.8μM, between 0.7μM and 0.9μM, between 0.8μM and l.0μM, between 0.9μM and l.lμM, between l.0μM and 1.2μM, between 1.1 μM and 1.3μM, between 1.2μM and 1.4μM, between 1.3μM and 1.5μM, between 1.4μM and 1.6μM, between 1.5μM and 1.7μM, between 1.6μM and 1.8μM, between 1.7μM and 1.9μM, or between 1.8μM and 2.0μM .

[0112] In certain embodiments, the second incubation step (second amplification) comprises a probe at a concentration of at or about 0.lnM, 0.2nM, 0.3nM, 0.4nM, 0.5nM, 0.6nM, 0.7nM, 0.8nM, 0.9nM, l.0nM, 2.0nM, 3.0nM, 4.0nM, 5.0nM, 6.0nM, 7.0nM, 8.0nM, 9.0nM, 10nM, 20nM, 30nM, 40nM, 50nM, 60nM, 70nM, 80nM, 90nM, 100nM, 110nM, 120nM, 130nM, 140nM, 150nM, 160nM, 170nM, 180nM, 190nM, 200nM, 210nM, 220nM, 230nM, 240nM, 250nM, 260nM 270nM, 280nM, 290nM, 300nM, 310nM, 320nM, 330nM, 340nM, 350nM, 360nM, 370nM, 380nM, 390nM or 400nM, or any value between any of the foregoing. In certain embodiments, the second incubation step comprises a probe at a concentration between 0.1nM and 5nM, l.0nM and 10nM, 10nM and 30nM, 20nM and 40nM, 30nM and 50nM, 40nM and 60nM, 50nM and 70nM, 60nM and 80nM, 70nM and 90nM, 80nM and 100nM, 90nM and 110nM, 100nM and 120nM, 110nM and 130nM, 120nM and 140nM, 130nM and 150nM, 140nM and 160nM, 150nM and 170nM, 160nM and 180nM, 170nM and 190nM, 180nM and 200nM, 190nM and 210nM, 200nM and 220nM, 210nM and 230nM, 220nM and 240nM, 230nM and 250nM, 240nM and 260nM, 250nM and 270nM, 260nM and 280nM, 270nM and 290nM, 280nM and 300nM, 290nM and 310nM, 300nM and 320nM, 310nM and 330nM, 320nM and 340nM, 330nM and 350nM, 340nM and 360nM, 350nM and 370nM, 360nM and 380nM, 370nM and 390nM, or 380nM and 400nM.

[0113] In some embodiments, the PCR amplification of the second incubation include PCR cycling conditions involving an initial denaturation step, which can be performed by heating the PCR reaction mixture to a temperature ranging from about 80°C to about 105°C for times ranging from about 1 to 30 seconds, such as for 10-30 seconds. Heat denaturation is typically followed by a number of cycles, ranging from about 20 to about 50 cycles, each cycle usually comprising an initial denaturation step, followed by a primer annealing/primer extension step. Enzymatic extension of the primers by the nucleic acid polymerase, e.g. TAQ polymerase, produces copies of the template that can be used as templates in subsequent cycles.

[0114] The duration and temperature of each phase in the second incubation, i.e. denaturation, annealing and extension, may vary based on the DNA polymerase and sample material used. However, a skilled person of the art can choose appropriate PCR reaction conditions and thus the present method is not limited to any certain cycle pattern. The amplification in the second incubation can include an initial denaturation step, which can be performed by heating the PCR reaction mixture to a temperature ranging from about 80°C to about 105°C, such as 90°-98°C for a duration of 1-30 second, such as 10-30 seconds, e.g. at or about 20 seconds. The second incubation amplification also can include a thermal cycling reaction for denaturation, annealing and extension for a number of cycles, ranging from about 20 to about 50 cycles. In some embodiments, the thermal cycling reaction includes heating the PCR reaction mixture to a temperature ranging from about 90°-98°C for a duration of 1- 30 second, such as for 1-10 seconds, followed by annealing and extension. The annealing and extension can occur as separate steps (three-step PCR) or in a combined step (two-step PCR). In some embodiments, the PCR includes an annealing step at 45-60°C for 10-30 seconds and an extension step at 60-72°C for 10-30 seconds. In some embodiments, the PCR is a two- step PCR in which the thermal cycling (repeated 20-50 cycles, such as 30-40 times) includes denaturation by heating the PCR reaction mixture to a temperature ranging from about 90°- 98°C for a duration of 1-30 seconds, such as for 1-10 seconds, followed by incubation at 60- 72°C, such as at or about 60 °C, for 10-60 seconds, such as for at or about 10-30 seconds.

[0115] In some embodiments, the initial denaturation step in the second incubation is for a duration of 5 second, 6 seconds, 7 seconds, 8 seconds, 9 seconds, 10 seconds, 11 seconds, 12 seconds, 13 seconds, 14 seconds, 15 seconds, 16 seconds, 17 seconds, 18 seconds, 19 seconds, 20 seconds, 21 seconds, 22 seconds, 23 seconds, 24 seconds, 25 seconds, 26 seconds, 27 seconds, 28 seconds, 29 seconds or 30 seconds. In certain embodiments, the initial denaturation step in the second incubation is between 1 5 seconds and 30 seconds, 10 seconds and 30 seconds, or 15 seconds and 20 seconds. In a specific embodiment, the initial denaturation step in the second incubations step is for a duration of 20 seconds. In certain embodiments, the temperature held during the initial denaturation step of the second incubation step is at or about 85 °C, 86 °C, 87 °C, 88 °C, 89 °C, 90 °C, 91 °C, 92 °C, 93 °C, 94 °C, 95 °C. 96 °C. 97 °C, 98 °C, 99 °C, or 100 °C. In certain embodiments, the temperature held during the initial denaturation step during the second incubation is between 85 °C to 91 °C, 90 °C to 96 °C, or 95 °C to 100 °C. In a specific embodiment, the temperature held during the initial denaturation step during the second incubation is 95 °C.

[0116] In certain embodiments, prior to the initial denaturation in the second incubation, the reaction mixture is first held at a lower temperature for a period of time. In some embodiments, the period of time is for a duration between 30 seconds and 5 minutes, 1 minute and 5 minutes, 1 minute and 4 minutes, or 2 minutes and 3 minutes. In some embodiments, the period of time is for a duration of at or about 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes or 5 minutes. In some embodiments, the period of time is for at or about 2 minutes. In some embodiments, the temperature is initially held at or about 40 °C,

41 °C, 42 °C, 43 °C, 44 °C, 45 °C, 46 °C, 47 °C, 48 °C, 49 °C, 50 °C. 51 °C. 52 °C, 53 °C, 54 °C, 55 °C. 56 °C, 57 °C, 58 °C, 59 °C, or 60 °C. In certain embodiments, the temperature is initially held between 40 °C to 46 °C, 45 °C to 51 °C, 50 °C to 56 °C, or 55 °C to 60 °C. In a specific embodiment, the temperature at the beginning of the second period is initially held at 50 °C.

[0117] In some embodiments, the thermal cycling of the second incubation includes denaturation by heating the PCR reaction mixture to a temperature ranging from about 85 °C- 100°C, such as from 90°-98°C for a duration of 1-10 seconds, followed by incubation at a temperature ranging from 50°C-72°C, such as 60-72°C, for a duration of 10-30 seconds. In some embodiments, the thermal cycling of the second incubation is repeated for at least 20 cycles. In some embodiments, the number of cycles is 30 cycles, 31 cycles, 32 cycles, 33 cycles, 34 cycles, 35 cycles, 36 cycles, 37 cycles, 38 cycles, 39 cycles, 40 cycles, 41 cycles,

42 cycles, 43 cycles, 44 cycles, 45 cycles, 46 cycles, 47 cycles, 48 cycles, 49 cycles, or 50 cycles. In a specific embodiment, the number of cycles is 40 cycles. In a specific embodiment, the cycling period comprises 40 cycles of amplification at 95 °C for 1 second and 60°C for 20 seconds.

[0118] An exemplary thermal profile for the second incubation includes: 95°C for 20 seconds, then 40 cycles of amplification (95°C for 1 second and 60°C for 20 seconds). In some embodiments, the second incubation first begins with an incubation at 40-60°C, such as at or about 50°C for 1-3 minutes. For instance, an exemplary thermal profile for the second incubation includes: an incubation at 50°C for 2 minutes, 95°C for 20 seconds, then 40 cycles of amplification (95 °C for 1 second and 60°C for 20 seconds).

[0119] In certain embodiments, the amplification efficiency of the second incubation step is between 85% to 110%. In certain embodiments, the amplification efficiency of the second incubation step is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, or 110%. In certain embodiments, the amplification efficiency of the second incubation step is between 85% and 87%, 86% and 88%, 87% and 89%, 88% and 90%, 89% and 91%, 90% and 92%, 91% and 93%, 92% and 94%, 93% and 95%, 94% and 96%, 95% and 97%, 96% and 98%, 97% and 99%, 98% and 100%, 99% and 101%, 100% and 102%, 101% and 103%, 102% and 104%, 103% and 105%, 104% and 106%, 105% and 107%, 106% and 108%, 107% and 109%, or 108% and 110%. In a specific embodiment, the amplification efficiency of the second incubation step is between 90% and 110%.

.7. Detection a net Diagnosis

[0120] The method provided herein can be used to detect the presence of HPV in a sample after first performing the pre-amplification as described above and then performing Real-Time PCR (quantitative PCR or qPCR) with a reaction mixture comprising the first preamplification reaction product or portion thereof in a second incubation under conditions for amplification of the HPV 16 ampliconas described above. In certain embodiments, qPCR can be performed to determine whether an HPV 16 amplicon of interest is detected in a given sample. In some embodiments, the reaction is carried out under conditions in which a detectable signal is produced if the HPV 16 amplicon is present. For instance, in qPCR methods with a detectable probe the DNA polymerase will digest each probe during amplification to dissociate the detectable moieity (e.g. fluorophore) from said quencher molecule so that a change of the detectable signal (e.g. fluorescence) upon dissociation of the detectable moiety (e.g. fluorophore) and the quencher can be detected. Typically, the change of the detectable signal (e.g. fluorescence) corresponds to the occurrence of nucleic acid amplification and indicates that the sample is positive for the HPV. In some embodiments, a subject is diagnosed with an HPV infection if an HPV 16 amplicon is detected in a sample by the provided methods.

[0121] In some embodiments, a detectable signal from the probe can be detected. Any method able to detect the detectable signal from the detectable moity can be used.

[0122] In some embodiments, the detectable moiety is a fluorophore and the change in fluorescence can be detected. In some embodiments, fluorescence can be detected by an automated fluorometer. In some embodiments, the fluorometer may be one that is designed to perform real-time PCR having the following features: a method of excitation to excite the fluorophore of the fluorescent probe, a means for heating and cooling PCR reaction mixtures and a means for detecting a change in fluorescence. This combination of features, when performed by a single real-time PCR instrument, allows real-time detection of PCR amplicons, which allows confirmation of PCR product amplification through examination of the kinetics of the fluorescence increase in real-time. Automated fluorometers for performing real time PCR reactions are known in the art and can be adapted for use in this specific assay, for example, the iCycler® from Bio-Rad Laboratories (Hercules, CA), the Mx3000P™, the MX3005P™ and the MX4000® from Stratagene (La Jolla, CA), the ABI PRISM® 7300, 7500, 7700, and 7900 Sequence Detection Instruments (Applied Biosystems, Foster City, CA), the SmartCycler® and the Gene Xpert® System (Cepheid, Sunnyvale, CA) and the LightCycler® (Roche Diagnostics Corp., Indianapolis, IN).

[0123] In some embodiments, the method can include DNA quantification of the HPV16 amplicon by qPCR by plotting the detectable signal (e.g. fluorescence) against the number of cycles on a logarithmic scale. A threshold for detection of the detectable signal may be set at a threshold level that is set slightly above background. In some embodiments, the number of cycles at which the fluorescence exceeds the threshold is called the quantification cycle (Cq). In some embodiments, the method further comprises determining the Cq value (quantification cycle) which refers to the number of cycles required for the PCR signal to reach the threshold level, e.g. level above background.

[0124] For instance, in a qPCR method of the second incubation, the primers and probes can be added to the pre-amplification reaction product (the DNA to be amplified). As the reaction commences under the thermal profile conditions of the second incubation, both probe and primers anneal to the DNA target during the annealing stage. Polymerizaiton of a new DNA strand is initiated from the primers, and once the polymerase reaches the probe, its 5’-3’ exonuclease degrades the probe, physically separating the detectable moiety (e.g. fluorphore) from the quencher, resulting in an increase in detectable signal (e.g. fluorescence). Fluorescence may then be detected and measured in a cycler, and its geometric increase corresponding to exponential increase of the product is used to determine the Cq in each reaction.

[0125] An amplicon is detected if the reaction reaches a fluorescent intensity above background levels. In certain embodiments, the detection of the HPV16 amplicon is determined by measurement of the Cq value. Lower Cq values indicate high amounts of the target amplicon. Higher Cq values indicate lower amounts of the target amplicon. In certain embodiments, the Cq value is determined as the cycle wherein the fluorescence emitted by the second reaction product is greater than the threshold. [0126] In some aspects, the method provided herein involves analyzing, e.g., detecting or determining, the presence or absence of HPV16 E7 amplicon in a sample from a subject. The method herein comprises the generation of a first pre- amplification reaction product. In certain embodiments, the generation of a first pre- amplification reaction product allows for greater sensitivity of amplicon detection as compared to assay methods that do not comprise the generation of a first pre- amplification reaction product. As used herein, the sensitivity of the assay refers to the percent of HPV samples correctly diagnosed. Increasing the sensitivity of amplicon detection is preferable when the sample from the subject is a non-optimal sample. Increasing detection sensitivity is also ideal when detecting an amplicon in smaller RNA.

[0127] Non-optimal samples can comprise samples that contain RNA that is fragmented, degraded, in low abundance, or otherwise low quality. In certain embodiments, degradation of the RNA is caused by heat or enzymatic degradation. In pathology archives, many tissue samples rely on the formalin-fixed and paraffin-embedded (FFPE) method for preservation. In the FFPE method, fixation delay, fixation process, tissue preparation, paraffin embedding, and archival preservation are some of the processes that may lead to fragmentation of the RNA.

