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Summer Research Fellowship Programme of India's Science Academies

Genotyping of HPV in cervical cancer samples using the type-specific bead-based multiplex genotyping method

H K Shreyas1

3rd year Integrated M.Sc, Molecular Biology, Yuvaraja's College (Autonomous), University of Mysore, JLB road, Mysuru 5700 05

Pillai Radhakrishna

Director, Rajiv Gandhi Center For Biotechnology, Poojapura, Thycaud, Thiruvananthapuram, Kerala 695014

Anantharaman Devasena

Scientist EII, Rajiv Gandhi Center For Biotechnology, Poojapura, Thycaud, Thiruvananthapuram, Kerala 695014

Abstract

Human papillomavirus is the primary cause for cervical cancer. The high risk types mainly, HPV-16 and HPV-18 are associated with carcinoma of cervix. Upon infection, the viral transcripts are translated into early and late proteins, of which proteins like E6 and E7 are known to hijack basic mechanisms like the cell cycle regulation and apoptosis which eventually leads to development of cancer. Vaccination against the virus, using vaccines such as Gardasil and Ceravrix in females has provided protection in women against the quadrivalent infection and cervical cancer development associated with it. The current study includes Multiplex bead-based genotyping of 21 HPV types in 30 cervical samples that include both vaccinated and unvaccinated samples and gives a comparison of effectiveness of vaccine against the infection. Also, the study describes the genotyping method and results for HPV-16/E7 developed in-house.

Keywords: cervical cancer, Human papillpomavirus, E6 and E7, multiplex bead-based genotyping

Abbreviations

TAble 2
In-house
HPV-16/E7 genotyping results
 Sl. No Individual HPV type/ controlMFI 
 1.HPV-16  1209.5 (+ve)
2.  Luminex control2.0 

INTRODUCTION

Cervical Cancer

Cervical cancer refers to cancers affecting the cervix or the lower part of the female reproductive tract, that secretes mucus against the pathogens entering the uterus. As per the WHO’s findings, in 2018, around 96,922 new cases of cervical cancer were reported in India, ranking second in relation to the incidence rate in the entire world (1). Various epidemiological examinations have shown HPV, as the sole cause of this condition which may be influenced by several other secondary factors (2). Common secondary risk factors include lifestyle changes, smoking, use of other tobacco-based products, reproductive factors and sexual habits all of which alter the genetic and epigenetic factors resulting in carcinoma of the cervix (3,4).

Objectives of the Research

     i.  Testing thirty cervical samples (vaccinated and unvaccinated) using the established high throughput type-specific E7 bead-based multiplex genotyping method.

    ii.  Development of the modified, in-house bead-based multiplex genotyping method. Specifically, to standardize the coupling of microsphere polystyrene beads with type-specific amine modified oligonucleotide probes developed in-house and to synthesize a vector based in-vitro system to establish the sensitivity of the method.

LITERATURE REVIEW

HPV and Cervical Cancer

HPV is a DNA based non-enveloped virus belonging to Alphapapillomavirus genera that comprises around 120 well characterised types, classified as the high-risk (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, 82), putative (26, 53, 66) or the low-risk (6, 11, 40, 42, 43, 44, 70) types. (5) The high risk (Hr) types infect the mucosal layer causing cervical, head and neck cancer and the other types cause cutaneous infections including genital warts, skin warts and skin cancer. The high-risk types are commonly associated with cervical cancer, and of these Hr-types, 70% of the cervical carcinoma is due to HPV-16 infection. (HPV oncoproteins pathways to transformation). However, it is known that several genetic alterations mediated by viral genes and viral factors are essential for tumour progression and malignancy.

