A method of identifying HPV biomarkers, a method of preparing a lateral flow device, said device and a diagnostic kit

Abstract

The present invention describes a method of identifying HPV biomarkers which comprises the use of peptides capable of recognising molecular markers in high-risk HPV strains. The high specificity and affinity of said peptides to molecular targets allows them to be used in immunochromatographic and immunoenzymatic assays. The present invention has applications in diagnostic medicine and the pharmaceutical industry, disclosing methods for preparing lateral flow devices, and said devices and diagnostic kits capable of detecting the presence of an HPV viral infection, as well as detecting whether the infection is at an early stage, advanced neoplasia stage or even cancer.

Description

“METHOD OF IDENTIFYING HPV BIOMARKERS, METHOD OF PREPARING A LATERAL FLOW DEVICE, SAID DEVICE AND DIAGNOSTIC KIT” Field of the Invention:

[0001] The present invention relates to the biochemical analysis of biological material, more specifically to a method of identifying HPV biomarkers, useful for the detection of human papillomavirus (HPV) in a biological sample.

[0002] The present invention is applicable to pharmacy and diagnostic medicine, primarily related to the construction of diagnostic devices and kits for individuals in need thereof. Fundamentals of the invention:

[0003] Cervical cancer is the fourth most common neoplasm among women worldwide, accounting for approximately 8-12% of all neoplasms, and infection with the human papillomavirus, HPV, is known to be the greatest risk factor for neoplastic transformation of the cervical epithelium.

[0004] From a taxonomic point of view, all human papillomaviruses belong to the Papillomaviridae family, divided into 37 genera and comprising 240 virus types. Of these, twelve to fifteen are considered cancer-causing or high-risk genotypes (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82), depending on the authors consulted, and are responsible for more than 90% of cases of cervical cancer, in addition to other types of cancer.

[0005] The HPV genome is traditionally divided into three regions: the "Long control region" (LCR), which regulates viral expression and replication; the "Early region" (E), which encodes the virus's functional proteins; and the "Late region" (L), which encodes structural proteins. Among the proteins in the E region, E5, E6, and E7 are known to be carcinogenic. The L region of the HPV genome encodes two structural proteins, L1 and L2, which form the viral capsid. During productive infection, with the release of viral particles, cervical tissue cells are stimulated by the functional "E" proteins to remain in a state of constant resumption of the S phase of mitosis. This type of regulation contributes to viral genome amplification and consequent viral replication. Additionally, in the superficial layers of the tissue, the structural proteins, L1 and L2, are transcribed, and complete viral particles are released.On the other hand, when HPV infection leads to more advanced stages of neoplasia and cancer, a non-productive phase of infection is observed, in which there is little or no release of viral particles. In this case, the expression of structural proteins is delayed and limited to small areas of the tissue surface, which restricts viral production. At the same time, viral DNA integrates into the host genome and the expression of E6 and E7 proteins increases, which contributes to this. in generating greater errors in the replication of the host genome.

[0006] The conventional technique for morphological analysis of cervical epithelial cells through the Pap smear has been used since the 1940s. However, the methodology has several limitations. Examples include collection and slide preparation errors, subjectivity in result interpretation, variability in sensitivity and specificity estimates, the need for qualified professionals for collection and analysis, low patient adherence to follow-up and return visits for results evaluation, and logistical factors such as the availability of tests in less-developed regions and the transportation of biological samples.

[0007] Technological advances have ensured methodological updates, such as ultrafast staining techniques and liquid-based cytology using commercial platforms capable of offering greater uniformity and diagnostic accuracy, such as the ThinPrep® Pap test (Hologic, Inc., USA) and SurePath™ (BD Diagnostics, USA). However, sensitivity remains insufficient, producing atypical squamous cells of undetermined significance, which may require further referrals for collection and analysis.

[0008] On the other hand, more modern techniques, such as those based on DNA analysis to identify the presence of strains considered high risk in samples of Cervical cells contribute to overcoming several challenges associated with the Pap smear, particularly regarding high accuracy and sensitivity. Since 2001, the relevant regulatory agency in the United States, the FDA (Food and Drug Administration), has approved five tests based on this type of technology: the Hybrid Capture 2 HPV DNA Test (Qiagen, Germany), the Cervista HPV HR Test (Hologic, USA), the Cobas 4800 HPV Test (Roche, Switzerland), the Aptima HPV Assay (Gen Probe / Hologic, USA), and the BD Onclarity HPV Assay (Becton Dickinson, USA). Other examples are also already available in commercial formats. However, the need for analysis in specialized laboratories and the high unit cost make this method prohibitive for public screening policies, especially in underdeveloped or developing countries, in addition to the test being limited to only a few viral types.

[0009] Additionally, detecting high-risk strains does not necessarily indicate the development of cervical cancer, since most HPV infections regress naturally, as do some cases of early-stage neoplastic development. Therefore, detecting lesions whose infections can generate a high risk of progression to more severe cancers becomes more important than simply detecting the virus itself.

[0010] In contrast, immunochromatographic and immunoenzymatic assays allow the detection of diverse samples (proteins, microorganisms, drugs, etc.) through fast, simple, low-cost and easy-to-interpret methodologies, thus eliminating the need for professionals specifically qualified for sample processing and method development.

[0011] These characteristics facilitate their application in home or field tests (immunochromatographic) or in laboratories with simpler analysis equipment (immunoenzymatic), compared to more sophisticated techniques involving the genetic analysis of specific strains. These tests often offer a favorable cost-benefit ratio to be used in public health policies, enabling the provision of reliable tests that would not be available in other formats for more isolated or difficult-to-access regions.

[0012] Several proteomic studies have demonstrated the differential expression of specific viral proteins depending on the stage of disease progression. The use of these markers, alone or in combination, may therefore be a promising tool for early diagnosis and identification of patients at risk for cancer.

[0013] The HPV biomarker identification method developed in the present invention allows the analysis and determination of consensus sequences in groups of high-risk HPV strains, promising for use as biological markers. For these potential diagnostic targets, peptides with high affinity and specificity, which can be combined with specific markers for use in immunochromatographic and enzyme-linked immunosorbent assays. The results obtained were successful and showed promise for use in the development of diagnostic platforms for the presence of HPV and, if positive, the probable stage of the disease.