[0128] In certain embodiments, the generation of a first pre-amplification reaction product allows for the reduction of the required sample input for the second incubation step. In certain embodiments, the generation of a first pre-amplification reaction product results in Cq values in the second incubation step to be shifted earlier (lower Cq values) due to the enrichment achieved during pre-amplification. In certain embodiments, the enrichment achieved during the first incubation step allows for the detection of amplicon that may be undetected using other assays and methods.

[0129] In certain embodiments, the amplicon is said to be detected if the Cq value for the second incubation step 15 of greater. For instance, an amplicon is said to be detected if the Cq value is a.

[0130] The method provided herein can be used to detect HPV 16 amplicon in a sample with a greater degree of specificity than other assays and methods. Specificity, as used herein, refers to the percent of non-HPV samples correctly diagnosed. In some aspects, the method provided herein provides for greater specificity (i.e. fewer false positive results) than alternate assays. [0131] In some aspects, the method provided herein involves diagnosing human papillomavirus in a subject. In certain embodiments, a subject is diagnosed with human papillomavirus if HPV16 amplicon is detected in the secondary reaction product following the second incubation.

II. METHODS OF TREATMENT AND USES IN THERAPY

[0132] In certain embodiments, a subject diagnosed with human papillomavirus in accord with the provided methods can be treated with therapeutic methods known in the field.

[0133] In certain embodiments, provided herein are methods for treating a subject having been diagnosed with HPV using the method provided herein. In certain embodiments, subjects are selected for treatment if the HPV 16 amplicon is detected in a sample using the method provided herein. In certain embodiments, the subject having been diagnosed with HPV is administered a therapeutic to treat the HPV infection. Any therapy known to a skilled artisan for treating HPV infection can be administered to a subject diagnosed with an HPV infection in accord with the provided methods.

[0134] In some embodiments, the methods may include steps or features to identify a subject who has, is suspected to have, or is at risk for developing an HPV 16-associated disease or disorder if an HPV 16 amplicon is detected in a biological sample from the subject in accord with the provided methods. In some embodiments, the subject to be treated may be a subject identified to have or to be so at risk for having or developing such HPV-associated disease or condition or cancer if an HPV 16 amplicon is detected in a biological sample from the subject in accord with the provided methods. Hence, provided in some aspects are methods for identifying subjects with diseases or disorders associated with HPV 16, such as associated with HPV16 E7 expression, and selecting such subjects for treatment and/or treating such subjects, e.g., selectively treating such subjects.

[0135] For example, a subject may be screened by the provided methods to detect the presence of HPV16 amplicon, e.g. HPV16 E7 amplicon, in a biological sample from the subject in order to diagnose the presence or likely presence of a disease or disorder associated with HPV 16, such as an HPV 16 cancer, in the subject. In some embodiments, the methods include screening for or detecting the presence of an HPV 16 associated disease, e.g. a tumor. Thus, in some aspects, a sample may be obtained from a patient suspected of having a disease or disorder associated with HPV 16 and assaying a sample from the subject for an HPV16 amplicon by the provided methods. In some aspects, a subject who tests positive for an HPV 16 amplicon is selected for treatment by the present methods. In some aspects, subjects treated by methods provided herein have been selected or tested positive for HPV according to such methods, e.g., prior to initiation of or during treatment.

[0136] Hence, provided are methods of administering and uses, such as therapeutic and prophylactic uses, for treating a subject having a disease, condition, or disorder expressing or associated with HPV, e.g., HPV 16, and/or in which cells or tissues in the subject are infected with HPV, e.g. HPV 16. In some embodiments, the a therapy is administered in an effective amount to effect treatment of the disease or disorder. Uses include uses of the therapy in such methods and treatments, and in the preparation of a medicament in order to carry out such therapeutic methods. In some embodiments, the methods are carried out by administering the therapy, or compositions comprising the same, to the subject selected as having or suspected of having a disease or condition associated with HPV infection. In some embodiments, the methods thereby treat the disease or condition or disorder in the subject.

[0137] As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to complete or partial amelioration or reduction of a disease or condition or disorder, or a symptom, adverse effect or outcome, or phenotype associated therewith. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. The terms do not imply complete curing of a disease or complete elimination of any symptom or effect(s) on all symptoms or outcomes.

[0138] As used herein, “delaying development of a disease" means to defer, hinder, slow, retard, stabilize, suppress and/or postpone development of the disease (such as cancer). This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, a late stage cancer, such as development of metastasis, may be delayed.

[0139] “Preventing,” as used herein, includes providing prophylaxis with respect to the occurrence or recurrence of a disease in a subject that may be predisposed to the disease but has not yet been diagnosed with the disease. In some embodiments, the provided molecules and compositions are used to delay development of a disease or to slow the progression of a disease.

[0140] As used herein, to “suppress” a function or activity is to reduce the function or activity when compared to otherwise same conditions except for a condition or parameter of interest, or alternatively, as compared to another condition. For example, a recombinant TCR or composition or cell which suppresses tumor growth reduces the rate of growth of the tumor compared to the rate of growth of the tumor in the absence of the recombinant TCR or composition or cell.

[0141] An “effective amount” of a therapy or therapeutic agent in the context of administration, refers to an amount effective, at dosages/amounts and for periods of time necessary, to achieve a desired result, such as a therapeutic or prophylactic result.

[0142] A “therapeutically effective amount” of a therapy or therapeutic agent refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result, such as for treatment of a disease, condition, or disorder, and/or pharmacokinetic or pharmacodynamic effect of the treatment. The therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the subject. In some embodiments, the provided methods involve administering the therapy or therapeutic agent at effective amounts, e.g., therapeutically effective amounts.

[0143] A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0144] As used herein, a “subject” is a mammal, such as a human or other animal, and typically is human.

[0145] Among the diseases to be treated are cancers. In some embodiments, the cancer is an HPV-associated cancers, and any HPV-associated, e.g., HPV 16-associated, diseases or conditions or diseases or conditions in which an HPV oncoprotein, e.g., E7, such as an HPV 16 oncoprotein, e.g., HPV 16 E7 is expressed. In certain diseases and conditions, the viral protein such as the oncoprotein such as the HPV 16 E7 is expressed in or by malignant cells and cancers, and/or a peptide epitope thereof is expressed on such malignant cancers or tissues, such as by way of MHC presentation. In some embodiments, the disease or condition is an HPV 16-expressing cancer. In some embodiments, the cancer is a carcinoma, melanoma or other precancerous or cancerous state caused by or otherwise associated with HPV, such as HPV-16. In some embodiments, the carcinoma can be a squamous cell or adenocarionma. In some embodiments, the disease or condition can be characterized by an epithelial cell abnormality associated with oncogenic HPV infection, such as koilocytosis; hyperkeratosis; precancerous conditions encompassing intraepithelial neoplasias or intraepithelial lesion; high-grade dysplasias; and invasive or malignant cancers. Among the HPV 16-associated diseases or conditions that can be treated include, but are not limited to, cervical cancer, uterine cancer, anal cancer, colorectal cancer, vaginal cancer, vulvar cancer, penile cancer, oropharyngeal cancers, tonsil cancer, pharyngeal cancers (pharynx cancer), laryngeal cancer (larynx cancer), oral cancer, skin cancer, esophageal cancer, head and neck cancer such as a squamous cell carcinoma (SCC) head and neck cancer, or small cell lung cancer. In some embodiments, the disease or condition is a cervical cancer. In some embodiments, the disease or condition is a cervical carcinoma.

[0146] In certain embodiments, the therapy for treating an HPV infection is selected from the group that includes, but is not limited to, vaccines that induce or boost HPV T cell adaptive immunity, adoptive cell therapy, therapeutic antibodies, antiviral therapeutics, immune response modifier compounds, proteasome inhibitors, HD AC inhibitors, and drugs targeting HPV genes. Exemplary treatments include, but are not limited to, therapeutic vaccines such as Cervarix™ and Gardasil™, vector vaccines (e.g. bacterial and viral vectors), peptide and protein vaccines, DNA vaccines, immune checkpoint inhibitors such as anti-PD- 1 (e.g. Nivolumab, cemiplimab, ), anti-PD-Ll (e.g. pembrolizumab, durvalumab, atezolizumab) or anti-CTLA-4 (e.g. ipilimumab, tremelimumab) antibodies, or cell-based therapies. In some embodiments, cell-based therapies include, but are not limited to, HPV- specific T cell therapies such as engineered TCR T cell therapies. In some aspects, an additional therapeutic may be administered, such as IL-2. In some aspects, the additional therapeutic agent is aldesleukin (Proleukin).

[0147] Therapies for treating HPV infections are known and described, for example, in U.S. Patent No. 7,172,870; U.S. Patent No. 8,663,964; published PCT Appl. No. WO 2014/165291; published PCT Appl. No. WO 2019/195486; published PCT Appl. No. WO 2019/070541; published PCT Appl. No. WO 2018/067618, published PCT appl. No. W02015009606; published PCT Appl. No. WO 2015/184228; published PCT Appl. No. WO2021/155518; U.S. Patent publication No. US2004/0214158; U.S. patent publication No. US2021/0330779; U.S. patent publication No. US2021/0038709; U.S. patent publication No. US2021/0121559; Stem et al. Vaccine 2012 30:F71-F82; Droran et al. J. Clin. Oncol., 2018 37:2759-2768; Fakhr et al. Immunology, 2021 163:33-345.

[0148] In some embodiments, the treatment for HPV is an adoptive cell therapy. In some embodiments, a method of treating a subject having been diagnosed with HPV include the administration of genetically engineered T cells expressing a recombinant T cell receptor (TCR). In some embodiments, the cell therapy is a T cell therapy engineered with a recombinant TCR that binds to a peptide epitope derived from HPV 16 E6 or E7 protein and/or to a peptide epitope expressed on a cell infected with HPV. In some embodiments, the recombinant TCR binds to an epitope or region of HPV 16 E7 or HPV 16 E6, such as a peptide epitope containing an amino acid sequence set forth in any of SEQ ID NOS: 18 and 19-25, and as shown below in Table 1. In some aspects, the recombinant TCR binds to or recognizes a peptide epitope of human papillomavirus (HPV), such as an epitope of HPV 16 E7.

[0149] Exemplary T cell therapies engineered with a recombinant anti-HPV TCR that bind to a peptide epitope of HPV 16 include those described in WO 2019/195486, WO 2019/070541, WO 2018/067618, W02015009606, or WO 2015/184228.

[0150] In some embodiments, the methods include adoptive cell therapy, whereby genetically engineered cells expressing the provided recombinant TCRs are administered to subjects. Such administration can promote activation of the cells (e.g., T cell activation) in an HPV 16-targeted manner, such that the cells of the disease or disorder are targeted for destruction. [0151] Thus, the provided methods and uses include methods and uses for adoptive cell therapy. In some embodiments, the methods include administration of the cells or a composition containing the cells to a subject, tissue, or cell, such as one having, at risk for, or suspected of having the disease, condition or disorder. In some embodiments, the cells, populations, and compositions are administered to a subject having the particular disease or condition to be treated, e.g., via adoptive cell therapy, such as adoptive T cell therapy. In some embodiments, the cells or compositions are administered to the subject, such as a subject having or at risk for the disease or condition. In some aspects, the methods thereby treat, e.g., ameliorate one or more symptom of the disease or condition, such as by lessening tumor burden in an HPV 16 E7-expressing cancer.

Exemplary TCR Cell Therapy

[0152] In some embodiments, the provided methods of treatment include administering to a subject diagnosed with having a disease or condition associated with an HPV infection a cell therapy that is a T cell therapy engineered with a recombinant TCR that binds to a peptide epitope derived from HPV 16 E7. In some embodiments, the epitope is or contains E7(l 1-19) YMLDLQPET (SEQ ID NO: 18). In some embodiments, the TCR recognizes or binds HPV 16 E7(l 1-19) in the context of an MHC, such as an MHC class I. In some aspects, the MHC Class I molecule is a human leukocyte antigen (HLA)-A2 molecule, including any one or more subtypes thereof, e.g. HLA-A*0201, *0202, *0203, *0206, or *0207. In some cases, there can be differences in the frequency of subtypes between different populations. For example, in some embodiments, more than 95% of the HLA-A2 positive Caucasian population is HLA-A*0201, whereas in the Chinese population the frequency has been reported to be approximately 23% HLA-A*0201, 45% HLA-A*0207, 8% HLA-A*0206 and 23% HLA-A*0203. In some embodiments, the subject is selected for treatment based on being positive for HLA-02. In some embodiments, the subject is selected for treatment based on being positive for HLA-A*0201. In some embodiments, the subject is selected for treatment based on being positive for HLA-A*0202.

[0153] Unless otherwise stated, the term “TCR” should be understood to encompass full TCRs as well as antigen-binding portions or antigen-binding fragments thereof. In some embodiments, the TCR is an intact or full-length TCR, such as a TCR containing the a chain and P chain. In some embodiments, the TCR is an antigen-binding portion that is less than a full-length TCR but that binds to a specific peptide bound in an MHC molecule, such as binds to an MHC-peptide complex. In some cases, an antigen-binding portion or fragment of a TCR can contain only a portion of the structural domains of a full-length or intact TCR, but yet is able to bind the peptide epitope, such as MHC-peptide complex, to which the full TCR binds. In some cases, an antigen-binding portion contains the variable domains of a TCR, such as variable a (Va) chain and variable P (VP) chain of a TCR, or antigen-binding fragments thereof sufficient to form a binding site for binding to a specific MHC-peptide complex.