 

All HPVs consists of a circular genome which is of 8kb in size. The entire genome is divided into ‘early’(E) and ‘late’(L) regions based on the time of expression and function. The early region encodes for 6 major proteins regulating viral genome replication and transcription, cell cycle and host-cell immune modulation. The late region codes for 2 proteins L1and L2 that are specifically expressed in the outermost epithelial layer at a later stage of infection (6,7). However, studies have shown that the isoforms of E6 (FLE6, E6I, E6II, E6X) in HPV-16 and HPV-18 cases, have vital importance in tumorigenesis (8). For example, E6I isoform is said to have a role in the regulation of procaspase 8 of the extrinsic apoptotic pathway and in cellular protein degradation (8). The viral transcription factor E2 is said to regulate viral copy number (6). The following figure depicts the HPV genome regions and their corresponding functions.

image 1.png
    Genomic organisation and function of HPV (Riemer et al., 2010 )

    Life Cycle

    The key to understanding tumorigenesis by crucial viral oncogenes viz., the E6/E7 lies in the unusual life cycle of the virus. While in most other viruses infecting a cell, replication of the virus occurs from the same infected cell but in case of HPV, the virus-host cell interaction plays a crucial role in its life cycle. The keratinocytes of the basal layer of the epithelium which is exposed to micro-wounds are more susceptible to HPV infection. Such infected cells can have HPV genomes either as episomes or as integrated genomes. The viral genomes replicate in parallel with the cellular DNA. The resulting mitotic division of infected cells results in one of the daughter cells to move away from the basal layer and unequal division. This differentiation induces the productive phase of the viral life cycle which requires DNA synthesis machinery. At this stage, the expression of viral oncogenes E6 and E7 are expressed which dismantles the delicate balance in cell-cycle control and forces the differentiated cells back to S-phase facilitating viral genome replication following which the expression L1 and L2 genes encapsulate the viral genomes which finally exit from the uppermost layers of the epithelium (6, 9, 10).

    Molecular Basis of Pathogenesis in Cervical Cancer

    As stated earlier, E6 and E7 are two viral oncoproteins involved in primary tumorigenesis of HPV infected cervical epithelial cells (11). As per the literature review, E6 (17kDa) and E7 (73k Da) are said to be nuclear proteins while some state that E7 is present in the cytoplasm of infected cells and E6 in both, nucleus and cytoplasm (12, 13). It is also debated that expression of E7 alone is sufficient to immortalize keratinocytes, but the combination being more efficient in altering the host cellular regulation promote carcinoma development (14, 15, 16). The nature of existence of viral genome also has a correlation to pathogenesis. In pre-cancerous lesions, if the viral genome is present in an extrachromosomal form, the E2 protein is expressed with the early genes including E6 and E7 as its direct target and represses their expression and in turn, the proliferative ability of host cell but upregulates the replication mechanism to increase the copy number (9). This explains the importance of E2 as a replication factor. Upon integration with genomic unstable regions, the expression of E2 is disrupted leading to deregulated expression of early genes promoting uncontrolled cell proliferation.

    image 3.png
      HPV mediated cervical cancer progression: A diagrammatic representation of existence of viral genome and expression of early and late viral oncogenes in various grades of cervical neoplasia (Woodman et al., 2007)

      The induction of hyperproliferation by the E7 protein triggers apoptosis, which is blocked by the actions of the E6 protein. Collectively E6 and E7 targets the regulatory factors controlling proliferation, apoptosis, immortalization and genomic stability in turn promoting transformation of cells with altered growth leading to malignancy.

      image 4.png
        Common molecular mechanisms that serve as the target for HPV E6 and E7 oncoproteins (Moody et al., 2010)

        The E6 targets p53-tumour suppressor protein directly. E7 targets and degrades Rb-family of proteins that regulate cell differentiation and maintains genome stability through ubiquitin mediated pathway (6), which eventually reduces cell growth and apoptosis by p53 dependent pathway. Due to this altered cell programming, over time, mutations accumulate leading to cancer.

        Immortalization of cells is a characteristic feature of cancer. Analysis of chromosomes has shown that the lifespan of a cell is attributed to the length of telomeric sequences (17) and the enzyme Telomerase Reverse Transcriptase (TERT) that replicate such sequences. Hence, this enzyme is an important target for the viral oncoproteins. High-risk E6 activates transcription of TERT, which is supplemented by E7 mediated-Rb inactivation. (6, 13).