[0014] Other methods and tests for HPV detection are also known in the state of the art. International application WO09151632 describes methods, polyclonal antibodies, monoclonal antibodies, assays, and kits for detecting HPV infection, including infection by various HPV genotypes, and early and / or late HPV-associated or -specific proteins or antibodies. However, the aforementioned document describes in detail platforms based on immunocytochemical analyses that involve the observation of specific markers in infected cells through analysis of infected tissue slides or flow cytometry. In both cases, specialized analysts are required to develop a more laborious and costly method that allows for individual and, depending on the method, subjective analysis of a given sample.

[0015] The document mentions the possibility of detection through ELISA, but describes the method superficially and states the need for more samples for analysis. It also describes detection through lateral flow, but mentions the use of polyclonal antibodies, a characteristic that could hinder test reproducibility and reduce sensitivity, in addition to increasing the cost of producing the inputs. Unlike the aforementioned document, the present invention offers both synthetic peptides and modified monoclonal bacteriophages capable of recognizing molecular targets (consensus sequences in HPV proteins). Both options allow for greater ease of production and lower costs compared to the production of the antibodies proposed in the aforementioned document. Furthermore, the present invention addresses only monoclonal options, which ensures greater reproducibility and specificity against the target of interest.

[0016] Patent application US2020088727 discloses a lateral flow test strip based on the interaction between antibodies (not defined) and HPV proteins (not clearly defined). This application reports the use of antibodies capable of recognizing HPV proteins; however, it fails to describe which proteins are used as markers, how many of these proteins there are, how many antibodies are used, and whether they are monoclonal or polyclonal. At some points, it also leaves unclear whether the antibodies are capable of recognizing viral proteins or proteins produced by the host itself. These parameters are directly related to the specificity, reproducibility, and sensitivity of the test.

[0017] In contrast, the present invention clearly defines both the peptides (and also the monoclonal bacteriophages) and their respective molecular targets, a fact which ensures greater reproducibility for the test, in addition to contributing to other parameters, such as specificity and sensitivity. Additionally, the present invention provides for the use of two diagnostic platforms—lateral flow and ELISA—compared to only one proposed in the aforementioned document—lateral flow.

[0018] At the same time, there is already at least one commercially available test based on an immunochromatographic platform. The Onco E6 Cervical Test (Arbor Vita Corporation, USA), not yet approved by the FDA, allows the detection of the E6 protein of HPV 16 and HPV 18. Since this protein is predominantly expressed in patients with cervical cancer and its expression varies according to the patient's neoplastic status, being more common in more advanced neoplasms and less common in milder cases, the test aims to identify those patients with a higher risk of developing cancer. The Onco E6 test is a qualitative test based on the use of monoclonal antibodies conjugated to an alkaline phosphatase, which recognize and detect "high levels" of the E6 oncoprotein expressed in cells infected with Human Papillomavirus types 16 and / or 18.

[0019] However, the Onco E6 Cervical test uses only one molecular marker (E6), which compromises the identification of the presence of high-risk viral strains if the infection stage is early and, as it identifies proteins from just two viral strains, is even more restricted than tests based on genetic analysis, which are capable of identifying several strains and are also limited to high-risk strains.

[0020] It is worth noting that the present invention reveals the use of two molecular markers, E6 and L1, which would allow not only the detection of the disease but also the inference of its stage. Furthermore, the present invention describes the use of synthetic peptides and monoclonal bacteriophages, whose production is simpler and less costly, in addition to relying on two diagnostic platforms—lateral flow and ELISA—compared to only one proposed in the aforementioned document—lateral flow.

[0021] Another international application, WO10127326, discloses methods for affinity purification of human papillomavirus (HPV) E6 polypeptides using E6-binding protein peptides. This method, therefore, is an expensive methodology that requires specialized equipment and professionals, since it requires purification of the target protein (E6) for subsequent confirmation of its presence.

[0022] Additionally, the application uses only one molecular marker (E6), which compromises the detection of HPV infection in the early stages of the disease. The method mentions the use of fused peptides, but does not specify whether these are novel sequences, which suggests that they are sequences already described in the literature. as described in the attached table in the document itself. Although the fusion of the fragments forms a new product, the fragments themselves are already described in the current literature.

[0023] Furthermore, the method developed in the present invention enables the direct detection of molecular markers in biological samples without the need for prior purification, which would optimize both the time spent on diagnosis and the need for specialized labor for the development of the lateral flow device. Additionally, the method of the present invention reveals the use of two molecular markers, E6 and L1, which would allow not only the detection of the disease but also the inference of its stage.

[0024] Finally, international application WO10123949 describes a method for detecting the E6 oncoprotein of Human Papillomavirus types 16 and / or 18 using a combination of E6-detecting antibodies, particularly monoclonal antibodies. Such a combination of antibodies would result in an increased signal-to-noise ratio, while enhancing detection capacity. Furthermore, the inventors of the application mention the use of animals such as rabbits, mice, or rats, or the use of cell culture and hybridomas, which are costly methods that require extensive infrastructure.

[0025] In contrast, the present invention reveals the use of synthetic peptides and bacteriophages monoclonal antibodies, whose production is simple and less expensive, in addition to having two molecular markers, E6 and L1, which allows not only the detection of the disease, but also allows inferring its stage.

[0026] Therefore, the method, device, and kit of the present invention allow us to determine whether or not a patient is infected with the virus and, if so, whether there is a higher or lower risk of neoplastic cell development. This information is useful for directing the patient to annual monitoring only or to more in-depth analysis and, if necessary, surgical intervention, considering that early diagnosis provides a high chance of cure for the patient undergoing this type of treatment.

[0027] It should be noted, therefore, that no prior art document discloses a method for identifying HPV biomarkers, its device, and diagnostic kit like that of the present invention, capable of detecting two viral proteins (E6 and L1) by means of synthetic peptides and monoclonal bacteriophages. Brief Description of the Invention:

[0028] The present invention relates to the biochemical analysis of biological material, more specifically to a method of identifying HPV biomarkers, useful for the detection of human papillomavirus (HPV) in a biological sample.