[0154] In some embodiments, the variable domains of the TCR contain complementarity determining regions (CDRs), which generally are the primary contributors to antigen recognition and binding capabilities and specificity of the peptide, MHC and/or MHC-peptide complex. In some embodiments, a CDR of a TCR or combination thereof forms all or substantially all of the antigen-binding site of a given TCR molecule. The various CDRs within a variable region of a TCR chain generally are separated by framework regions (FRs), which generally display less variability among TCR molecules as compared to the CDRs (see, e.g., lores et al., Proc. Nat'l Acad. Sci. U.S.A. 57:9138, 1990; Chothia et al., EMBO J. 7:3745, 1988; see also Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). In some embodiments, CDR3 is the main CDR responsible for antigen binding or specificity, or is the most important among the three CDRs on a given TCR variable region for antigen recognition, and/or for interaction with the processed peptide portion of the peptide-MHC complex. In some contexts, the CDR1 of the alpha chain can interact with the N-terminal part of certain antigenic peptides. In some contexts, CDR1 of the beta chain can interact with the C-terminal part of the peptide. In some contexts, CDR2 contributes most strongly to or is the primary CDR responsible for the interaction with or recognition of the MHC portion of the MHC-peptide complex. In some embodiments, the variable region of the P-chain can contain a further hypervariable region (CDR4 or HVR4), which generally is involved in superantigen binding and not antigen recognition (Kotb (1995) Clinical Microbiology Reviews, 8:411-426).

[0155] In some embodiments, the TCRa chain and/or TCRP chain also can contain a constant domain, a transmembrane domain and/or a short cytoplasmic tail (see, e.g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 3^rd Ed., Current Biology Publications, p. 4:33, 1997). In some aspects, each chain (e.g. alpha or beta) of the TCR can possess one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminal end. In some embodiments, a TCR, for example via the cytoplasmic tail, is associated with invariant proteins of the CD3 complex involved in mediating signal transduction. In some cases, the structure allows the TCR to associate with other molecules like CD3 and subunits thereof. For example, a TCR containing constant domains with a transmembrane region may anchor the protein in the cell membrane and associate with invariant subunits of the CD3 signaling apparatus or complex. The intracellular tails of CD3 signaling subunits (e.g. CD3y, CD36, CD3s and CD3^ chains) contain one or more immunoreceptor tyrosine-based activation motif or IT AM and generally are involved in the signaling capacity of the TCR complex.

[0156] The various domains or regions of a TCR can be identified. In some cases, the exact locus of a domain or region can vary depending on the particular structural or homology modeling or other features used to describe a particular domain. It is understood that reference to amino acids, including to a specific sequence set forth as a SEQ ID NO: used to describe domain organization of a TCR are for illustrative purposes and are not meant to limit the scope of the embodiments provided. In some cases, the specific domain (e.g. variable or constant) can be several amino acids (such as one, two, three or four) longer or shorter. In some aspects, residues of a TCR are known or can be identified according to the International Immunogenetics Information System (IMGT) numbering system (see e.g. www.imgt.org; see also, Lefranc et al. (2003) Developmental and Comparative Immunology, 27(l);55-77; and The T Cell Factsbook 2nd Edition, Lefranc and LeFranc Academic Press 2001). Using this system, the CDR1 sequences within a TCR Va region and/or VP region correspond to the amino acids present between residue numbers 27-38, inclusive, the CDR2 sequences within a TCR Va region and/or VP region correspond to the amino acids present between residue numbers 56-65, inclusive, and the CDR3 sequences within a TCR Va region and/or VP region correspond to the amino acids present between residue numbers 105-117, inclusive.

[0157] In some embodiments, an exemplary recombinant TCR contains a TCRa chain comprising TCR Va region and a TCRP chain comprising a VP region in which: (1) the Va region comprises a complementarity determining region 1 (CDR-1) comprising the sequence of SEQ ID NO:26 or a sequence that comprises at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto; a complementarity determining region 1 (CDR-2) comprising the sequence of SEQ ID NO:27 or a sequence that comprises at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto; a complementarity determining region 1 (CDR-3) comprising the sequence of SEQ ID NO:28 or a sequence that comprises at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto; and (2) the VP region comprises a complementarity determining region 1 (CDR-1) comprising the sequence of SEQ ID NO:30 or a sequence that comprises at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, a complementarity determining region 1 (CDR-2) comprising the sequence of SEQ ID NO:31 or a sequence that comprises at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, and a complementarity determining region 1 (CDR-3) comprising the sequence of SEQ ID NO:32 or a sequence that comprises at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto.

[0158] In some embodiments, the Va region comprises a CDR-1 comprising the sequence of SEQ ID NO:26, a CDR-2 comprising the sequence of SEQ ID NO:27, and a CDR-3 comprising the sequence of SEQ ID NO:28; and the VP region comprises a CDR-1 comprising the sequence of SEQ ID NO:30, a CDR-2 comprising the sequence of SEQ ID NO:31, and a CDR-3 comprising the sequence of SEQ ID NO:32.

[0159] In some embodiments, the TCR or antigen-binding fragment includes a Va region that contains a CDR-1, a CDR-2, and a CDR-3, respectively comprising the CDR-1, the CDR-2, and the CDR-3 amino acid sequences set forth in Table 2 and a VP region that contains a CDR-1, a CDR-2, and a CDR-3, respectively comprising the CDR-1, the CDR-2, and the CDR-3 amino acid sequences set forth in Table 2. Exemplary TCRs containing such CDRs, or their modified versions as described elsewhere herein, also are set forth in the Table 2. Also among the provided TCRs are those containing sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.

[0160] In some embodiments, the Va region comprise the sequence of SEQ ID NO:29, or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:29; and the VP region comprise the sequence of SEQ ID NO:1, or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:33. [0161] In some embodiments, the Va region comprise the sequence of SEQ ID NO:29; and the VP region comprise the sequence of SEQ ID NO:33.

[0162] In some embodiments, the TCRa chain and/or TCRP chain each further contain a constant domain. In some embodiments, the a chain constant domain (Ca) and P chain constant domain (CP) individually are mammalian, such as is a human or murine constant domain. In some embodiments, the constant domain is adjacent to the cell membrane. For example, in some cases, the extracellular portion of the TCR formed by the two chains contains two membrane-proximal constant domains, and two membrane-distal variable domains, which variable domains each contain CDRs.

[0163] In some embodiments, the TCR may be a heterodimer of two chains TCRa and TCRP that are linked, such as by a disulfide bond or disulfide bonds. In some embodiments, the constant domain of the TCR may contain short connecting sequences in which a cysteine residue forms a disulfide bond, thereby linking the two chains of the TCR. In some embodiments, a TCR may have an additional cysteine residue in each of the TCRa and TCRP chains, such that the TCR contains two disulfide bonds in the constant domains. In some embodiments, each of the constant and variable domains contains disulfide bonds formed by cysteine residues.

[0164] In some embodiments, the TCR can contain an introduced disulfide bond or bonds. In some embodiments, the native disulfide bonds are not present. In some embodiments, the one or more of the native cysteines (e.g. in the constant domain of the a chain and P chain) that form a native interchain disulfide bond are substituted to another residue, such as to a serine or alanine. In some embodiments, an introduced disulfide bond can be formed by mutating non-cysteine residues on the alpha and beta chains, such as in the constant domain of the a chain and P chain, to cysteine. Opposing cysteines in the TCRa and TCRP chains provide a disulfide bond that links the constant regions of TCRa and TCRP chains of the substituted TCR to one another and which is not present in a TCR comprising the unsubstituted constant region in which the native disulfide bonds are present, such as unsubstituted native human constant region or the unsubstituted native mouse constant region. In some embodiments, the presence of non-native cysteine residues (e.g. resulting in one or more non-native disulfide bonds) in a recombinant TCR can favor production of the desired recombinant TCR in a cell in which it is introduced over expression of a mismatched TCR pair containing a native TCR chain. In some embodiments, the TCRa and/or TCRP chain and/or a TCRa and/or TCRP chain constant domains are modified to replace one or more non-cysteine residues to a cysteine.

[0165] In some embodiments, the one or more non-native cysteine residues are capable of forming non-native disulfide bonds, e.g., between the recombinant TCRa and TCRP chain encoded by the transgene. In some embodiments, the cysteine is introduced at one or more of residue Thr48, Thr45, Tyrl0, Thr45, and Serl5 with reference to numbering of a TCRa constant domain (Ca) set forth in SEQ ID NO: 34. In certain embodiments, cysteines can be introduced at residue Ser57, Ser77, Serl7, Asp59, of Glul5 of the TCRP chain with reference to numbering of TCRP constant domain (CP) set forth in SEQ ID NO: 35. Exemplary non- native disulfide bonds of a TCR are described in W02006/000830, WO 2006/037960 and Kuball et al. (2007) Blood, 109:2331-2338. In some embodiments, the transgene encodes a portion of a TCRa chain and/or a TCRa constant domain containing one or more modifications to introduce one or more disulfide bonds. In some embodiments, cysteines can be introduced or substituted at a residue corresponding to Thr48 of the Ca chain and Ser57 of the CP chain, at residue Thr45 of the Ca chain and Ser77 of the CP chain, at residue Tyrl0 of the Ca chain and Serl7 of the CP chain, at residue Thr45 of the Ca chain and Asp59 of the CP chain and/or at residue Serl5 of the Ca chain and Glul5 of the CP chain with reference to numbering of a Ca set forth in SEQ ID NO:34, or a CP set forth in SEQ ID NO:35.

[0166] In some embodiments, the TCRa constant domain (Ca) of the recombinant TCR encoded by the transgene contains a cysteine at a position corresponding to position 48 with numbering as set forth in SEQ ID NO: 34. In some embodiments, the TCRa constant domain has an amino acid sequence set forth in any of SEQ ID NOS: 37, 40-45, 46, 47, and 48-51, or a sequence of amino acids that has, has about, or has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% sequence identity thereto containing one or more cysteine residues capable of forming a non-native disulfide bond with a TCRP chain.

[0167] In some embodiments, the TCRP constant domain (CP) of the recombinant TCR encoded by the transgene contains a cysteine at a position corresponding to position 57 with numbering as set forth in SEQ ID NO: 35. In some embodiments, the TCRP constant domain has an amino acid sequence set forth in any of SEQ ID NOS: 36, 55, 35, 56, 57, 58, and 59, or a sequence of amino acids that has, has about, or has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% sequence identity thereto containing one or more cysteine residues capable of forming a non-native disulfide bond with a TCRa chain. [0168] In some embodiments, the Ca region comprises a sequence selected from any one of SEQ ID NOS: 37, 40-45, 46, 47, and 48-51 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS: 37, 40-45, 46, 47, and 48-51 and/or the CP region comprises a sequence selected from any one of SEQ ID NOS:36, 55, 35, 56, 57, 58, and 59 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS: 36, 55, 35, 56, 57, 58, and 59. In some embodiments, the Ca region comprises a sequence selected from any one of SEQ ID NOS: 37, 40-45, 46, 47, and 48-51; and the CP region comprises a sequence selected from any one of SEQ ID NOS:36, 55, 35, 56, 57, 58, and 59.

[0169] In some embodiments, the Ca region comprises the sequence of SEQ ID NO:37, or a sequence that has at least at or about 90% sequence identity to SEQ ID NO:37; and the CP region comprises the sequence of SEQ ID NO:36, or a sequence that has at least at or about 90% sequence identity to SEQ ID NO:36. In some embodiments, the Ca region comprises the sequence of SEQ ID NO:37; and the CP region comprises the sequence of SEQ ID NO:36.

[0170] Exemplary TCRs or antigen-binding fragments include those set forth in Table 3, such as in each row therein. In some embodiments, the Va and VP regions contain the amino acid sequences corresponding to the SEQ ID NOs: set forth in Table 3, such as in each row therein. In some embodiments, the Va and VP regions contain the CDR-1, the CDR-2 and the CDR-3 sequences contained within the Va and VP regions set forth in Table 3, such as in each row therein. In some aspects, the TCR contains constant alpha and constant beta region sequences, such as those corresponding to the SEQ ID NOs: set forth in Table 3, such as in each row therein. In some cases, the TCR contains a full sequence comprising the variable and constant chain, such as a sequence corresponding to the SEQ ID NOs: set forth in Table 3 (“Full”), such as in each row therein. Also among the provided TCRs are those containing sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences. Exemplary TCRs containing such sequences, or their modified versions as described elsewhere herein, also are set forth in the Table 3, respectively, such as in each row therein.

[0171] In particular embodiments, an engineered TCR therapy is a T cell therapy engineered with a recombinant TCR containing a TCRa chain with a Va having a CDR1, 2 and 3 set forth in SEQ ID NOS: 26, 27, and 28, respectively, and a TCRP chain with a VP having a CDR1, 2, and 3 set forth in SEQ ID NOS: 30, 31 and 32, respectively. In some embodiments, the recombinant TCR contains a Va set forth in SEQ ID NO: 29 and a VP set forth in SEQ ID NO: 33. In some embodiments, the recombinant TCR contains a TCRa chain and a TCRP chain that further contains a Ca region and a CP region, respectively. In some embodiments, the Ca and CP are human constant regions or are functional variants thereof. In some embodiments, the recombinant TCR contains a TCRa chain containing a Ca human constant region or a variant thereof containing a non-native cysteine replacement, such as any described herein. In some embodiments, the recombinant TCR contains a TCRa chain containing a Ca constant region set forth in SEQ ID NO:37. In some embodiments, the recombinant TCR contains a TCRP chain containing a CP human constant region or a variant thereof containing a non-native cysteine replacement, such as any described herein. In some embodiments, the recombinant TCR contains a TCRP chain containing a CP region set forth in SEQ ID NO:36. In some embodiments, the recombinant TCR contains an alpha chain set forth in SEQ ID NO:52 and a beta chain set forth in SEQ ID NO:53. In some embodiments, the recombinant TCR contains a TCRa chain set forth in SEQ ID NO:39 and a TCRP chain set forth in SEQ ID NO:38. In some embodiments, the TCR is encoded by a polynucleotide that encodes the sequence set forth in SEQ ID NO:54. Also among the provided TCRs are those containing sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to any of such sequences.

[0172] In some embodiments, the cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is carried out by autologous transfer, in which the cells are isolated and/or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, following isolation and processing are administered to the same subject. [0173] In some embodiments, the cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is carried out by allogeneic transfer, in which the cells are isolated and/or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject. In such embodiments, the cells then are administered to a different subject, e.g., a second subject, of the same species. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.