        As mentioned earlier the alterations brought about by E6/E7 proteins are not sufficient to cause cervical malignancy, several genetic changes like mutations at the genetic level and centromeric abnormalities like aneuploidy and translocations at the chromosome level promote malignancy. Hr-E7 inactivates RB family of proteins leading to G1-S arrest, thus promoting hyperproliferation of cells. The cellular expression is totally de-mantled due to interaction of E7 with NA binding proteins like Histone Deacetylases (HDACs). The cell cycle control is further deregulated due to E7 mediated inactivation of cyclin dependent kinase (CDK) inhibitors (CKIs) like p21 and p27, thereby stimulating cyclins by direct CDK2 activation. Due to this E7 mediated CDK2 activation, an abnormal centrosome synthesis is stimulated altering the delicate genomic balance existing inside the cell. This genomic instability is further increased due to micro-filament instability, especially γ-tubulin due to viral E7 protein. p53 mediated apoptosis is another major target of E6-E7 co-expression which ultimately results accumulation of genomic instability caused due to an imbalance in the cell’s replication system despite the existing DNA damage (6). In the cytoplasm, p600 and clathrin form a meshwork structure acting like a cytoskeletal organization and is said to play a role in membrane morphogenesis. Reduced p600 expression has shown to prevent interferon mediated survival pathways and promoting cells to detach from the extracellular matrix scaffold thus making the cells free (18). Having this known, it is reported that E7 acts on p600, promoting anchorage independency facilitating the malignant progression (19). E7 also acts on Interferon Receptor Factor (IRF), helping HPV infected cells escape immune surveillance which can be one of the reasons for HPV persistent infections.

        image 5.png
          Common molecular mechanisms that serve as the target for HPV E6 and E7 oncoproteins (Moody et al., 2010)

          HPV Vaccination

          With HPV as the major cause of cervical cancer, the need for the development of vaccines against HPV becomes more evident. In this context, two vaccines were recently introduced, Gardasil (Merck Sharp and Dohme, Whitehouse Station, NJ, USA) and Cervarix (Glaxo, Smith, Kline). Gardasil is a quadrivalent prophylactic vaccine given against HPV-6, HPV-11, HPV-16 and HPV-18 infections in females aged from 10–18 years presumably, above the age of 15 years. This vaccine when given in 3 doses on day 1, day 60 and day 180 or later has shown a significant reduction in the incidence of HPV infections, specifically HPV-16 and 18 infections. However, the WHO recommends a 2-dose vaccination (day1 and day 180 or greater) with a gap of at least 6 months in young girls as sufficient dose against Hr-HPV types, pointing out that single dose of the HPV vaccine has further scope in terms of efficacy in preventing incident HPV infections. In support to this, a recent Indian based multi-centric randomized cohort study have the findings stating that 2-dose vaccine administration within a period of more than 180 days has non-inferior results in terms of immunogenicity in comparison to 3-doses administration against the incident and HPV-quadrivalent infections. Additionally, the 2-dose vaccination has been found successful in reducing non-targeted HPV types also. The study also states that even when a single dose was administered, detectable amounts of neutralizing antibodies were produced against the quadrivalent types, but in a lower concentration indicating further scope of study in this direction (19, 20, 21).

          METHODOLOGY

          Type-specific E7 Bead-based Multiplex genotyping from Cervical Samples

          DNA isolation

          Around 30 cervical samples from the ongoing multi-centric HPV vaccination study were included and DNA was extracted from the samples using EZ-1 extraction robot (QIAGEN, Germany).

          Multiplex-PCR amplification

          PCR was performed using 21 HPV-E7 type-specific primers for 21 types. The reverse primers were biotinylated. The PCR was carried out for 45 cycles at an annealing temperature of 63 degree Celsius with Beta-globin as the internal positive control. PCR was conducted using Applied Biosystems Veriti Thermal Cycler, USA.