[0029] The method of the present invention was developed based on the differential expression of viral proteins to select peptides capable of recognizing and binding with high affinity and specificity to the viral proteins L1 and E6. High-risk HPV strains were separated into two groups according to genetic similarities for each diagnostic target, and peptides against conserved regions of L1 and E6 were selected in both groups. These peptides were capable of recognizing important diagnostic targets that can be used in both immunochromatographic and immunoenzymatic assays.

[0030] The method of the present invention comprises the following steps: A) Identification of conserved regions, through in silico analysis, in the form of peptide sequences present in HPV types considered high risk; B) Chemically synthesize the selected molecular targets: Target 1. AlaAlaLysGlnArgPheHisAsnIleAlaGlyArgTrpThrGlyArgCys AAKQRFHNIAGRWTGR Target 2. AlaAlaArgArgProTyrLysLeuProAspLeuCysThrAlaLeuAsn AARRPYKLPDLCTALN Target 3. AlaAlaGlnTyrArgValPheArgIleAlaLeuProAspProAsnLysPheGly PheProAsp AAQYRVFRIALPDPNKFGFPD Target 4. AlaAlaThrGlnArgLeuValTrpAlaCysAlaGlyValGluIleGlyArgGly GlnProLeuGlyValGlyLeuSerGlyHisPro AATQRLVWACAGVEIGRGQPLGVGLSGHP C) select peptide phages, through the phage display technique, capable of recognizing and binding with high affinity and specificity to the molecular targets previously determined;D) chemically synthesize selected peptides capable of recognizing the diagnostic targets: GlySerProCysTyrLysCys GSPCYKC SEQ ID NO:1 MetGlyCysGlyGlnCysThr MGCGQCT SEQ ID NO:2 MetHisValGlnThrLeuAla MHVQTLA SEQ ID NO:3 CysGluLeuCysHisTrpPro CELCHWP SEQ ID NO:4 LysCysCysTyrValThrPro KCCYVTP SEQ ID NO:5 ThrIleLeuAsnTyrHisIle TILNYHI SEQ ID NO:6 LysCysCysPheProValIle KCCFPVI SEQ ID NO:7 PheAlaGlnAspTrpHisVal FAQDWHV SEQ ID NO:8 GluTrpTyrTrpTrpSerTyr EWYWWSY SEQ ID NO:9 TyrHisTyrThrProTyrAsn YHYTPYN SEQ ID NO:10 LeuMetValSerProSerLeu LMVSPSL SEQ ID NO:11 AsnTrpSerSerLeuLeuGln NWSSLLQ SEQ ID NO:12 E) evaluate phages and synthetic peptides for their binding capabilities to diagnostic targets via ELISA and lateral flow platforms. ;

[0031] The present invention further discloses a method of preparing lateral flow devices comprising the steps of: i) a nitrocellulose membrane labeled with the peptides of the present invention is mounted on a plastic support of a membrane card-type platform; ii) the absorption and conjugation pads are mounted superimposed on the nitrocellulose membrane; iii) the sample pad is also fixed on the membrane card superimposed on the conjugation pad, so that the assembly presents an adequate capillary flow.

[0032] The diagnostic devices and kits derived from the revealed method are capable of detecting the presence of more than one diagnostic target in different viral strains, with an emphasis on high-risk strains, from a biological sample obtained from an individual. Simultaneous labeling of E6 and L1 would indicate a productive phase of the infection, with a low probability of neoplastic development of the disease. On the other hand, labeling of only E6 would indicate the need for patient monitoring, as there is a greater chance of neoplastic progression of the disease. Brief Description of the Figures:

[0033] To assist in identifying the main features of the present invention and its technical results and effects, the figures to which reference is made are presented as follows:

[0034] Figure 1 represents a flowchart with the main steps of the method developed in the present invention for selection and synthesis of phages and peptides of interest.

[0035] Figure 2 shows the results of ELISA tests of monoclonal phages with the lowest concentration values ​​at which there was a significant difference between the target and the control (double alanine conjugated to biotin, similar to the targets). The phages were named according to the target protein against which they were selected (F1, target 1; F3, target 3; and F4, target 4). The following number indicates different clones, and the numbers in brackets [] indicate the target / control concentration used in the assay. The results were revealed with anti-M13 antibody conjugated to HRP and TMB / H2O2 substrate in a plate reader set at 450 nm.

[0036] Figure 3 shows the graph showing the results of the ELISA tests for the synthetic peptides conjugated to BSA. These peptides were named after their respective phages, replacing "F" with "PB". A double alanine conjugated to biotin was also used as a negative control. The results were revealed with anti-biotin antibody conjugated to HRP and TMB / H2O2 substrate in a plate reader set at 450 nm (A 450 ).

[0037] Figure 4 shows the results of the qualitative lateral flow tests for (a) enzymatic reaction with TMB, (b) labeling with adsorbed colloidal gold, and (c) conjugation with gold particles. As this is qualitative analyses, examples of positive results are illustrated in the figure.

[0038] Figure 5 shows the graph with the results of the ELISA tests with native HPV16 and HPV18 viruses and phages. As a negative control, wells sensitized with BSA were used, and as a positive control, wells sensitized with the phages themselves (F1.1 and F3.1) were used. The results were revealed with anti-M13 antibody conjugated to HRP and TMB / H2O2 substrate in a plate reader set at 450 nm (A450). Detailed Description of the Invention:

[0039] The present invention relates to the biochemical analysis of biological material, more specifically to a method of identifying HPV biomarkers, useful for the detection of human papillomavirus (HPV) in a biological sample.

[0040] To develop the method of the present invention, we chose to use the L1 and E6 proteins as targets for constructing a diagnostic platform. Due to the relatively low genetic similarity between the various HPV types, and in order to obtain conserved regions, we opted to use small fragments of these proteins as molecular targets. For practical reasons, we opted for chemical synthesis of these targets. Additionally, in order to work with the viral types responsible for the largest number of cervical cancer cases, the present invention focused on viral types. considered high risk. The main selection steps of the developed method are represented in the flowchart in Figure 1.