[0174] In some embodiments, the subject, to whom the cells, cell populations, or compositions are administered, is a primate, such as a human. In some embodiments, the primate is a monkey or an ape. The subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. In some embodiments, the subject is a non-primate mammal, such as a rodent. In some examples, the patient or subject is a validated animal model for disease, adoptive cell therapy, and/or for assessing toxic outcomes such as cytokine release syndrome (CRS).

[0175] The provided cells expressing a recombinant TCRs or antigen-binding fragments thereof, and cells expressing the same, can be administered by infusion, such as intravenous infusion.

[0176] For the prevention or treatment of disease, the appropriate dose of cells expressing the recombinant TCR may depend on the type of disease to be treated, the severity and course of the disease, whether the recombinant TCR is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the recombinant TCR, and the discretion of the attending physician. The cells may in some embodiments suitably administered to the patient at one time or over a series of treatments.

[0177] In certain embodiments, in the context of genetically engineered cells expressing a recombinant TCR, a subject is administered the range of at or about one million to at or about 200 billion cells, such as, e.g., 1 million to at or about 50 billion cells (e.g., at or about 5 million cells, at or about 25 million cells, at or about 500 million cells, at or about 1 billion cells, at or about 5 billion cells, at or about 20 billion cells, at or about 30 billion cells, at or about 40 billion cells, or a range defined by any two of the foregoing values), or such as at or about 10 million to at or about 100 billion cells (e.g., at or about 20 million cells, at or about 30 million cells, at or about 40 million cells, at or about 60 million cells, at or about 70 million cells, at or about 80 million cells, at or about 90 million cells, at or about 10 billion cells, at or about 25 billion cells, at or about 50 billion cells, at or about 75 billion cells, at or about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases at or about 100 million cells to at or about 50 billion cells (e.g., at or about 120 million cells, at or about 250 million cells, at or about 350 million cells, at or about 450 million cells, at or about 650 million cells, at or about 800 million cells, at or about 900 million cells, at or about 3 billion cells, at or about 30 billion cells, at or about 45 billion cells) or any value in between these ranges, and/or such a number of cells per kilogram of body weight of the subject. In some embodiments, in the context of genetically engineered cells comprising the recombinant TCR, a subject is administered at or about 10 million cells, at or about 100 million cells, at or about 1 billion cells, at or about 10 billion cells, at or about 100 billion cells, or any value in between these ranges and/or per kilogram of body weight.

[0178] Dosages may vary depending on attributes particular to the disease or disorder and/or patient and/or other treatments. In some embodiments, such values refer to numbers of recombinant receptor-expressing cells.

[0179] In some embodiments, the dose of genetically engineered T cells comprises between at or about 3 x 10⁷ recombinant TCR-expressing T cells and at or about 3 x 10¹⁰ recombinant TCR-expressing T cells, inclusive. In some embodiments, the dose of genetically engineered T cells comprises between at or about 1 x 10⁸ recombinant TCR- expressing T cells and at or about 1 x 10¹⁰ recombinant TCR-expressing T cells, inclusive. In some embodiments, the dose of genetically engineered T cells comprises between at or about 1 x 10⁸ recombinant TCR-expressing T cells and at or about 1 x 10⁹ recombinant TCR- expressing T cells, inclusive.

[0180] In some embodiments, the dose of genetically engineered T cells comprises: at or about 1 x 10⁸ recombinant TCR-expressing T cells; at or about 3 x 10⁸ recombinant TCR- expressing T cells; at or about 1 x 10⁹ recombinant TCR-expressing T cells; at or about 3 x 10⁸ recombinant TCR-expressing T cells; or at or about 1 x 10¹⁰ recombinant TCR- expressing T cells.

[0181] In some embodiments, the dose of genetically engineered T cells comprises at or about 1 x 10⁸ recombinant TCR-expressing T cells. In some embodiments, the dose of genetically engineered T cells comprises at or about 3 x 10⁸ recombinant TCR-expressing T cells. In some embodiments, the dose of genetically engineered T cells comprises at or about 1 x I0⁹ recombinant TCR-expressing T cells. In some embodiments, the dose of genetically engineered T cells comprises at or about 3 x 10⁹ recombinant TCR-expressing T cells. In some embodiments, dose of genetically engineered T cells comprises at or about 1 x I0¹⁰ recombinant TCR-expressing T cells. In some embodiments, the dose of genetically engineered T cells comprises CD4+ T cells and/or CD 8+ T cells.

[0182] In some embodiments, for example, where the subject is a human, the dose includes fewer than about 3 x 10¹¹ total recombinant TCR-expressing cells, e.g., in the range of from at or about 1 x 10⁶ to at or about 1.5 x 10¹¹ total of such cells, such as at or about 1 x 10⁷, 3 x 10⁷, 1 x 10⁸, 5 x 10⁸, 1 x 10⁹, 1 x I0¹⁰, 5 x I0¹⁰, 1 x 10¹¹, 1.25 x 10¹¹, 2 x 10¹¹ total such cells, or the range between any two of the foregoing values. In some embodiments, for example, where the subject is a human, the dose includes more than at or about 1 x 10⁷ total recombinant TCR-expressing cells, and fewer than at or about 1 x 10¹¹ total recombinant TCR-expressing cells, e.g., in the range of at or about 1 x 10⁷ to at or about 1 x 10¹¹ such cells, such as at or about 5 x 10⁷, 1 x 10⁸, 5 x 10⁸, 1 x 10⁹, 1 x I0¹⁰, 5 x I0¹⁰, 7.5 x I0¹⁰, 1 x 10¹¹ total of such cells, or the range between any two of the foregoing values.

[0183] In some embodiments, the dose of genetically engineered cells comprises from at or about 1 x 10⁶ to at or about 2 x 10¹¹ total TCR-expressing cells, from at or about 1 x 10⁶ to at or about 1.5 x 10¹¹ total TCR-expressing cells, from at or about 1 x 10⁶ to at or about 1 x 10¹¹ total TCR-expressing cells, from at or about 1 x 10⁶ to at or about 5 x I0¹⁰ total TCR- expressing cells, from at or about 1 x 10⁶ to at or about 1 x I0¹⁰ total TCR-expressing cells, from at or about 1 x 10⁶ to at or about 1 x 10⁹ total TCR-expressing cells, from at or about 1 x 10⁶ to at or about 5 x 10⁸ total TCR-expressing cells, from at or about 1 x 10⁶ to at or about 1 x 10⁸ total TCR-expressing cells, from at or about 1 x 10⁶ to at or about 5 x 10⁷ total TCR- expressing cells, from at or about 1 x 10⁶ to at or about 1 x 10⁷ total TCR-expressing cells, from at or about 1 x 10⁶ to at or about 5 x 10⁶ total TCR-expressing cells, from at or about 1 x 10⁶ to at or about 2.5 x 10⁶ total TCR-expressing cells, from at or about 1 x 10⁶ to at or about

2 x 10⁶ total TCR-expressing cells, from at or about 2 x 10⁶ to at or about 2 x 10¹¹ total TCR- expressing cells, from at or about 2.5 x 10⁶ to at or about 2 x 10¹¹ total TCR-expressing cells, from at or about 5 x 10⁶ to at or about 2 x 10¹¹ total TCR-expressing cells, from at or about 1 x 10⁷ to at or about 2 x 10¹¹ total TCR-expressing cells, from at or about 3 x 10⁷ to at or about 2 x 10¹¹ total TCR-expressing cells, from at or about 1 x 10⁸ to at or about 2 x 10¹¹ total TCR-expressing cells, from at or about 5 x 10⁸ to at or about 2 x 10¹¹ total TCR- expressing cells, from at or about 1 x 10⁹ to at or about 2 x 10¹¹ total TCR-expressing cells, from at or about 1 x I0¹⁰ to at or about 2 x 10¹¹ total TCR-expressing cells, from at or about 5 x I0¹⁰ to at or about 2 x 10¹¹ total TCR-expressing cells, from at or about 1 x 10⁶ to at or about 2 x 10¹¹ total TCR-expressing cells, from at or about 1.5 x I0¹⁰ to at or about 2 x 10¹¹ total TCR-expressing cells. In some embodiments, the dose of genetically engineered cells comprises from or from about 1 x 10⁷ to at or about 1 x 10¹¹ total TCR-expressing cells, such as from or from about 1 x 10⁹ to or to about 1 x I0¹⁰ total TCR-expressing cells.

[0184] In some embodiments, the dose of genetically engineered cells comprises less than at or about 2 x 10¹¹ TCR-expressing cells, less than at or about 1.75 x 10¹¹ TCR-expressing cells, less than at or about 1.5 x 10¹¹ TCR-expressing cells, less than at or about 1.25 x 10¹¹ TCR-expressing cells, less than at or about 1 x 10¹¹ TCR-expressing cells, less than at or about 7.5 x I0¹⁰ TCR-expressing cells, less than at or about 5 x I0¹⁰ TCR-expressing cells, less than at or about 2.5 x I0¹⁰ TCR-expressing cells, less than at or about 1 x I0¹⁰ TCR- expressing cells, less than at or about 5 x 10⁹ TCR-expressing cells, less than at or about 1 x 10⁹ TCR-expressing cells, less than at or about 5 x 10⁸ TCR-expressing cells, less than at or about 6 x 10⁷ TCR-expressing cells, less than at or about 3 x 10⁷ TCR-expressing cells, less than at or about 1 x 10⁷ TCR-expressing cells, less than at or about 5 x 10⁶ TCR-expressing cells, less than at or about 1 x 10⁶ TCR-expressing cells.

[0185] In some embodiments, the dose of genetically engineered cells comprises at or about 3 x 10¹¹ TCR-expressing cells, at or about 2 x 10¹¹ TCR-expressing cells, at or about 1.75 x 10¹¹ TCR-expressing cells, at or about 1.5 x 10¹¹ TCR-expressing cells, at or about 1.25 x 10¹¹ TCR-expressing cells, at or about 1 x 10¹¹ TCR-expressing cells, at or about 7.5 x I0¹⁰ TCR-expressing cells, at or about 5 x I0¹⁰ TCR-expressing cells, at or about 2.5 x I0¹⁰ TCR-expressing cells, at or about 1 x I0¹⁰ TCR-expressing cells, at or about 5 x 10⁹ TCR- expressing cells, at or about 1 x 10⁹ TCR-expressing cells, at or about 5 x 10⁸ TCR- expressing cells, at or about 1 x 10⁸ TCR-expressing cells, at or about 5 x 10⁷ TCR- expressing cells, at or about 3 x 10⁷ TCR-expressing cells, at or about 1 x 10⁷ TCR- expressing cells, at or about 5 x 10⁶ TCR-expressing cells, at or about 1 x 10⁶ TCR- expressing cells.

[0186] In some embodiments, the dose of cells comprises between at or about 2 x 10⁵ of the cells/kg and at or about 2 x 10⁶ of the cells/kg, such as between at or about 4 x 10⁵ of the cells/kg and at or about 1 x 10⁶ of the cells/kg or between at or about 6 x 10⁵ of the cells/kg and at or about 8 x 10⁵ of the cells/kg. In some embodiments, the dose of cells comprises no more than 2 x 10⁵ of the cells (e.g. recombinant TCR-expressing cells) per kilogram body weight of the subject (cells/kg), such as no more than at or about 3 x 10⁵ cells/kg, no more than at or about 4 x 10⁵ cells/kg, no more than at or about 5 x 10⁵ cells/kg, no more than at or about 6 x 10⁵ cells/kg, no more than at or about 7 x 10⁵ cells/kg, no more than at or about 8 x 10⁵ cells/kg, no more than at or about 9 x 10⁵ cells/kg, no more than at or about 1 x 10⁶ cells/kg, or no more than at or about 2 x 10⁶ cells/kg. In some embodiments, the dose of cells comprises at least at or about 2 x 10⁵ of the cells (e.g. recombinant TCR-expressing cells) per kilogram body weight of the subject (cells/kg), such as at least at or about 3 x 10⁵ cells/kg, at least at or about 4 x 10⁵ cells/kg, at least at or about 5 x 10⁵ cells/kg, at least at or about 6 x 10⁵ cells/kg, at least at or about 7 x 10⁵ cells/kg, at least at or about 8 x 10⁵ cells/kg, at least at or about 9 x 10⁵ cells/kg, at least at or about 1 x 10⁶ cells/kg, or at least at or about 2 x 10⁶ cells/kg.

[0187] In some embodiments, the populations or sub-types of cells, such as CD8⁺ and CD4⁺ T cells, are administered at or within a tolerated difference of a desired dose of total cells, such as a desired dose of T cells. In some aspects, the desired dose is a desired number of cells or a desired number of cells per unit of body weight of the subject to whom the cells are administered, e.g., cells/kg. In some aspects, the desired dose is at or above a minimum number of cells or minimum number of cells per unit of body weight. In some aspects, among the total cells, administered at the desired dose, the individual populations or subtypes are present at or near a desired output ratio (such as CD4⁺ to CD8⁺ ratio), e.g., within a certain tolerated difference or error of such a ratio.

[0188] In some embodiments, the cells are administered at or within a tolerated difference of a desired dose of one or more of the individual populations or sub-types of cells, such as a desired dose of CD4+ cells and/or a desired dose of CD8+ cells. In some aspects, the desired dose is a desired number of cells of the sub-type or population, or a desired number of such cells per unit of body weight of the subject to whom the cells are administered, e.g., cells/kg. In some aspects, the desired dose is at or above a minimum number of cells of the population or sub-type, or minimum number of cells of the population or sub-type per unit of body weight.

[0189] Thus, in some embodiments, the dosage is based on a desired fixed dose of total cells and a desired ratio, and/or based on a desired fixed dose of one or more, e.g., each, of the individual sub-types or sub-populations. Thus, in some embodiments, the dosage is based on a desired fixed or minimum dose of T cells and a desired ratio of CD4⁺ to CD8⁺ cells, and/or is based on a desired fixed or minimum dose of CD4⁺ and/or CD8⁺ cells.