          Table 2. PCR standardized conditions (Schmitt et al., 2006)

          PCR standardized conditions (Schmitt et al., 2006)
          Steps involved Temperature in degree Celsius Time
          Initial denaturation 95 15 minutes (1X)
          Final denaturation 94 30 seconds
          Annealing 63 3 seconds 45 cycles
          Extension 72 1 minute 30 seconds
          Final extension 72 10 minutes

          Hybridization assay and Multiplex genotyping

          Following PCR amplification, the PCR products were hybridized to previously available amine-modified oligonucleotide probes-coupled microsphere polystyrene beads. Each bead set had around 2000 beads for each HPV type containing a specific fluorescent dye ratio. The hybridization efficiency of a particular type to the coupled probe was computed in terms of reporter median fluorescence intensity of any 100 beads from each bead set upon addition of streptavidin-R-phycoerythrin conjugate to each bead set (for detailed procedure of hybridization refer Appendix 1) through the type-specific bead-based multiplex genotyping assay. The genotyping was done using the Bio-Plex200 Luminex platform. This method is a high throughput method which allows simultaneous analysis of absorption spectra of around 100 different types in a single reaction.

          ​Statistics

          Validation of the obtained result was done by using Chi-square test. The efficiency of vaccination against specific HPV types was determined in terms of percentage.

          Inhouse HPV-16 E7 genotyping

          Cell line maintenance

          SiHa, HeLa and CaSki were used in the experimental procedures. SiHa is a single copy number HPV16 positive cell line. HeLa is HPV-18 positive and CaSki is HPV-16 and 18 positive respectively. Cells were maintained according to the guidelines of the American Type Culture Collection (ATCC). The cells were cultured on a 10 cm Petri plate in Dulbecco Modified Eagle’s Medium (DMEM) containing 10% FBS in 5% CO2 incubator at 37 °C until 80% confluency. SiHa cells were further used for genotyping.

          ​DNA isolation and PCR amplification

          DNA was isolated from cultured SiHa cells using the standard CSHL (Cold Spring Harbour Laboratory) protocol (refer appendix 2). PCR was performed targeting the E7 region using type-specific primers with the reverse primer being biotinylated. The PCR conditions followed were similar to those discussed in Table 1 of section 3.1.i. The PCR products after amplification were confirmed on a 1% agarose gel.

          Coupling and hybridization

          Initial bead count was determined so as to get 2000 beads per reaction from each bead sort. The inhouse developed 5| amino-modifier C-12 linked oligonucleotide probes specific for HPV-16 E7 region were coupled to carboxylated beads through a carbodiimide bond formation. Every combination of probe and bead set had 2.5 million carboxylated beads (xMAP; Luminex corp., Austin, TX) to which 25µl of 0.1 M coupling buffer (2-( N-Morpholino) ethansulphonic acid; pH 4.5) was added. To this bead mixture, 400pmol of probes and 200µl (100mg/ml stock) of (1-(3-dimethylaminopropyl)-3-ethyl carbodiimide) or EDC was added and mixed properly prior to use. The beads were incubated for half an hour at ̴ 350rpm in dark, with a vortex step at in the middle of incubation. The steps from EDC addition till incubation was repeated once again after which 1ml of 0.02% Tween-20 (0.2g/L) and 1ml of 0.1% Sodium Dodecyl Sulphate (0.1g/L) was added and the beads were centrifuged at 13000rpm for 2 minutes. The supernatant was discarded and the oligonucleotide-coupled beads were resuspended in 100µl of TE (23, 24).

          Hybridization of HPV-16 E7 PCR product to the inhouse designed oligonucleotide probes was performed as discussed in section 3.1.i.c and Appendix 1 followed by the which HPV-16/E7 Uni plex-genotyping was performed using the Bio-Plex200 Luminex platform.

          image 6.jpg
            Diagrammatic representation of principle and working of type- specific bead-based genotyping(Schmitt et al., 2006)  

            Picture3.png
              xMap-Luminex assay (a. Bio-Plex200 Luminex platform b. carboxylated micro-bead c. 96 well-plate d. bead-based fluorescence detection)

              Cloning

              To evaluate the sensitivity and specificity of the assay developed, plasmid clones of the HPV-16 was used. In this assay, sensitivity refers to the lowest limit of detection of a particular HPV type. Specificity is the ability to detect the required HPV determinant type.