[0041] For the E6 protein, to ensure greater sequence similarity, high-risk viral types were divided into two groups. Types 51 and 56 were excluded due to low similarity; however, these types account for only approximately 1% of cancer cases.

[0042] The first group included viral types 16, 31, 33, 35, 52, and 58, for which a 15-amino acid residue sequence was selected, based on HPV16, due to the highest incidence within the group. Target 1 was established as AAKQRFHNIAGRWTGRC, based on the following regions: HPV16-E6. KQRFHNIRGRWTGRC HPV35-E6. KKRFHNIGGRWTGRC HPV31-E6. KKRFHNIGGRWTGRC HPV33-E6. NKRFHNISGRWAGRC HPV58-E6. NKRFHNISGRWTGRC HPV52-E6. NKRFHNIMGRWTGRC

[0043] The second group included viral types 18, 39, 45, and 59, for which a 14-amino acid residue sequence was selected, based on HPV18, due to the higher incidence within the group. Target 2 was established as AARRPYKLPDLCTALN, based on the following regions: HPV18-E6.1 RRPYKLPDLCTELN HPV45-E6. QRPYKLPDLCTELN HPV39-E6. ERPYKLPDLCTTLD HPV59-E6. QRPYKLPDLSTTLN

[0044] To increase the similarity between the sequences for each target, amino acid residues with very different characteristics were replaced with alanine. For technical reasons, due to the strategy used to select peptides capable of recognizing and binding to the diagnostic targets, these were labeled with biotin in the amino-terminal region. To avoid folding and / or undesirable interactions with biotin, two alanine residues were added at the beginning of each sequence. Although the similarity is greater within the same group, there is still some similarity between targets when dealing with different groups. For this reason, it was decided to work with conserved regions in different positions in the two groups, since the targets determined for one group may also be recognized, with less specificity, in the other group.

[0045] For the L1 protein, due to the greater similarity observed between the high-risk strains, there was no need to divide it into groups. However, it was decided to maintain the standard of two sequences per target protein, one based on HPV16 and the other on HPV18, due to the higher incidence of these two strains in cancer cases.

[0046] Thus, target 3 was established as AAQYRVFRIALPDPNKFGFPD, based on HPV16 and the following regions: HPV16-L1. QYRVFRIHLPDPNKFGFPD HPV35-L1. QYRVFRVKLPDPNKFGFPD HPV31-L1. QYRVFRVRLPDPNKFGFPD HPV33-L1. QYRVFRVRLPDPNKFGFPD HPV58-L1. QYRVFRVRLPDPNKFGFPD HPV52-L1. QYRVFRIKLPDPNKFGFPD HPV18-L1.1 QYRVFRVQLPDPNKFGLPD HPV45-L1. QYRVFRVALPDPNKFGLPD HPV59-L1. QYRVFRVKLPDPNKFGLPD HPV39-L1. QYRVFRVTLPDPNKFSIPD HPV51-L1. QYRVFRVQLPDPNKFGLPD HPV56-L1. QYRVFRVRLPDPNKFGLPD

[0047] The second sequence disregards types 51 and 56 in order to maintain greater similarity. However, even with both types remaining, there is still reasonable similarity.

[0048] Target 4 was established as AATQRLVWACAGVEIGRGQPLGVGLSGHP, based on HPV18 and the following regions: HPV16-L1. TQRLVWACVGVEVGRGQPLGVGISGHP HPV35-L1. SQRLVWACTGVEVGRGQPLGVGISGHP HPV31-L1. TQRLVWACVGLEVGRGQPLGVGISGHP HPV33-L1. TQRLVWACVGLEIGRGQPLGVGISGHP HPV58-L1. TQRLVWACVGLEIGRGQPLGVGVSGHP HPV52-L1. TQRLVWACTGLEIGRGQPLGVGISGHP HPV18-L1.1 TQRLVWACAGVEIGRGQPLGVGLSGHP HPV45-L1. TQRLVWACVGMEIGRGQPLGIGLSGHP HPV59-L1. SQRLVWACVGVEIGRGQPLGVGLSGHP HPV39-L1. TQRLVWACVGVEVGRGQPLGVGISGHP

[0049] Once the diagnostic targets were determined and taking advantage of their relatively small sizes, they were obtained by chemical synthesis with a purity level greater than 95% (Genone, Brazil). Briefly, a microwave-assisted solid-phase synthesis (fMoc) and amino acid coupling method was used. Subsequently, the peptides were purified by high-performance liquid chromatography (HPLC) and analyzed by electrospray ionization (ESI) and / or matrix-assisted laser desorption / ionization (MALDI) mass spectrometry on ESI-Q-ToF Micromass Microrm (Micromass, Manchester, UK) and MALDI-ToF / ToFMS (Applied Biosystems, USA) mass spectrometers.

[0050] To select peptides capable of recognizing and binding to each target, the phage display technique was used. The commercial library Ph.D.-7 (New England Biolabs, USA) was used, with a repertoire of approximately 10 9independent clones. The system is based on a modified M13 vector for pentavalent peptide presentation at the N-terminal end of the viral capsid protein pIII. Due to the small size of each target, a solution capture technique was chosen to avoid steric hindrance. The library was incubated with each target in solution, and specific phages were captured using a streptavidin-based magnetic system (New England Biolabs, USA), according to the library and capture system manuals.

[0051] In detail, 20 mL of culture medium Luria Bertani flasks supplemented with 20 mg / L tetracycline (LB / Tet) were transferred to a 250 mL Erlenmeyer flask and infected with Escherichia coli ER2738. Additionally, a 50 mL Falcon tube containing 10 mL of the infected medium was prepared for phage titration. Both were incubated on a shaker set at 37°C and 180 rpm. Simultaneously, 50 μL of an aqueous suspension of streptavidin-labeled affinity beads was transferred to a 2 mL microtube, to which 1 mL of TBS buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl) was added to suspend the resin. The beads were captured using a magnetic capture system (New England Biolabs, USA), and the supernatant was removed. The resin was resuspended in 1 mL of blocking buffer (0.1 M NaHCO3, pH 8.6 supplemented with 5 mg / mL BSA) and incubated for 60 minutes at 4°C, with shaking every 15 minutes. The resin was captured again and washed 4 times with 1 mL of TBS buffer plus 0.2% Tween-20 (TBST).Simultaneously, a 10 μL aliquot of the library was mixed with each target peptide (final concentration of approximately 1 μM) for a total of 200 μL. Each mixture was incubated for 20 minutes at room temperature and transferred to the microtube containing the washed resin. These were incubated for 15 minutes at room temperature, with shaking every 5 minutes, and the resin was captured again. The supernatant was discarded and the resin was washed 10 times with 1 mL of TBST buffer.