[0190] In some embodiments, the cells are administered at or within a tolerated range of a desired output ratio of multiple cell populations or sub-types, such as CD4⁺ and CD8⁺ cells or sub-types. In some aspects, the desired ratio can be a specific ratio or can be a range of ratios, for example, in some embodiments, the desired ratio (e.g., ratio of CD4⁺ to CD8⁺ cells) is between at or about 5:1 and at or about 5:1 (or greater than about 1:5 and less than about 5:1), or between at or about 1:3 and at or about 3:1 (or greater than about 1:3 and less than about 3:1), such as between at or about 2:1 and at or about 1:5 (or greater than about 1:5 and less than about 2:1, such as at or about 5:1, 4.5:1, 4:1, 3.5:1, 3:1, 2.5:1, 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9: 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5. In some aspects, the tolerated difference is within about 1%, about 2%, about 3%, about 4% about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50% of the desired ratio, including any value in between these ranges.

[0191] In some embodiments, the dose of genetically engineered T cells comprises CD4+ T cells and CD8+ T cells, and the percentage of CD4+ T cells in the dose is between at or about 20% and at or about 80%, or at or about 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the total cells in the dose; and/or the percentage of CD8+ T cells in the dose is between at or about 20% and at or about 80%, or at or about 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the total cells in the dose.

[0192] In some embodiments, the dose of genetically engineered T cells comprises CD4+ T cells and CD8+ T cells, and the percentage of CD4+ T cells in the dose is between at or about 20% and at or about 80%, or at or about 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the total cells in the dose; and the percentage of CD8+ T cells in the dose is between at or about 20% and at or about 80%, or at or about 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the total cells in the dose.

[0193] In some embodiments, the dose of genetically engineered T cells comprises CD4+ T cells and CD8+ T cells, and the percentage of CD4+ T cells in the dose is at or about 50% of the total cells in the dose; and the percentage of CD8+ T cells in the dose is at or about 50% of the total cells in the dose. [0194] In some embodiments, the dose of genetically engineered T cells comprises CD4+ T cells and CD8+ T cells, and the ratio of CD4+ T cells to CD8+ T cells is from at or about 1:3 to at or about 3:1. In some embodiments, the dose of genetically engineered T cells comprises CD4+ T cells and CD8+ T cells, and the ratio of CD4+ T cells to CD8+ T cells is at or about 1:1.

[0195] In particular embodiments, the numbers and/or concentrations of cells refer to the number of recombinant receptor-expressing cells. In other embodiments, the numbers and/or concentrations of cells refer to the number or concentration of all cells, T cells, or peripheral blood mononuclear cells (PBMCs) administered.

[0196] In some embodiments, the dose of cells, e.g., recombinant TCR-expressing T cells, is administered to the subject as a single dose or is administered only one time within a period of two weeks, one month, three months, six months, 1 year or more. In some embodiments, the subject is administered one or more doses.

[0197] In some embodiments, the recombinant TCRs or cells are administered as part of a combination treatment, such as simultaneously with or sequentially with, in any order, another therapeutic intervention, such as another TCR, antibody or engineered cell or receptor or agent, such as a cytotoxic or therapeutic agent.

[0198] The cells or antibodies in some embodiments are co-administered with one or more additional therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order. In some contexts, the cells are coadministered with another therapy sufficiently close in time such that the cell populations enhance the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the cells or antibodies are administered prior to the one or more additional therapeutic agents. In some embodiments, the cells or antibodies are administered after to the one or more additional therapeutic agents.

[0199] Once the cells are administered to a mammal (e.g., a human), the biological activity of the engineered cell populations and/or recombinant TCRs in some aspects is measured by any of a number of known methods. Parameters to assess include specific binding of an engineered or natural T cell or other immune cell to antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J. Immunological Methods, 285(1): 25-40 (2004). In certain embodiments, the biological activity of the cells also can be measured by assaying expression and/or secretion of certain cytokines, such as CD 107a, IFNy, IL-2, and TNF. In some aspects the biological activity is measured by assessing clinical outcome, such as reduction in tumor burden or load.

III. KITS

[0200] Also provided herein are kits, for example comprising one or more oligonucleotide primers disclosed herein, one or more oligonucleotide probes, and reagents for performing the methods provided herein, for example reagents required for one or more steps comprising sample preparation, RNA extraction and purification, reverse-transcription, PCR, or qPCR as described herein. The various components of the kit may be present in separate containers or certain compatible components may be pre-combined into a single container. In some embodiments, the kits further contain instructions for using the components of the kit to practice the provided methods.

[0201] In some embodiments, the kits can contain reagents and/or consumables required for performing one or more steps of the provided methods. In some embodiments, the kits optionally contain other components, for example primers, enzymes, buffers, nucleotides, modified nucleotides, and reagents for additional assays.

[0202] In some embodiments, the kit can further comprise a pair of PCR primers specific for the gene of interest. In some embodiments, the kit can comprise a buffer. In certain embodiments, the kit can comprise deoxy nucleoside triphosphates or deoxynucleotide triphosphates (dNTPs), a thermostable DNA polymerase such as Taq polymerase, and all other reagents necessary for performing disclosed method, including but not limited to a buffer reagent, additional dNTP, and sequence specific amplification primers. In addition, the kit may comprise reagents necessary for detection of the amplicon during qPCR such as at least one fluorescently labeled hybridization probe, or a doubles stranded fluorescent dye.

IV. DEFINITIONS

[0203] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0204] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, “a” or “an” means “at least one” or “one or more.” It is understood that aspects and variations described herein include “consisting” and/or “consisting essentially of’ aspects and variations.

[0205] The term “about” as used herein refers to the usual error range for the respective value readily known. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”. In some embodiments, “about” may refer to ±25%, ±20%, ±15%, ±10%, ±5%, or ±1%.

[0206] The term “detectable moiety” as used herein refers to any atom or molecule which can be used to provide a detectable (optionally quantifiable) signal, and which can be attached to a nucleic acid or protein. Detectable moieties may provide signals detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, and the like.

[0207] The term “fluorescent moiety” as used herein refers to a chemical moiety that can emit light under conditions appropriate for the particular moiety. Typically, a particular fluorescent moiety can emit light of a particular wavelength following absorbance of light of shorter wavelength. The wavelength of the light emitted by a particular fluorescent moiety is characteristic of that moiety. Thus, a particular fluorescent moiety can be detected by detecting light of an appropriate wavelength following excitation of the fluorescent moiety with light of shorter wavelength.

[0208] As used herein, a “subject” is a mammal, such as a human or other animal, and typically is human.

[0209] As used herein, “amplicon” refers to an amplified nucleic acid product of a PCR reaction or other nucleic acid amplification process. For instance, an amplicon is produced by PCR amplification of a sample comprising nucleic acid in the presence of a nucleic acid polymerase and a specific primer pair. For purposes herein, the amplicon is composed of a nucleotide sequence derived from reverse-transcribed DNA from a self-inactivating viral nucleic acid, such as retroviral nucleic acid. For instance, the amplicon is not produced from viral vector transduction residuals, such as plasmids, that are episomal in a cell transduced with a viral vector. Typically, an amplicon is a DNA amplicon generated by PCR, such as RT-PCR.

[0210] As used herein, the term “oligonucleotide” refers to linear oligomers of natural or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, and the like, capable of specifically binding to a target polynucleotide by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type base pairing. For purposes herein, the term oligonucleotide includes both oligonucleotide probes and oligonucleotide primers.

[0211] As used herein, "oligonucleotide set" refers to a grouping of a pair of oligonucleotide primers and an oligonucleotide probe that hybridize to a specific nucleotide sequence the same gene. For instance, an oligonucleotide set is composed of: (a) a forward primer that hybridizes to a first location of a nucleic acid sequence of an HPV16 gene, such as HPV16 E7; (b) a reverse primer that hybridizes to a second location of the nucleic acid sequence of the HPV16 gene downstream of the first location and (c) a detectable probe labeled with a detectable moiety, such as a fluorphore, and, in some cases, a quencher, which hybridizes to a location of the nucleic acid sequence of the HPV gene between the primers. In some embodiments, an oligonucleotide set is composed of a pair of PCR primers capable of initiating synthesis of an amplicon specific to an HPV 16 gene, such as HPV 16 E7, and a fluorescent probe which hybridizes to the amplicon.

[0212] As used herein, the term “primer” refers to an oligonucleotide that hybridizes to the template strand of a nucleic acid and initiates synthesis of a nucleic acid strand complementary to the template strand when placed under conditions in which synthesis of a primer extension product is induced. Such conditions include the presence of nucleotides and a polymerization-inducing agent such as a DNA or RNA polymerase and at suitable temperature, pH, metal concentration, and salt concentration. For instance, a “primer” is complementary to a template, and complexes by hydrogen bonding or hybridization with the template to give a primer/template complex for initiation of synthesis by a polymerase, which is extended by the addition of covalently bonded bases linked at its 3' end complementary to the template in the process of DNA or RNA synthesis. As employed herein, an oligonucleotide primer can be naturally occurring, as in a purified restriction digest, or can be produced synthetically. The primer is preferably single-stranded for maximum efficiency in amplification. In some cases, a primer may alternatively be double- stranded. If doublestranded, the primer can first be treated to separate its strands before being used to prepare extension products. This denaturation step is typically effected by heat, but may alternatively be carried out using alkali, followed by neutralization.

[0213] As used herein a “pair of PCR primers”, is composed of a forward amplification primer and a reverse amplification primer. As used herein, “forward amplification primer” refers to a polynucleotide used for PCR amplification that is complementary to the sense (plus) strand of the target nucleic acid. “Reverse amplification primer” refers to a polynucleotide used for PCR amplification that is complementary to the antisense (minus) strand of the target nucleic acid. For a given target, a forward and reverse amplification primer are used to amplify the DNA in PCR. As used herein, “primer pair” refers to two primers, a forward primer and a reverse primer, that are capable of participating in PCR amplification of a segment of nucleic acid in the presence of a nucleic acid polymerase to produce a PCR amplicon. It is understood that the orientation of the primers is the direction in which the elongation of the primer in DNA synthesis occurs. Since DNA synthesis is 5' to 3' , the 3' ends of a PCR primer set (e.g. forward primer and reverse primer) point towards each other, when they are annealed to their template strand, and the primers anneal on opposite strands of the PCR template. For instance, the forward primer may anneal to (i.e. is complementary to) the minus template minus (-) strand and the reverse primer anneals to (i.e. is complementary to) the template (+) strand.

[0214] As used herein, a “detectable moiety” or “detectable label” refers to a molecule capable of detection, including, but not limited to, radioactive isotopes, fluorophores, chemiluminescers, chromophores, enzymes, enzyme substrates, enzyme co factors, enzyme inhibitors, semiconductor nanoparticles, dyes, metal ions, metal sols, ligands (e.g., biotin, streptavidin or haptens) and the like. The term "fluorescent moiety" refers to a substance or a portion thereof which is capable of exhibiting fluorescence in the detectable range. As used herein, “fluorophore” refers to a fluorescent reporter molecule which, upon excitation with a laser, tungsten, mercury or xenon lamp, or a light emitting diode, releases energy in the form of light with a defined spectrum. Through the process of fluorescence resonance energy transfer (FRET), the light emitted from the-fluorophore-can excite a second molecule whose excitation spectrum overlaps the emission spectrum of the fluorophore. The transfer of emission energy of the fluorophore to another molecule quenches the emission of the fluorophore. The second molecule is known as a quencher molecule. The term "fluorophore" is used interchangeably herein with the term "fluorescent reporter".

[0215] As used herein "quencher" or "quencher molecule" refers to a molecule that, when linked to a fluorescent probe comprising a fluorophore, is capable of accepting the energy emitted by a fluorophore, thereby quenching the emission of the fluorophore. A quencher can be fluorescent, which releases the accepted energy as light, or non-fluorescent, which releases the accepted energy as heat, and can be attached at any location along the length of the probe.

[0216] As used herein, “probe” refers to an oligonucleotide that is capable of forming a duplex structure with a sequence in a target nucleic acid, due to complementarity of at least one sequence of the probe with a sequence in the target region, or region to be detected. The term “probe” includes an oligonucleotide as described above, with or without a fluorophore and a quencher molecule attached. The term “fluorescent probe” refers to a probe comprising a fluorophore and a quencher molecule.

[0217] As used herein. “Cq” refers to quantification cycle values calculated from the record fluorescence measurements of the real time quantitative PCR. “Cq” refers to the number of cycles required for the PCR signal to reach a threshold level. In some embodiments, the threshold level is set at or above the baseline, such as within about 10 times the standard deviation of the noise of the baseline.

[0218] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range. V. EXEMPLARY EMBODIMENTS

[0219] Among the provided embodiments are:

1. A method of detecting human papillomavirus (HPV) in a subject, the method comprising (a) incubating a template DNA obtained from a biological sample in a first incubation with (i) an oligonucleotide set comprising a forward oligonucleotide primer, a reverse oligonucleotide primer, and an oligonucleotide probe and (ii) a DNA polymerase to produce a first pre-amplification reaction product; (b) incubating the first pre-amplification reaction product or sample thereof in a second incubation with the (i) the oligonucleotide set comprising the forward oligonucleotide primer, the reverse oligonucleotide primer, and the oligonucleotide probe and (ii) a DNA polymerase to produce a secondary reaction product, wherein the incubating in (a) and the incubating in (b) is under conditions sufficient for amplification of a HPV 16 amplicon if present in the sample; and (c) detecting the generated HPV 16 amplicon.

2. A method of amplifying a HPV 16 amplicon, the method comprising: (a) incubating a template DNA obtained from a biological sample in a first incubation with (i) an oligonucleotide set comprising a forward oligonucleotide primer, a reverse oligonucleotide primer, and an oligonucleotide probe, and (ii) a DNA polymerase to produce a first preamplification reaction product; (b) incubating the first pre-amplification reaction product or sample thereof in a second incubation with (i) the oligonucleotide set comprising the forward oligonucleotide primer, the reverse oligonucleotide primer, and the oligonucleotide probe, and (ii) a DNA polymerase to produce a secondary reaction product, wherein the incubating in (a) and the incubating in (b) is under conditions sufficient for amplification of a HPV 16 amplicon if present in the sample.

3. The method of embodiment 1 or embodiment 2, wherein the HPV 16 amplicon is an HPV 16 E7 amplicon.

4. The method of any of embodiments 1-3, wherein the first incubation and the second incubation individually is performed by polymerase chain reaction (PCR).