              RESULTS

              Type-Specific E7 Bead-Based Multiplex Genotyping from Cervical Samples

              a.     Multiplex-Genotyping results

              Picture4.png
                Genotyping results of thirty cervical samples: The samples collected includes 15 unvaccinated and vaccinated samples. The samples were considered to be positive for any one of the 21 HPV types if the reported median fluorescence intensity was above a particular lower limit of background intensity. From the above table it is clear that, except for one case, majority of the vaccinated individuals show almost nil detection for the presence of 21 HPV types. On the other hand, most of the unvaccinated samples were found to be positive for more than one of the detected 21 HPV types which clearly depicts the efficiency of protection due to vaccination against HPV mediated cervical carcinoma in females.

                b.     Interpretation of the Luminex data

                The Luminex assay is a qualitative assay with five controls (refer Appendix 1). Of the controls used comparison and determination of HPV positivity from a particular sample is determined using the negative control (usually the Luminex control) having the highest MFI. The bead background cut-off for the control is calculated using equation,

                Bead background cut-off = (MFI of control used) 1.1+5

                If any of the samples included in the study had MFI value greater than the mentioned cut-off of the included 21 HPV types, then such samples were considered to be positive for that HPV type.

                Inhouse HPV-16 E7 Genotyping

                a. Cell line maintenance: SiHa (HPV-16) and CaSki (HPV-16 and 18) that were cultured are shown below.

                Picture5.png
                  a. SiHa b. CaSki

                  b. PCR of E7 HPV-16 SiHa template

                  image 9.png
                    Lane 1&2- SiHa template (238bp),Lane 3&4 –Clinical samples, Lane 5 and 6 –Negative control, Lane 7-100bp ladder

                    c. In-house HPV-16 (SiHa) genotyping Luminex assay results

                    TAble 2
                    In-house
                    HPV-16/E7 genotyping results
                     Sl. No Individual HPV type/ controlMFI 
                     1.HPV-16  1209.5 (+ve)
                    2.  Luminex control2.0 

                    On comparison with Luminex control, it is clear that the median fluorescence intensity for the hybridized HPV-16/E7 PCR products with the specific oligonucleotide probe, is significantly higher to consider that SiHa cells cultured are positive for HPV-16.

                    d. PCR protocol standardization for cloning primers

                    Gradient PCR for HPV-16 type-specific primers was performed over a range of ten annealing temperatures differing by 2 °C. The annealing temperature for the primers used was found to be 58 °C.

                    Picture1.jpg
                      Gradient PCR for HPV-16 SiHa template, Lane1-520C, Lane 2-540C, Lane 3- 560C, Lane 4-580C, Lane 5-600C, Lane 6-620C, Lane 7-100bp ladder

                      e. LB agar plates containing colonies after transformation of the recombinant plasmid to DH5α: The ligated products were transformed into DH5α cells and were plated on LB agar plates containing Ampicillin antibiotics. Plasmids were isolated from the plates using QIAGEN plasmid Midi-Kit (Germany).

                      Picture2.png 12.png
                        Plating after transformation of the plasmid to DH5α: The colonies that have acquired the HPV-16 E7 region for ampicillin-resistant gene of pUC18 backbone is grown as discrete colonies after plating (b and c). Fig 9.a. negative control-pUC18 without insert.

                        DISCUSSION AND CONCLUSION

                        Human papillomaviruses are said to be the prominent cause of cervical carcinoma. As discussed earlier, E7 as well as E6 viral oncoproteins have a complementary effect in promoting hyperproliferation, immortalization of cells and genomic instability and distortion of balance in cell division and apoptosis in turn promoting carcinoma and malignancy of cervical epithelium.