[0052] The phages that remained attached to the resin were then eluted for 10 minutes with 1 mL of elution buffer (0.2 M Glycine-HCl pH 2.2 supplemented with 1 mg / mL BSA). The resin was captured again and the supernatant was transferred to a new microtube containing 150 μL of 1 M Tris-HCl buffer, pH 9.1, for pH neutralization. This is the phage eluate obtained after each selection step.

[0053] The titer of eluted phages was estimated by infecting the bacteria incubated in the Falcon tube. One hundred and ninety microliters of bacteria in logarithmic phase of growth (OD600 0.4 - 0.6) were transferred to microtubes, which were infected with 10 μL of eluted phages after preparing a serial dilution ranging from 10 to 10 4 . Each medium was incubated for 5 minutes at room temperature and 25 μL were transferred to petri dishes containing LB agar medium supplemented with IPTG (50 μg / mL) and Xgal (40 μg / mL). The plates were incubated for 16 hours at 37°C and the number of isolated colonies was evaluated.

[0054] The eluted phages were amplified by infecting the bacteria incubated in the Erlenmeyer flask (DO 6000.01–0.05) with the remaining eluted phages. The culture was incubated on a shaker set at 37°C and 180 rpm for 4.5 hours and subsequently centrifuged for 10 minutes at 12,000 xg (4°C). The supernatant was transferred to a new tube, and the centrifugation step was repeated. Sixteen milliliters was transferred to a new tube, to which 2.75 mL of a 20% polyethylene glycol 8,000 plus 2.5 M NaCl (PEG / NaCl) solution was added. This was incubated for 2 hours at 4°C to precipitate the phages, which were separated by centrifugation at 12,000 xg for 15 minutes (4°C). The supernatant was discarded, and the sample was centrifuged under the same conditions for 5 minutes. The residual supernatant was removed with a pipette, and the pellet was resuspended in 1 mL of TBS buffer and transferred to a 2 mL microtube. Cell debris was removed by centrifugation for 5 minutes at 12,000 xg (room temperature). An aliquot for maintenance at -80°C was prepared after adding glycerol to a final concentration of 50%, and the remainder was stored at 4°C. The titer of amplified phages was determined similarly to that described in the previous paragraph, varying the serial dilution (10 8 to 10 11 ).

[0055] From the amplified phage titer, these were diluted to values ​​close to 10 11pfu / mL and used in subsequent selection rounds for a total of 4 rounds against each target. To increase astringency, the Tween-20 concentration in the wash steps was increased to 0.5%, and the number of washes was increased to 15, 20, and 20. From the second round onward, negative selection was performed to avoid obtaining phages capable of recognizing the resin itself, biotin, or other interferents. To this end, the phages used at the beginning of each round, before being incubated with the resin bound to each target, were incubated with the resin bound to a biotin-labeled alanine dipeptide. The supernatant was collected and used in subsequent steps.

[0056] After the third and fourth rounds of selection, isolated clones were picked from the titration plates (blue colonies) and used to construct master plates. Wells of a 96-well culture plate were seeded with 150 μL of LB / Tet medium infected with E. coli ER2738 (DO 600 0.01-0.05) and reinfected with isolated clones collected from petri dishes. The cultures were incubated on a shaker for 4.5 hours at 37°C and 180 rpm. 50 μL of an 80% glycerol solution in LB medium was added to each well, and the plates were stored at -80°C.

[0057] For the sequencing step, a culture of E. coli ER2738 was grown for 16 hours in LB / Tet medium on a shaker set at 37°C and 180 rpm. The culture was diluted 1:100 in the same medium and distributed into 2 mL microtubes containing 1 mL of medium. Sterile tips were used to pick isolated clones from the master plates into each microtube, which were incubated on a shaker, under the same conditions, for 4.5 hours. The samples were centrifuged for 30 seconds at 12,000 xg, and 500 μL of each supernatant was transferred to new microtubes. 200 μL of the PEG / NaCl solution was added, and the samples were incubated for 15 minutes at room temperature. The microtubes were centrifuged at 12,000 xg for 10 minutes (4°C) and the supernatants discarded. Each precipitate, sometimes not visible, was resuspended in 100 μL of iodide buffer (100 mM Tris-HCl pH 8.0, 1.0 mM EDTA, 4.0 M NaI) and 250 μL of ethanol to each microtube. After incubation for 15 minutes at room temperature, the microtubes were centrifuged at 12,000 xg for 10 minutes (4°C), and the supernatants were discarded. The precipitates were washed with 500 μL of 70% ethanol kept at -20°C, centrifuged under the same conditions, and the supernatants were discarded. The microtubes were dried in a vacuum system, and the precipitates were resuspended in 30 μL of ultrapure water. The genetic material was confirmed by agarose gel electrophoresis and quantified in a spectrophotometer adjusted for DNA quantification (Thermofisher, USA). Sequencing was performed by a third-party company (Helixxa, Brazil).

[0058] Four selection rounds were performed for each target, which were monitored by ELISA in order to evaluate the increase in specificity throughout the selection.

[0059] Polyclonal assays were conducted, in which the phages eluted after each round of selection were amplified and subjected to ELISA assays. As expected, the phages from the fourth round demonstrated greater specificity against their respective targets. These phages were isolated, amplified, and subjected to monoclonal ELISA assays. The clones that performed best were amplified and subjected to subsequent ELISA tests, in addition to genetic sequencing to determine the presence of consensus sequences. The appearance of repeated sequences within a universe of 10 9 independent clones suggests a selection process satisfactory.