5. The method of embodiment 4, wherein the PCR is real-time PCR (RT-PCR).

6. The method of any of embodiments 1-5, wherein the template DNA is complementary DNA (cDNA). 7. The method of any of embodiments 1-6, wherein prior to the incubating in (a), the method comprises synthesizing the template DNA by reverse-transcribing an RNA template from the biological sample into cDNA.

8. The method of any of embodiments 1-7, wherein the DNA polymerase is a Taq DNA polymerase.

9. The method of any of embodiments 1-8, wherein the HPV16 amplicon is less than 120 base pairs.

10. The method of any one of embodiments 1-9, wherein the HPV16 amplicon is between 60 and 119 base pairs, inclusive.

11. The method of any one of embodiments 1-10, wherein the HPV16 amplicon is between 70 and 119 base pairs, inclusive.

12. The method of any one of embodiments 1-11, wherein the HPV16 amplicon is between 80 and 119 base pairs, inclusive.

13. The method of any one of embodiments 1-12, wherein the HPV16 amplicon is between 80 and 117 base pairs, inclusive.

14. The method of any one of embodiments 1-13, wherein the (i) the forward oligonucleotide primer comprises 15-30 nucleotides that are complementary to a plus strand of HPV16 E7 template DNA, and (ii) the reverse oligonucleotide primer comprises 15-30 nucleotides that are complementary to a minus strand of HPV16 E7 template DNA.

15. The method of any one of embodiments 1-14, wherein the (i) the forward oligonucleotide primer comprises 18-25 nucleotides that are complementary to a plus strand of HPV16 E7 template DNA, and (ii) the reverse oligonucleotide primer comprises 18-25 nucleotides that are complementary to minus strand of HPV16 E7 template DNA.

16. The method of any one of embodiments 1-15, wherein the forward oligonucleotide primer comprises a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to any one of SEQ ID NOs: 3, 6, 9, 12 or 15.

17. The method of any one of embodiments 1-16, wherein the forward oligonucleotide primer comprises the sequence set forth in any of SEQ ID NOs: 3, 6, 9, 12 or 15.

18. The method of any one of embodiments 1-17, wherein the reverse oligonucleotide primer comprises a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to any one of SEQ ID NOs: 4, 7, 10, 13 or 16. 19. The method of any one of embodiments 1-18, wherein the reverse oligonucleotide primer comprises the sequence set forth in any of SEQ ID NOs: 4, 7, 10, 13 or 16.

20. The method of any of embodiments 1-19, wherein the oligonucleotide probe comprises a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to any one of SEQ ID NOs: 5, 8, 11, 14 or 17.

21. The method of any of embodiments 1-20, wherein the oligonucleotide probe comprises one of any of the sequences as set forth in SEQ ID NOs: 5, 8, 11, 14 or 17.

22. The method of any one of embodiments 1-21, wherein the HPV16 amplicon detected is at or about 75-90 base pairs, optionally at or about 80 base pairs.

23. The method of any one of embodiments 1-22, wherein the forward oligonucleotide primer is set forth in SEQ ID NO: 9 and the reverse oligonucleotide primer is set forth in SEQ ID NO: 10.

24. The method of any one of embodiments 1-23, wherein the forward oligonucleotide primer is set forth in SEQ ID NO: 9, the reverse oligonucleotide primer is set forth in SEQ ID NO: 10 and the oligonucleotide probe is set forth in SEQ ID NO: 11.

25. The method of any one of embodiments 1-22, wherein the forward oligonucleotide primer is set forth in SEQ ID NO: 12 and the reverse oligonucleotide primer is set forth in SEQ ID NO: 13.

26. The method of any one of embodiments 1-22 and 25, wherein the forward oligonucleotide primer is set forth in SEQ ID NO: 12, the reverse oligonucleotide primer is set forth in SEQ ID NO: 13 and the oligonucleotide probe is set forth in SEQ ID NO: 14.

27. The method of any one of embodiments 1-22, wherein the forward oligonucleotide primer is set forth in SEQ ID NO: 15 and the reverse oligonucleotide primer is set forth in SEQ ID NO: 16.

28. The method of any one of embodiments 1-22 and 27, wherein the forward oligonucleotide primer is set forth in SEQ ID NO: 15, the reverse oligonucleotide primer is set forth in SEQ ID NO: 16 and the oligonucleotide probe is set forth in SEQ ID NO: 17.

29. The method of any one of embodiments 1-21, wherein the HPV16 amplicon is 95 to 105 base pairs, optionally at or about 100 base pairs. 30. The method of any one of embodiments 1-21 and 29, wherein the forward oligonucleotide primer is set forth in SEQ ID NO: 6 and the reverse oligonucleotide primer is set forth in SEQ ID NO: 7.

31. The method of any one of embodiments 1-21, 29 and 30, wherein the forward oligonucleotide primer is set forth in SEQ ID NO: 6, the reverse oligonucleotide primer is set forth in SEQ ID NO: 7 and the oligonucleotide probe is set forth in SEQ ID NO:8.

32. The method of any one of embodiments 1-21, wherein the HPV16 the amplicon is at or about 110 to 140 base pairs, optionally at or about 117 base pairs.

33. The method of any one of embodiments 1-21 and 32, wherein the forward oligonucleotide primer is set forth in SEQ ID NO: 3 and the reverse oligonucleotide primer is set forth in SEQ ID NO: 4.

34. The method of any one of embodiments 1-21, 32 and 33, wherein the forward oligonucleotide primer is set forth in SEQ ID NO: 3, the reverse oligonucleotide primer is set forth in SEQ ID NO: 4 and the oligonucleotide probe is set forth in SEQ ID NO:5.

35. The method of any of embodiments 1-34, wherein the oligonucleotide probe comprises a detectable moiety.

36. The method of embodiment 35, wherein the detectable moiety is a fluorescent moiety.

37. The method of embodiment 35 and 36, wherein the HPV16 amplicon is detected by measuring a detectable signal emitted from the detectable moiety of the second reaction product.

38. The method of embodiment 37, wherein the detectable signal is a fluorescent signal.

39. The method of embodiment 37 and 38, detecting the HPV16 amplicon comprises determining the quantification cycle (Cq) at which the detectable signal, optionally the fluorescent signal, exceeds background fluorescence.

40. The method of any one of embodiments 1-39, wherein the concentration of the oligonucleotide set in the first incubation step is less than the concentration in the second incubation step.

41. The method of embodiment 40, wherein the concentration of the oligonucleotide set in the first incubation is step is between 0.05-fold and 0.001-fold of the concentration in the second incubation step. 42. The method of embodiment 40 or embodiment 41, wherein the concentration of the oligonucleotide set in the first incubation is step is between 0.01 -fold and 0.002-fold of the concentration in the second incubation step.

43. The method of embodiment 40 or embodiment 41, wherein the concentration of the oligonucleotide set in the first incubation is step is at or about 0.0025-fold the concentration in the second incubation step.

44. The method of any one of embodiments 1-43, wherein the first incubation step comprises thermal profile conditions set forth as: 10 seconds at 95 °C followed by 10 cycles of amplification (95°C for 15 seconds and 60°C for 4 minutes).

45. The method of any one of embodiments 1-44, the second incubation step comprises thermal profile conditions set forth as: 50°C for 2 minutes, 95°C for 20 seconds, then 40 cycles of amplification comprising 95°C for 1 second and 60°C for 20 seconds.

46. The method of any one of embodiments 1-45, wherein the subject has, or is suspected of having head and neck cancer.

47. The method of embodiment 46, wherein the head and neck cancer is squamous cell carcinoma (HNSCC).

48. The method of any one of embodiments 1-47, wherein the biological sample is a tissue, a buccal sample, a saliva sample, or a blood sample.

49. The method of embodiment 48, wherein the tissue sample is a tumor tissue.

50. The method of any one of embodiments 1-49, wherein the sample is a tissue sample that has been formalin fixed and paraffin embedded.

51. A method of diagnosing human papillomavirus (HPV) in a subject, the method comprising:

(i) detecting a generated HPV 16 amplicon by the method of any of embodiments 1- 50; and

(ii) diagnosing a subject with HPV if a detectable amount of HPV is present in the sample.

52. The method of embodiment 51, wherein the detectable amount of HPV is determined by the quantification cycle (Cq).

53. An oligonucleotide set comprising a primer pair, comprising (i) a forward oligonucleotide primer comprising a nucleic acid sequence set forth in any of SEQ ID NOs: 6, 9, or 12, and (ii) a reverse oligonucleotide primer comprising a nucleic acid sequence set forth in any of SEQ ID NOs: 7, 10, or 13.

54. The oligonucleotide set of embodiment 53, further comprising an oligonucleotide probe set forth in any one of SEQ ID NOs: 5, 8, 11, 14 or 17.

55. The oligonucleotide set of embodiment 53 or embodiment 54, wherein (i) the forward oligonucleotide primer comprises a nucleic acid sequence set forth in SEQ ID NO: 6, and (ii) the reverse oligonucleotide primer comprises a nucleic acid sequence set forth in SEQ ID NO: 7.

56. The oligonucleotide set of embodiment 55, comprising the oligonucleotide probe is set forth in SEQ ID NO:8.

57. The oligonucleotide set of embodiment 53 or embodiment 54, wherein (i) the forward oligonucleotide primer comprises a nucleic acid sequence set forth in SEQ ID NO: 9, and (ii) the reverse oligonucleotide primer comprises a nucleic acid sequence set forth in SEQ ID NO: 10.

58. The oligonucleotide set of embodiment 57, comprising the oligonucleotide probe is set forth in SEQ ID NO: 11.

59. The oliognucleotide set of embodiment 53 or embodiment 54, wherein (i) the forward oligonucleotide primer comprises a nucleic acid sequence set forth in SEQ ID NO: 12, and (ii) the reverse oligonucleotide primer comprises a nucleic acid sequence set forth in SEQ ID NO: 13.

60. The oligonucleotide set of embodiment 59, comprising the oligonucleotide probe is set forth in SEQ ID NO: 14.

61. The oligonucleotide set of embodiment 53 or embodiment 54, wherein (i) the forward oligonucleotide primer comprises a nucleic acid sequence set forth in SEQ ID NO: 15, and (ii) the reverse oligonucleotide primer comprises a nucleic acid sequence set forth in SEQ ID NO: 16.

62. The oligonucleotide set of embodiment 61, comprising the oligonucleotide probe is set forth in SEQ ID NO: 17.

63. The oligonucleotide set of embodiment 53 or embodiment 54, wherein (i) the forward oligonucleotide primer comprises a nucleic acid sequence set forth in SEQ ID NO: 3, and (ii) the reverse oligonucleotide primer comprises a nucleic acid sequence set forth in SEQ ID NO: 4. 64. The oligonucleotide set of embodiment 63, comprising the oligonucleotide probe is set forth in SEQ ID NO:5.

65. The oligonucleotide set of any of embodiments 53-64, wherein the oligonucleotide probe comprises a detectable moiety.

66. The oligonucleotide set of embodiment 65, wherein the detectable moiety is a fluorescent moiety.

67. The oligonucleotide set of embodiment 65 or embodiment 66, wherein the detectable moiety is at the 5’ end of the oligonucleotide probe.

68. The oligonucleotide set of any one of embodiments 65-67, wherein the detectable moiety is selected from FAM, HEX, FITC, Texas Red, TET, JOE, VIC, NED, TAMRA, ROX, ABY, PET, JUN, LIZ, Cy3, or Cy5.

69. The oligonucleotide set of any one of embodiments 54-68, wherein the oligonucleotide probe comprises a minor groove binder (MGB) moiety, optionally wherein the MGB is at the 3’ end.

70. The oligonucleotide set of any of embodiments 54-69, wherein the oligonucleotide probe comprises a nonfluorescent quencher (NFQ), optionally wherein the NFQ is at the 3’ end.

71. The oligonucleotide set of any one of embodiments 65-70, wherein the detectable moiety is FAM-MGB.

72. A kit, comprising the oligonucleotide set of any of embodiments 53-71 and one or more reagents for carrying out a polymerase chain reaction.

73. The kit of embodiment 72, wherein the one or more reagents comprises a DNA polymerase.

74. The kit of embodiment 73, wherein the DNA polymerase is a Taq polymerase.

75. A method of treatment comprising administering a therapeutic to a subject in need thereof for treating an human papillomavirus (HPV) infection, wherein: the subject is selected for treatment if an HPV 16 amplicon is detected in a sample obtained from said subject using the method of any one of embodiments 1-52.

76. A method of treatment, the method comprising: selecting a subject in which an HPV16 amplicon is detected according to any of methods of embodiments 1-52; and administering to the selected subject a therapeutic agent for treating an human papillomavirus (HPV) infection. 77. A method of treatment comprising administering a therapeutic to a subject in need thereof for treating an human papillomavirus (HPV) infection, wherein the subject is diagnosed with an HPV infection by the method of embodiment 75.

78. The method of any of embodiments 75-77, wherein the therapeutic for treating an HPV infection is selected from the group consisting of vaccines that induce or boost HPV T cell adaptive immunity, adoptive cell therapy, therapeutic antibodies, antiviral therapeutics, immune response modifier compounds, proteasome inhibitors, HD AC inhibitors, and drugs targeting HPV genes.

79. The method of any of embodiments 75-78, wherein the therapeutic is a T cell therapy comprising a T cells expressing a recombinant antigen receptor specific to an HPV16 epitope.

80. The method of embodiment 79, wherein the HPV 16 epitope is an HVP16 E7 epitope.

81. The method of embodiment 80, wherein the HPV 16 eptiope is HPV E7 (11- 19).

82. The method of any of embodiments 75-81, wherein the subject has or is suspected of having a cancer.

83. The method of any one of embodiments 75-82, wherein the subject has, or is suspected of having head and neck cancer.

84. The method of embodiment 83, wherein the head and neck cancer is squamous cell carcinoma (HNSCC).

VI. EXAMPLES

[0220] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1 Development of a Real-Time RNA-PCR Assay for Detection of HPV in Patient Samples

[0221] Real-time HPV- 16 RNA-PCR-based assay methods were developed to identify, assess, and/or diagnose HPV status or positivity in samples. Five custom real-time RNA-PCR assays were designed and developed for detection of HPV 16 E7 transcript. Primers and probes targeting the HPV- 16 E7 transcript were designed using the NCBI (National Center for Biotechnology Information) reference sequence NC_001526.4:7604-7900 Human papillomavirus type 16. The amplification products were designed to be <120 bp in length to ensure optimal performance for highly fragmented RNA from formalin fixed paraffin embedded samples. The primer sequences, probe sequences, melting temperatures, and predicted amplicon lengths for each of these five RNA-PCR assay methods are provided in Table E1A. The designed primers and probes were in a premixed 20x or 40x solution. Each probe was prepared as a 6-carboxyfluoroscein (FAM)-minor groove binder moiety (FAM- MGB) derivative.

Table E1A

[0222] For each assay, a NCBI Primer-B FAS T/Nucleo tide BLAST analysis was performed in order to design the primer and probe sequences listed in Table El A. The RefSeq Representative Genome Database (organism limited to viruses and Homosapiens) was used for analysis (including identifying the conservative regions of E7 gene among the HPV16 strains, design of primer and probe sequences, and analyzing the binding specificity of the designed primer or probes to the target regions). For all primers and probes, exact matches were found for HPV16 E7. Some homology with non-target genes was identified for the HPV16e7_l, HPV16e7_3 and HPV16e7_5 primer and probe sets; however these were deemed to be not significant due to the length of the expected amplicon that would be obtained from these non-target genes (>1500 bp), which exceeds the amplicon lengths that would be obtained from the target sequences, as well as the number of nucleotide differences when compared with the original primer sequence.

[0223] After extraction of RNA from five sections of 5pm thickness using the High Pure FFPE RNA Isolation kit (Roche, Indianapolis, IN), reverse transcription reactions were conducted using the High Capacity RNA to cDNA Reverse Transcription Kit (Thermo Fisher, Foster City, CA) with 30 ng RNA in a 20 pL reaction volume. Approximately, 6.25 pL of cDNA was used for a pre-amplification step performed in a 25 pL reaction volume including lx TaqMan® PreAmp Master Mix (Thermo Fisher, Foster City, CA) and 0.0025x primer/probe, followed by thermal profiling protocol: 95 °C for 10 seconds and 10 cycles of amplification (95°C for 15 seconds and 60°C for 4 minutes).

[0224] Real-time PCR (RT-PCR; aka quantative PCR (qPCR)) was then carried out in a l0pL reaction volume, which included lx TaqMan® Fast Advanced Master Mix (Thermo Fisher, Foster City, CA), lx primer/probe and 2.5pL of a 1:5 dilution of the pre-amplification step reaction product, using the following thermal profiling protocol: 50°C for 2 minutes, 95 °C for 20 seconds, then 40 cycles of amplification (95 °C for 1 second and 60°C for 20 seconds). Parallel assays to assess TOPI and P-actin as reference genes were used as internal controls for input RNA quality and quantity. Samples that did not generate any Cq value for either of the controls were considered invalid and were excluded from data analysis.

[0225] Results

[0226] The amplification efficiency for each of the five exemplary RNA-PCR methods was assessed using RNA from a cervical carcinoma cell line (SiHa, S 1988-00005). The PCR performance data was summarized in Table E1B, below.

Table E1B

* Optimal amplification efficiency range 90% to 110%. [0227] The amplicon size generated by each of the potential HPV16e7 assays was compared to the expected amplicon size based on the method design and is depicted in Table E1C, below.

Table E1C: Specificity results using the S1988-0005 SiHa cell line

Example 2 Comparison of detection sensitivity of the Real-Time RNA-PCR assay with composite p!6 immunohistochemistry (IHC), HPV DNA genotyping, and a reference RNA-PCR assay.

A. Overview

[0228] The analytical performance of the real-time HPV- 16 RNA-PCR-based assay method described in Example 1 was compared to a composite pl6 immunohistochemistry (IHC) assay, to HPV DNA genotyping, and to a reference RNA-PCR assay using archival formalin fixed paraffin embedded (FFPE) tissue samples of HNSCC.

B. Patients and Tumor Samples

[0229] A total of 95 archival FFPE tissue blocks of head and neck squamous cell carcinomas (HNSCC) obtained from patients were acquired (Avaden Biosciences, Seattle, WA). Demographic and pathological information (age, gender, sites of biopsy or resection, diagnosis, and staging) for each patient were also obtained and are provided in Table E2A with respect to the whole set of 95 samples and the set of 51 samples further assessed by pl6 IHC and HPV DNA genotyping (described below). As evident in Table E2A, no significant difference in age, gender, site of tumor, tumor grade, or tumor stage was observed between the whole set of samples or the 51 samples assessed by pl6 IHC and HPV DNA genotyping. The patient characteristics were also consistent with other information reported in the literature (Fonmarty D et al., Study of the concordance between pl6 immunohistochemistry and HPV-PCR genotyping for the viral diagnosis of oropharyngeal squamous cell carcinoma. 2015;132(3): 135-139). Table E2A: Characteristics of the patient cohort

[0230] In order to ensure sample quality control and assess tumor cellular content, a section of each FFPE block with a thickness of approximately four (4) micrometers (pm) was stained with hematoxylin and eosin (H&E) and reviewed by a pathologist. One of the samples was observed to contain no tumor content was therefore excluded from further analysis. The remaining 94 FFPE tissue block samples were then analyzed by performing the pl6 immunohistochemistry (IHC) and HPV DNA genotyping assays, described below, to identify and assess the initial HPV- 16 status of each tumor sample.

C. pl6 IHC Assay

[0231] An immunohistochemical assay for pl6 (pl6 IHC) on the 94 patient samples that were determined to contain tumor content was performed with a commercial kit (CINtec Histology (Roche, Tucson, AZ)) in a CLIA laboratory (NeoGenomics, FL) for assessment of the pl 6^INK4a protein in the FFPE HNSCC tissues on a VENTANA BenchMark ULTRA instrument (Roche, Tucson, AZ). A historical HNSCC sample with a high pl6 expression was used as a positive control. For a negative control, the primary antibody was omitted from the staining procedure. Positive and negative controls were included for each instrumental run. Each pl6 IHC slide was reviewed and scored by a pathologist. The assay was scored positive when the cells with diffused nuclear and cytoplasmic staining were greater than 75% in the tumor region.

[0232] Results

[0233] Using the pl6 IHC Assay, of the 94 samples analyzed, 42 of them (44.7%) were scored as positive for pl6 staining with a range of positively stained cells between approximately 75% to 100%; median of 80%. The remaining 52 of the 94 analyzed samples (55.3%) were scored as negative for pl6 staining (0 to 35% stained cells; median 3%). Four of the 42 samples that were scored and considered as positive samples were observed to exhibit a borderline stain result (75-80%). Representative pl6 IHC staining results are depicted in Figures 2A- 2C.

D. HPV DNA Genotyping Assay

[0234] An HPV- 16 DNA assessment of each FFPE tissue sample was performed in a CEIA laboratory (NeoGenomics, FE) with a CEIA-validated laboratory-developed assay (NeoGenomics, FL) for HPV DNA genotyping. Briefly, type-specific primers were designed to anneal to DNA in early protein genes (E5-E7), PCR was performed, and PCR products of various sizes for different HPV subtypes were generated. The HPV subtype was identified by determining the size of PCR products using AB 13730x1 Genetic Analyzer (Thermo Fisher, Foster City, CA). Six high risk types, 16, 18, 31, 33, 45, and 58, and one low risk type, 6/11, were detected by fragment analysis, which covers 95% of cancer-related HPV.

[0235] Results

[0236] Using the HPV DNA genotypic assay, eighty-one (81) of the 94 samples (86.2%) subjected to the HPV DNA genotyping assay exhibited valid HPV DNA genotyping results. Among these 81 samples, 36 samples (44.4%) were determined to be positive for HPV16 DNA, 45 samples were determined to be negative (55.5%). Thirteen (13) of the 94 samples subjected to the HPV DNA genotyping assay (13.8%) did not yield valid DNA results due to either inadequate amplification or quality control failure of internal controls, and were therefore excluded from further analysis. Of the 81 samples that had valid pl6 IHC and HPV DNA genotyping results, a total of 51 exhibited concordant positive or negative results from the two assays. These 51 samples exhibiting concordant positive or negative results for the p 16 and HPV DNA genotyping assays were therefore selected for further RNA-PCR analysis as outlined in the workflow diagram shown in Figure 1 and as described further below.

E. Reference RNA-PCR Assay

[0237] A reference RNA-PCR assay (Gao G et al., A novel RT-PCR method for quantification of human papillomavirus transcripts in archived tissues and its application in oropharyngeal cancer prognosis. Int J Cancer. 2013;132(4):882-890) was also performed to determine the detection sensitivity and specificity of HPV 16 E7 transcript in a sample. The oligonucleotide primers for the reference RNA-PCR assay were designed to have the following sequences (both forward and reverse primer sequences provided in 5’ to 3’ fashion):

HPV16 E7 forward primer (SEQ ID NO: 1):

HPV16 E7 reverse primer (SEQ ID NO: 2):

[0238] Both primers were purchased from Thermo Fisher (Foster City, CA).

[0239] After extraction of RNA from five sections of 5 pm thickness using the High Pure FFPE RNA Isolation kit (Roche, Indianapolis, IN), a reverse transcription reaction was done by using the High Capacity RNA to cDNA Reverse Transcription Kit (Thermo Fisher, Foster City, CA) with 30 ng RNA in a 20 pL reaction volume. Real-time PCR was then carried out with 2 pL cDNA by using Power SYBR Green PCR Master Mix (Thermo Fisher, Foster City, CA) and 500 nM each primer in a 10 pF reaction volume. The PCR assay conditions were as follows: 95°C for 10 minutes, followed by 40 cycles of amplification (each cycle as follows: 95°C for 10 seconds, 58°C for 15 seconds and 60°C for 15 seconds). A SYBR Green PCR assay for P-actin as a reference gene was used as the internal control for input RNA quality and quantity. Samples that did not generate any Cq value for the control were considered invalid and were excluded from data analysis.

[0240] Results

[0241] Using the reference RNA-PCR assay, detectable HPV16 E7 transcript was observed in 90.9% of the samples identified as positive by both pl6 IHC and HPV DNA genotyping (twenty (20) out of twenty-two (22)). One of the 29 negative samples exhibited an internal control failure (did not yield a Cq value for the internal control), and was therefore excluded from the data analysis. Detectable HPV16 E7 transcript was observed in five (5) of the remaining twenty-eight (28) negative samples (17.9%). These results are depicted in

Table E2B below.

Table E2B

F. Real-Time RNA-PCR assay method

[0242] Results for detection of HPV by each of the alternative assays described above was compared to the real-time RNA-PCR assay method described in Example 1, as further summarized below. For the comparisons, the HPV16e7_2 primer and probe set was selected to be used in the real-time RNA-PCR assay to serve as the comparator to the sensitivity and performance of the alternative assays.

Comparison with p!6 IHC and HPV DNA genotyping analysis

[0243] Results from the real-time RNA-PCR assay were compared to results from the pl6 IHC and HPV DNA genotyping analysis of the 51 samples in a manner analogous to the comparison with the reference RNA-PCR method described in Section E. The sample that exhibited an internal control failure with the reference RNA-PCR assay of Section E also exhibited an internal control failure using the real-time RNA-PCR assay method (did not yield a Cq value for the internal control), and was therefore again excluded from further data analysis.

[0244] Detectable HPV 16 E7 transcript was observed in 100% of the samples identified as positive by both pl6 IHC and HPV DNA genotyping (twenty-two (22) out of twenty-two (22)). Detectable HPV16 E7 transcript was also observed in 42.8% of the remaining twentyeight (28) negative samples (z.e., in twelve (12) of the 28). These results are depicted in Table E2C below.

Table E2C

Comparison with reference RNA-PCR assay

[0245] As above, the HPV16e7_2 primer and probe set was selected to be used in the real-time RNA-PCR assay to serve as a comparator to the sensitivity and performance of the reference RNA-PCR assay. A total of 25 out of the 50 samples were HPV16 E7 transcripts- positive for both RNA assays (Table E2D).

[0246] As part of the comparative analysis, Cq values were determined using the realtime RNA-PCR assay method and compared to those determined using the reference RNA- PCR assay method. As depicted in Figure 3, the distribution of the Cq values achieved by the real-time RNA-PCR assay method was highly correlated to those achieved with the reference assay (Pearson's R = 0.96). However, the Cq values achieved by the real-time RNA-PCR assay method were significantly lower than those of the reference RNA-PCR assay in the pair- wise comparison, as depicted in Figure 4 (p < 0.001). Furthermore, as indicated in Table E2D below, 100% of the samples (/'.<?., 25 out of 25) observed to be positive for HPV16 E7 transcripts using the reference RNA-PCR assay method were also observed to be positive using the real-time RNA-PCR assay method; however 36% of the samples (9 out of 25) that exhibited no amplification signal (and thus no significant HPV E7 transcript detection) by the reference RNA-PCR assay method were observed to be positive (yielding significant HPV E7 transcript detection) by the real-time RNA-PCR assay method.

Table E2D

[0247] Collectively, these results demonstrate that the real-time RNA-PCR assay method provide significantly greater performance, including increased sensitivity and specificity in identification and detection of HPV E7 transcripts thus in assessing HPV status, in samples compared to the reference RNA-PCR assay method. [0248] The present invention is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure.

SEQUENCES

Claims

1. A method of detecting human papillomavirus (HPV) in a subject, the method comprising

(a) incubating a template DNA obtained from a biological sample in a first incubation with (i) an oligonucleotide set comprising a forward oligonucleotide primer, a reverse oligonucleotide primer, and an oligonucleotide probe and (ii) a DNA polymerase to produce a first pre- amplification reaction product;

(b) incubating the first pre-amplification reaction product or sample thereof in a second incubation with (i) the oligonucleotide set comprising the forward oligonucleotide primer, the reverse oligonucleotide primer, and the oligonucleotide probe and (ii) a DNA polymerase to produce a secondary reaction product, wherein each of the incubating in (a) and the incubating in (b) is under conditions sufficient for amplification of a HPV 16 amplicon if present in the sample; and

(c) detecting the generated HPV 16 amplicon if present in the sample.