[0060] Selections against HPV 16- and 18-based E6 targets, targets 1 and 2, respectively, resulted in two consensus sequences for the first target and none for the second. Therefore, the results regarding selection against HPV 18-based E6, target 2, were unsatisfactory.

[0061] Selections against HPV 16 and 18-based L1 targets, targets 3 and 4, respectively, resulted in three consensus sequences for the former and two for the latter. The consensus sequences, as well as some others related to phages that performed well in monoclonal ELISA assays, are:

[0062] For target 1 (HPV 16-E6): Phage F1. 1: GSPCYKC (consensus), Phage F1. 2: MGCGQCT (consensus), Phage F1. 3: MHVQTLA, Phage F1. 4: CELCHWP,

[0063] For target 3 (HPV 16-L1): Phage F3.1: KCCYVTP (consensus), Phage F3.2: TILNYHI (consensus), Phage F3.3: KCCFPVI (consensus), Phage F3.4: FAQDWHV,

[0064] For target 4 (HPV 18-L1): Phage F4.1: EWYWWSY (consensus), Phage F4.2: YHYTPYN (consensus), Phage F4.3: LMVSPSL, Phage F4.4: NWSSLLQ.

[0065] Taking advantage of the small size of each variable region responsible for interaction with the target (7 amino acid residues, according to the Ph.D.-7 library), chemical synthesis of the peptides was chosen. Since each phage presents a linker (Gly-Gly-Gly-Ser) between the peptide and the capsid protein, pIII, this sequence was preserved during synthesis. The sequences were synthesized with a purity level greater than 90% and treated with trifluoroacetic acid (Genone, Brazil). To ensure greater testing options and to check for unwanted interactions and steric hindrances after fusion of the peptides with markers (enzymes, gold particles, etc.), each sequence was synthesized in two formats: with and without bovine serum albumin (BSA) at the carboxy-terminal region.

[0066] To verify specificity, each phage of interest was amplified and subjected to a monoclonal ELISA assay in which the concentrations of the target and control (biotin-conjugated alanine-alanine dipeptide, similar to the targets used in the selection steps) were increased. As expected, the results increased with increasing target concentrations and remained stable regardless of the control concentration. It is important to note that, because these are different clones, the sensitivities to their respective targets varied.

[0067] In greater detail, wells of a plate ELISAs were sensitized with 50 μL of a 2.5 μg / mL streptavidin solution prepared in 100 mM NaHCO3 buffer, pH 8.6. As a negative control, wells were sensitized with the buffer alone, and as a positive control, wells were sensitized with 5 μL of each phage plus 45 μL of TBS buffer. The plate was incubated for 16 hours at 4°C, and the contents of each well were discarded. Unbound sites were blocked with 250 μL / well of 0.1% bovine serum albumin (BSA) in TBS buffer, and the plate was incubated for 2 hours at 4°C. After blocking, the plate was washed 4 times with 0.5% TBST. Each target peptide, as well as the biotin-labeled double-alanine dipeptide, were added in increasing concentrations by a factor of 4x (0.005; 0.02; 0.08; 0.32; 1.28 and 5.12 μM), prepared in 0.1% TBST buffer, 50 μL / well, and the plate was incubated for 30 min at room temperature.

[0068] After a new washing step, as in the previous step, the respective phages for each target were added (25 μL of each phage plus 25 μL of BSA prepared in 0.5% TBST buffer), and the plate was incubated for 30 minutes at room temperature. After a new washing step, 50 μL of an anti-M13 antibody solution labeled with HRP (Santa Cruz Biotechnology, USA) diluted 1:7000 in 0.05% BSA prepared in 0.25% TBST was added, and the plate was incubated for another 30 minutes at room temperature.

[0069] The plate was washed again and developed with TMB solution (1.0 mL of 3,3′,5,5′-Tetramethylbenzidine and 5 μL of H2O2 diluted in 9.0 mL of 100 mM sodium citrate-phosphate buffer (pH 5.0), 100 μL / well. The plate was incubated at room temperature for 15 minutes and the reaction was stopped by adding 50 μL of H2SO42 M. The plate was then analyzed in an ELISA plate reader set to 450 nm.

[0070] Figure 2 shows the lowest concentrations at which there was a significant difference between the target and the control. A random protein (lytic polysaccharide monooxygenase, PnAA16a from Peniophora) was also tested, and the results were similar to those of the control, corroborating the specificity of the assay. The negative control, whose wells were sensitized with buffer only, yielded values ​​close to 0.050, and the positive control yielded values ​​above 1.00, which are not shown in the graph in Figure 2.

[0071] The synthetic peptides conjugated to BSA (named after their respective phages, replacing "F" with "PB") were subjected to ELISA tests to verify their ability to interact with their respective targets in order to optimize the ELISA and lateral flow assays. Unlike the previous assay, in this one, wells of an ELISA plate were sensitized with the BSA-conjugated peptides and treated with each target, which were labeled with biotin. As seen in Figure 3, those selected against targets 1 and 4 demonstrated satisfactory results, but not those selected against target 3 (considering that no peptides selected against target 2 were synthesized). As expected, no interaction was observed with the negative control (double alanine conjugated to biotin). Since the peptides not conjugated to BSA are very small, they were not used in ELISA assays due to impaired plate binding capacity. ELISA assays with synthetic peptides were performed similarly to that described for phages.