2. A method of amplifying a HPV 16 amplicon, the method comprising:

(a) incubating a template DNA obtained from a biological sample in a first incubation with (i) an oligonucleotide set comprising a forward oligonucleotide primer, a reverse oligonucleotide primer, and an oligonucleotide probe, and (ii) a DNA polymerase to produce a first pre-amplification reaction product;

(b) incubating the first pre-amplification reaction product or sample thereof in a second incubation with (i) the oligonucleotide set comprising the forward oligonucleotide primer, the reverse oligonucleotide primer, and the oligonucleotide probe, and (ii) a DNA polymerase to produce a secondary reaction product, wherein each of the incubating in (a) and the incubating in (b) is under conditions sufficient for amplification of a HPV 16 amplicon if present in the sample.

3. The method of claim 1 or claim 2, wherein the HPV16 amplicon is an HPV 16 E7 amplicon.

4. The method of any of claims 1-3, wherein the first incubation and the second incubation individually is performed by polymerase chain reaction (PCR).

5. The method of claim 4, wherein the PCR is real-time PCR (RT-PCR).

6. The method of any of claims 1-5, wherein the template DNA is complementary DNA (cDNA).

7. The method of any of claims 1-6, wherein prior to the incubating in (a), the method comprises synthesizing the template DNA by reverse-transcribing an RNA template from the biological sample into cDNA.

8. The method of any of claims 1-7, wherein the DNA polymerase is a Taq DNA polymerase.

9. The method of any of claims 1-8, wherein the HPV16 amplicon is less than 120 base pairs.

10. The method of any one of claims 1-9, wherein the HPV16 amplicon is between 60 and 119 base pairs, inclusive.

11. The method of any one of claims 1-10, wherein the HPV16 amplicon is between 70 and 119 base pairs, inclusive.

12. The method of any one of claim 1-11, wherein the HPV16 amplicon is between 80 and 119 base pairs, inclusive.

13. The method of any one of claims 1-12, wherein the HPV16 amplicon is between 80 and 117 base pairs, inclusive.

14. The method of any one of claims 1-13, wherein the (i) the forward oligonucleotide primer comprises 15-30 nucleotides that are complementary to a plus strand of HPV16 E7 template DNA, and (ii) the reverse oligonucleotide primer comprises 15-30 nucleotides that are complementary to a minus strand of HPV16 E7 template DNA.

15. The method of any one of claims 1-14, wherein the (i) the forward oligonucleotide primer comprises 18-25 nucleotides that are complementary to a plus strand of HPV16 E7 template DNA, and (ii) the reverse oligonucleotide primer comprises 18-25 nucleotides that are complementary to minus strand of HPV16 E7 template DNA.

16. The method of any one of claims 1-15, wherein the forward oligonucleotide primer comprises a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to any one of SEQ ID NOs: 3, 6, 9, 12 or 15.

17. The method of any one of claims 1-16, wherein the forward oligonucleotide primer comprises the sequence set forth in any of SEQ ID NOs: 3, 6, 9, 12 or 15.

18. The method of any one of claims 1-17, wherein the reverse oligonucleotide primer comprises a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to any one of SEQ ID NOs: 4, 7, 10, 13 or 16.

19. The method of any one of claims 1-18, wherein the reverse oligonucleotide primer comprises the sequence set forth in any of SEQ ID NOs: 4, 7, 10, 13 or 16.

20. The method of any of claims 1-19, wherein the oligonucleotide probe comprises a sequence that has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to any one of SEQ ID NOs: 5, 8, 11, 14 or 17.

21. The method of any of claims 1-20, wherein the oligonucleotide probe comprises one of any of the sequences as set forth in SEQ ID NOs: 5, 8, 11, 14 or 17.

22. The method of any one of claims 1-21, wherein the HPV16 amplicon detected is at or about 75-90 base pairs, optionally at or about 80 base pairs.

23. The method of any one of claims 1-22, wherein the forward oligonucleotide primer is set forth in SEQ ID NO: 9 and the reverse oligonucleotide primer is set forth in SEQ ID NO: 10.

24. The method of any one of claims 1-23, wherein the forward oligonucleotide primer is set forth in SEQ ID NO: 9, the reverse oligonucleotide primer is set forth in SEQ ID NO: 10 and the oligonucleotide probe is set forth in SEQ ID NO: 11.

25. The method of any one of claims 1-22, wherein the forward oligonucleotide primer is set forth in SEQ ID NO: 12 and the reverse oligonucleotide primer is set forth in SEQ ID NO: 13.

26. The method of any one of claims 1-22 and 25, wherein the forward oligonucleotide primer is set forth in SEQ ID NO: 12, the reverse oligonucleotide primer is set forth in SEQ ID NO: 13 and the oligonucleotide probe is set forth in SEQ ID NO: 14.

27. The method of any one of claims 1-22, wherein the forward oligonucleotide primer is set forth in SEQ ID NO: 15 and the reverse oligonucleotide primer is set forth in SEQ ID NO: 16.

28. The method of any one of claims 1-22 and 27, wherein the forward oligonucleotide primer is set forth in SEQ ID NO: 15, the reverse oligonucleotide primer is set forth in SEQ ID NO: 16 and the oligonucleotide probe is set forth in SEQ ID NO: 17.

29. The method of any one of claims 1-21, wherein the HPV16 amplicon is 95 to 105 base pairs, optionally at or about 100 base pairs.

30. The method of any one of claims 1-21 and 29, wherein the forward oligonucleotide primer is set forth in SEQ ID NO: 6 and the reverse oligonucleotide primer is set forth in SEQ ID NO: 7.

31. The method of any one of claims 1-21, 29 and 30, wherein the forward oligonucleotide primer is set forth in SEQ ID NO: 6, the reverse oligonucleotide primer is set forth in SEQ ID NO: 7 and the oligonucleotide probe is set forth in SEQ ID NO:8.

32. The method of any one of claims 1-21, wherein the HPV16 the amplicon is at or about 110 to 140 base pairs, optionally at or about 117 base pairs.

33. The method of any one of claims 1-21 and 32, wherein the forward oligonucleotide primer is set forth in SEQ ID NO: 3 and the reverse oligonucleotide primer is set forth in SEQ ID NO: 4.

34. The method of any one of claims 1-21, 32 and 33, wherein the forward oligonucleotide primer is set forth in SEQ ID NO: 3, the reverse oligonucleotide primer is set forth in SEQ ID NO: 4 and the oligonucleotide probe is set forth in SEQ ID NO:5.

35. The method of any of claims 1-34, wherein the oligonucleotide probe comprises a detectable moiety.

36. The method of claim 35, wherein the detectable moiety is a fluorescent moiety.

37. The method of claim 35 and 36, wherein the HPV 16 amplicon is detected by measuring a detectable signal emitted from the detectable moiety of the second reaction product.

38. The method of claim 37, wherein the detectable signal is a fluorescent signal.

39. The method of claim 37 and 38, wherein detecting the HPV 16 amplicon comprises determining the quantification cycle (Cq) at which the detectable signal, optionally the fluorescent signal, exceeds background fluorescence.

40. The method of any one of claims 1-39, wherein the concentration of the oligonucleotide set in the first incubation step is less than the concentration in the second incubation step.

41. The method of claim 40, wherein the concentration of the oligonucleotide set in the first incubation step is between 0.05-fold and 0.001-fold of the concentration in the second incubation step.

42. The method of claim 40 or claim 41, wherein the concentration of the oligonucleotide set in the first incubation step is between 0.01 -fold and 0.002-fold of the concentration in the second incubation step.

43. The method of claim 40 or claim 41, wherein the concentration of the oligonucleotide set in the first incubation step is at or about 0.0025-fold the concentration in the second incubation step.

44. The method of any one of claims 1-43, wherein the first incubation step comprises thermal profile conditions set forth as: 10 seconds at 95 °C followed by 10 cycles of amplification (95°C for 15 seconds and 60°C for 4 minutes).

45. The method of any one of claims 1-44, wherein the second incubation step comprises thermal profile conditions set forth as: 50°C for 2 minutes, 95°C for 20 seconds, then 40 cycles of amplification comprising 95°C for 1 second and 60°C for 20 seconds.

46. The method of any one of claims 1-45, wherein the subject has, or is suspected of having head and neck cancer.

47. The method of claim 46, wherein the head and neck cancer is squamous cell carcinoma (HNSCC).

48. The method of any one of claims 1-47, wherein the biological sample is a tissue, a buccal sample, a saliva sample, or a blood sample.

49. The method of claim 48, wherein the tissue sample is a tumor tissue.

50. The method of any one of claims 1-49, wherein the sample is a tissue sample that has been formalin fixed and paraffin embedded.

(i) detecting a generated HPV 16 amplicon by the method of any of claims 1-50 if present in the sample; and

52. The method of claim 51, wherein the detectable amount of HPV is determined by the quantification cycle (Cq).

54. The oligonucleotide set of claim 53, further comprising an oligonucleotide probe set forth in any one of SEQ ID NOs: 5, 8, 11, 14 or 17.

55. The oligonucleotide set of claim 53 or claim 54, wherein (i) the forward oligonucleotide primer comprises a nucleic acid sequence set forth in SEQ ID NO: 6, and (ii) the reverse oligonucleotide primer comprises a nucleic acid sequence set forth in SEQ ID NO: 7.

56. The oligonucleotide set of claim 55, wherein the oligonucleotide probe is set forth in SEQ ID NO:8.

57. The oligonucleotide set of claim 53 or claim 54, wherein (i) the forward oligonucleotide primer comprises a nucleic acid sequence set forth in SEQ ID NO: 9, and (ii) the reverse oligonucleotide primer comprises a nucleic acid sequence set forth in SEQ ID NO: 10.

58. The oligonucleotide set of claim 57, wherein the oligonucleotide probe is set forth in SEQ ID NO:11.

59. The oligonucleotide set of claim 53 or claim 54, wherein (i) the forward oligonucleotide primer comprises a nucleic acid sequence set forth in SEQ ID NO: 12, and (ii) the reverse oligonucleotide primer comprises a nucleic acid sequence set forth in SEQ ID NO: 13.

60. The oligonucleotide set of claim 59, wherein the oligonucleotide probe is set forth in SEQ ID NO: 14.

61. The oligonucleotide set of claim 53 or claim 54, wherein (i) the forward oligonucleotide primer comprises a nucleic acid sequence set forth in SEQ ID NO: 15, and (ii) the reverse oligonucleotide primer comprises a nucleic acid sequence set forth in SEQ ID NO: 16.

62. The oligonucleotide set of claim 61, wherein the oligonucleotide probe is set forth in SEQ ID NO: 17.

63. The oligonucleotide set of claim 53 or claim 54, wherein (i) the forward oligonucleotide primer comprises a nucleic acid sequence set forth in SEQ ID NO: 3, and (ii) the reverse oligonucleotide primer comprises a nucleic acid sequence set forth in SEQ ID NO: 4.

64. The oligonucleotide set of claim 63, wherein the oligonucleotide probe is set forth in SEQ ID NO:5.

65. The oligonucleotide set of any of claims 54-64, wherein the oligonucleotide probe comprises a detectable moiety.

66. The oligonucleotide set of claim 65, wherein the detectable moiety is a fluorescent moiety.

67. The oligonucleotide set of claim 65 or claim 66, wherein the detectable moiety is at the 5’ end of the oligonucleotide probe.

68. The oligonucleotide set of any one of claims 65-67, wherein the detectable moiety is selected from FAM, HEX, FITC, Texas Red, TET, JOE, VIC, NED, TAMRA, ROX, ABY, PET, JUN, LIZ, Cy3, or Cy5.

69. The oligonucleotide set of any one of claims 54-68, wherein the oligonucleotide probe comprises a minor groove binder (MGB) moiety, optionally wherein the MGB is at the 3’ end.

70. The oligonucleotide set of any of claims 54-69, wherein the oligonucleotide probe comprises a nonfluorescent quencher (NFQ), optionally wherein the NFQ is at the 3’ end.

71. The oligonucleotide set of any one of claims 65-70, wherein the detectable moiety is FAM-MGB .

72. A kit, comprising the oligonucleotide set of any of claims 53-71 and one or more reagents for carrying out a polymerase chain reaction.

73. The kit of claim 72, wherein the one or more reagents comprises a DNA polymerase.

74. The kit of claim 73, wherein the DNA polymerase is a Taq polymerase.

75. A method of treatment comprising administering a therapeutic to a subject in need thereof for treating an human papillomavirus (HPV) infection, wherein: the subject is selected for treatment if an HPV16 amplicon is detected in a sample obtained from said subject using the method of any one of claims 1-52.

76. A method of treatment, the method comprising: selecting a subject in which an HPV16 amplicon is detected according to the method of any of claims 1-52; and administering to the selected subject a therapeutic agent for treating an human papillomavirus (HPV) infection.

77. A method of treatment comprising administering a therapeutic to a subject in need thereof for treating an human papillomavirus (HPV) infection, wherein the subject is diagnosed with an HPV infection if HPV is detected in a sample from the subject by the method of any of claims 1-53.

78. The method of any of claims 75-77, wherein the therapeutic for treating an HPV infection is selected from the group consisting of vaccines that induce or boost HPV T cell adaptive immunity, adoptive cell therapy, therapeutic antibodies, antiviral therapeutics, immune response modifier compounds, proteasome inhibitors, HD AC inhibitors, and drugs targeting HPV genes.

79. The method of any of claims 75-78, wherein the therapeutic is a T cell therapy comprising T cells expressing a recombinant antigen receptor specific to an HPV16 epitope.

80. The method of claim 79, wherein the HPV16 epitope is an HVP16 E7 epitope.

81. The method of claim 80, wherein the HPV 16 epitope is HPV E7 (11-19).

82. The method of any of claims 75-81, wherein the subject has or is suspected of having a cancer.

83. The method of any one of claims 75-82, wherein the subject has, or is suspected of having head and neck cancer.

84. The method of claim 83, wherein the head and neck cancer is squamous cell carcinoma (HNSCC).