[0072] In summary, the method of the present invention comprises the following steps: A) identify conserved regions, through in silico analysis, in the form of peptide sequences present in HPV types considered high-risk. B) chemically synthesize the selected molecular targets: Target 1. AAKQRFHNIAGRWTGRC Target 2. AARRPYKLPDLCTALN Target 3. AAQYRVFRIALPDPNKFGFPD Target 4. AATQRLVWACAGVEIGRGQPLGVGLSGHP C) select phage-peptides, through the phage display technique, capable of recognizing and binding with high affinity and specificity to the molecular targets previously determined; D) chemically synthesize selected peptides, capable of recognizing the diagnostic targets: GSPCYKC (SEQ ID NO:1), MGCGQCT (SEQ ID NO:2), MHVQTLA (SEQ ID NO:3) and CELCHWP (SEQ ID NO:4); KCCYVTP (SEQ ID NO:5), TILNYHI (SEQ ID NO:6), KCCFPVI (SEQ ID NO:7) and FAQDWHV (SEQ ID NO:8); EWYWWSY (SEQ ID NO:9), YHYTPYN (SEQ ID NO:10), LMVSPSL (SEQ ID NO:11) and NWSSLLQ (SEQ ID NO:12); and E) evaluate the phages and synthetic peptides for their binding capabilities to diagnostic targets via ELISA and lateral flow platforms.

[0073] The phages of interest, as well as the synthetic peptides, were also evaluated through diagnostic tests, such as devices and lateral flow chromatography tests. A membrane card platform was used, consisting of a plastic support on which a nitrocellulose membrane (Hi-FLow Plus Membrane HF135 and HF075, Merck) was mounted. The membrane card has specific adhesive regions for attachment to the nitrocellulose membrane, in addition to other components, such as the absorption and conjugation pads used (GFDX Glass Fiber Conjugate Pads and C083 Cellulose Absorbent Sample Pad, Merck, from the "Sure Wick" line), superimposed on the nitrocellulose membrane.

[0074] The samples (synthetic targets or viral samples), as well as the other components (gold-labeled peptides, buffer, etc.) are added to the nitrocellulose membrane, sample pads, and conjugation pads. As different strategies were tested in the present invention, the components varied according to the strategies.

[0075] Thus, the method of preparing the lateral flow device comprises the following steps: i) a nitrocellulose membrane labeled with the peptides of the present invention is mounted on a plastic support of a membrane card-type platform; ii) the absorption and conjugation pads are mounted superimposed on the nitrocellulose membrane; iii) the sample pad is also fixed on the membrane card superimposed on the conjugation pad, so that the assembly presents an adequate capillary flow.

[0076] Different development strategies were evaluated, such as enzymatic development (TMB / HRP) or colloidal gold labeling through adsorption or conjugation using specific kits (Bioready 150 nm Carboxyl Gold Nanoshells, NanoComposix).

[0077] The synthetic peptides conjugated with BSA (PB1.1, PB1.2, PB3.1, PB3.2, PB4.1 and PB4.2) demonstrated positive results in a direct test, when sensitized against the membrane and revealed with TMB after application of each target and washing of the membrane (reaction with peroxidase-labeled antibiotin), which helps to confirm the binding capacity of each peptide against its respective target.

[0078] The strategy of labeling BSA with streptavidin, followed by sensitization of the membrane with the product, application of a mixture containing phages and respective targets, washing and development with TMB (reaction with anti-M13 labeled with peroxidase) was satisfactory for phages F1.1, F1.3, F3.2 and F4.1.

[0079] Sensitization of the membrane with the phages themselves returned positive results for F1.3, F3.3 and F4.1 after treatment with the respective targets and revelation with both TMB (reaction with peroxidase-labeled antibiotin) and conjugated gold (streptavidin conjugated with gold).

[0080] Sandwich assays with BSA-conjugated synthetic peptides (hereinafter referred to as AB), in which these were sensitized on the membrane, treated with their respective targets and revealed with other BSA-conjugated and gold-labeled synthetic peptides, were satisfactory for the AB3.2 / AB3.1 and AB4.1 / AB4.2 pairs. In similar assays with membrane sensitization with BSA-conjugated synthetic peptides and revelation with gold-labeled phages, the AB3.2 / F3.1; AB3.2 / F3.3 and AB4.1 / F4.2 pairs also showed positive results.

[0081] Since these are qualitative tests, Figure 4 presents examples of positive results for each of the development techniques evaluated, in the following order: enzymatic reaction with TMB, labeling with adsorbed colloidal gold, and conjugation with gold particles. For example, The synthetic peptides conjugated to BSA (PB1.1, 1.2, 3.1, 3.2, 4.1 and 4.2) demonstrated satisfactory results when sensitized against the membrane, which was treated with the respective targets conjugated to biotin and revealed with anti-biotin antibody conjugated to HRP in TMB / H2O2 substrate. The labeling of the membrane with streptavidin (conjugated to BSA), followed by treatment with phages and respective targets (conjugated to biotin) and revelation with anti-M13 antibody conjugated to HRP in TMB / H2O2 substrate returned positive results for phages F1.1, F1.3, F3.2 and F4.1. The sensitization of the membrane with phages followed by treatment with the respective targets (conjugated to biotin) returned positive results for F1.3, F3.3 and F4. 1 when revealed with either TMB (reaction with anti-biotin antibody conjugated to HRP) or gold (streptavidin conjugated to gold particles). The examples described above illustrate direct lateral flow tests.Additionally, the efficacy of indirect assays was evaluated by sensitizing the membrane with synthetic peptides conjugated to BSA, followed by treatment with gold-adsorbed phages and their respective targets. The results were satisfactory for the AB3.2 / F3.1; AB3.2 / F3.3; and AB4.1 / F4.2 pairs. This range of possibilities guarantees freedom of choice for different lateral flow strategies depending on the established parameters, such as sensitivity, specificity, production cost, among others.

[0082] As explained above and observed in Figure 4, the HPV biomarker identification method proved to be successful, since it was possible to produce phages capable of recognizing and binding with high specificity and sensitivity to the proposed diagnostic targets in both ELISA and lateral flow platforms.

[0083] Additionally, the sequences were chemically synthesized as peptides, which also demonstrated good binding capabilities to their respective targets. These sequences can then be used to develop commercial tests and kits for detecting viral particles of interest from a biological sample obtained from an individual, such as a swab or cervical mucus sample. These patients can then be referred for routine monitoring or more in-depth analysis of the lesion stage.

[0084] The applications of the objects of the present invention in all their modalities can be contemplated from the following embodiment examples. Example:

[0085] As previously demonstrated by ELISA, phages were able to recognize synthetic targets constructed from E6 and L1 consensus sequences of high-risk HPV strains.

[0086] In this example, we aimed to demonstrate that the selected monoclonal phages are also capable of recognizing the “native” proteins of viruses. To this end, we worked with samples of HPV 16 and HPV 18 cultured in CASKI and HELA cells, respectively.

[0087] Viruses were collected and treated with a denaturing solution containing 1% Triton X-100 to expose the molecular targets. These samples were then used to sensitize wells of an ELISA plate. As a negative control, wells were sensitized with BSA, and as a positive control, wells were sensitized with phages (only two positive controls, phages F1.1 and F3.1). After blocking and washing steps, the wells were treated with the phages and developed with anti-M13 antibody conjugated to HRP and TMB / H2O2 substrate. A negative control was also performed with wells sensitized with HPV viral samples only, not treated with phages in the subsequent step.

[0088] According to the results presented in Figure 5, the selected phages were able to recognize both viral samples (HPV 16 and HPV 18) with varying degrees of specificity depending on the phage and strain used. The negative results against BSA suggest that the phages are specific for their proposed targets. The negative results with HPV-sensitized, untreated wells demonstrate that viruses alone are not capable of generating positive results.

[0089] This experiment is evidence that the phages selected in the present invention are capable of recognizing the viral proteins E6 and L1 from sequences chemically synthesized conserved after in silico analysis of E6 and L1 from several high-risk HPV strains.

[0090] The present invention further comprises a diagnostic kit modality comprising synthetic peptides conjugated to BSA as obtained by the method described above, for detecting HPV E6 and L1 viral proteins from a biological sample of a smear or cervicouterine mucus obtained from an individual. For detection, the kit further comprises reagents in the form of a cell extraction / lysis buffer composed of 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1001% Triton X, 1 mM EDTA, and 1 mM PMSF.

[0091] In the lateral flow device, TMB development was ensured by adding a substrate solution (1.0 mL of 3,3′,5,5′-Tetramethylbenzidine and 5.0 μL of H2O2 diluted in 9.0 mL of 100 mM sodium citrate-phosphate buffer, pH 5.0) to the membrane, which is consumed by a peroxidase-type enzyme previously conjugated to one of the markers; gold development was developed by two methods: adsorption of colloidal gold to the selected markers, prepared with 40.01% AuCl and 1.0% sodium citrate, according to the protocol described by Anfossi et. al., (Development and application of a quantitative lateral flow assay for fumonisins in corn, 2010); and conjugation of colloidal gold to the selected markers, using the Bioready 150 nm Carboxyl Gold Nanoshells kit (NanoComposix).

Claims

1 / 3 CLAIMS 1. Method for identifying HPV biomarkers, characterized by the fact that it comprises: A) identifying conserved regions, through in silico analysis, in the form of peptide sequences present in HPV types considered high risk; B) chemically synthesizing the selected molecular targets: Target 1. AAKQRFHNIAGRWTGRC Target 2. AARRPYKLPDLCTALN Target 3. AAQYRVFRIALPDPNKFGFPD Target 4.AATQRLVWACAGVEIGRGQPLGVGLSGHP C) select peptide phages, through the phage display technique, capable of recognizing and binding with high affinity and specificity to the previously determined molecular targets; D) chemically synthesize selected peptides capable of recognizing the diagnostic targets: GSPCYKC (SEQ ID NO: 1), MGCGQCT (SEQ ID NO: 2), MHVQTLA (SEQ ID NO: 3) and CELCHWP (SEQ ID NO: 4); KCCYVTP (SEQ ID NO: 5), TILNYHI (SEQ ID NO: 6), KCCFPVI (SEQ ID NO: 7) and FAQDWHV (SEQ ID NO: 8); EWYWWSY (SEQ ID NO: 9), YHYTPYN (SEQ ID NO: 10), LMVSPSL (SEQ ID NO: 11) and NWSSLLQ (SEQ ID NO: 12); and E) evaluate synthetic phages and peptides for their binding capabilities to diagnostic targets via ELISA and lateral flow platforms. 2 / 3 2. Method according to claim 1, characterized in that in step D) the peptides are synthesized in the presence and absence of bovine serum albumin (BSA) in the carboxy-terminal region.

3. Method for preparing a lateral flow device, characterized in that it comprises the steps of: i) a nitrocellulose membrane labeled with the peptides obtained in claim 1 is mounted on a plastic support of a membrane card-type platform; ii) the absorption and conjugation pads are mounted superimposed on the nitrocellulose membrane; iii) the sample pad is also fixed to the membrane card superimposed on the conjugation pad, so that the assembly presents an adequate capillary flow. 4.Lateral flow device, obtained by the method defined in claim 3, characterized by the fact that it comprises the synthetic peptides conjugated with BSA obtained by the method of claim 1, for detecting HPV E6 and L1 viral proteins from a biological sample obtained from an individual.

5. Lateral flow device, according to claim 4, characterized by the fact that it is a chromatographic test comprising a membrane card-type platform. 3 / 3 6. Lateral flow device according to claim 5, characterized in that it detects HPV viral proteins through enzymatic reaction with TMB, labeling with colloidal gold through adsorption or conjugation with gold particles.

7. Lateral flow device according to claim 6, characterized in that the development with TMB occurs by adding a substrate solution containing 3,3′,5,5′-Tetramethylbenzidine and H2O2 diluted in 100 mM sodium citrate-phosphate buffer pH 5.0 to the membrane, which is consumed by a peroxidase-type enzyme previously conjugated to one of the markers. 8.Lateral flow device according to claim 6, characterized in that the gold development is performed by two methods: adsorption of colloidal gold to the selected markers, prepared with 0.01% AuCl4 and 1.0% sodium citrate, and conjugation of colloidal gold to the selected markers, through the Bioready 150 nm Carboxyl Gold Nanoshells kit.

9. Kit for detecting HPV viral proteins, characterized in that it comprises the lateral flow device, as defined in claim 4, and other reagents.

10. Kit, according to claim 9, characterized in that the reagents comprise cell extraction / lysis buffer, composed of 50 mM Tris-HCl pH 7.5, 150 mM NaCl, Triton X-1001%, 1 mM EDTA and 1 mM PMSF.

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