                        The present study aimed at detection of specific HPV types from thirty cervical samples collected which included fifteen vaccinated and rest fifteen unvaccinated samples using bead based multiplex genotyping as detection tool for 21 specific HPV types. It was clearly observed that HPV-16 was detected in five unvaccinated samples (33.33%), while only a single case of HPV-16 infection was detected from vaccinated samples (6.6%). Also, it was observed that multiple infections apart from major Hr-types (HPV-6, 11, 16 and 18) viz., HPV-51, 53, 70 and 73 were observed in unvaccinated samples (33.33%). Such multiple infections were found to be absent in vaccinated samples which clearly showed the efficiency of the vaccine not only against the quadrivalent Hr-types but also demonstrates the ability to provide cross-protection against other Hr-types which would paly a role in development and progression of cervical carcinoma. No significant changes were seen with respect to detection of low-risk types in comparison with vaccinated and unvaccinated samples (For a clear representation refer Appendix 3).

                        The second part of the study was to detect specific HPV types using inhouse Uni-plex genotyping method. The study designed included an amplification step followed by hybridization of the inhouse coupled beads and its detection using Uni-plex genotyping targeting the E7 region of the viral genome. Plasmid clones of HPV-16/E7 were done using which the specificity and sensitivity of the developed assay is being assessed.

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                        4.     Alfonso Duenas- Gonzalez, Marcela Lizano, Myrna Candelaria, Lucely Cetina, Claudia Arce, Eduardo Cervera. (2005). Epigenetics of cervical cancer. An overview and therapeutic perspectives. Molecular Cancer, p.1-24.

                        5.     Schmitt, M., Dondog, B., Waterboer, T., Pawlita, M., Tommasino, M. and Gheit, T. (2009). Abundance of Multiple High-Risk Human Papillomavirus (HPV) Infections Found in Cervical Cells Analyzed by Use of an Ultrasensitive HPV Genotyping Assay. Journal of Clinical Microbiology, 48(1), pp.143-149.

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                        11.  cLaughlin-Drubin, M. E. & Munger, K. (2009) Oncogenic activities of human papillomaviruses. Virus Res. 143, 195–208.

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                        14. Hawley-Nelson, P., Vousden, K. H., Hubbert, N. L., Lowy, D. R. & Schiller, J. T. HPV 16 (1989).E6 and E7 proteins cooperate to immortalize human foreskin keratinocytes. EMBO J. 8, 3905–3910.

                        15.  Munger, K., Phelps, W. C., Bubb, V., Howley, P. M. & Schlegel, R. (1989) The E6 and E7 genes of the human papillomavirus type 16 together are necessary and sufficient for transformation of primary human keratinocytes. J. Virol. 63, 4417–4421.

                        16.  Mirabello, L., Yeager, M., Yu, K., Clifford, G., Xiao, Y., Zhu, B., Cullen, M., Boland, J., Wentzensen, N., Nelson, C., Raine-Bennett, T., Chen, Z., Bass, S., Song, L., Yang, Q., Steinberg, M., Burdett, L., Dean, M., Roberson, D., Mitchell, J., Lorey, T., Franceschi, S., Castle, P., Walker, J., Zuna, R., Kreimer, A., Beachler, D., Hildesheim, A., Gonzalez, P., Porras, C., Burk, R. and Schiffman, M. (2017). HPV16 E7 Genetic Conservation Is Critical to Carcinogenesis. Cell, 170(6), pp.1164-1174.e6.

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                        18.  Nakatani, Y., Konishi, H., Vassilev, A., Kurooka, H., Ishiguro, K., Sawada, J., Ikura, T., Korsmeyer, S., Qin, J. and Herlitz, A. (2005). p600, a unique protein required for membrane morphogenesis and cell survival. Proceedings of the National Academy of Sciences, 102(42), pp.15093-15098.

                        19.  Huh, K., DeMasi, J., Ogawa, H., Nakatani, Y., Howley, P. and Munger, K. (2005). Association of the human papillomavirus type 16 E7 oncoprotein with the 600-kDa retinoblastoma protein-associated factor, p600. Proceedings of the National Academy of Sciences, 102(32), pp.11492-11497.

                        20.  Roden, R. and Wu, T. (2006). How will HPV vaccines affect cervical cancer? Nature Reviews Cancer, 6(10), pp.753-763.

                         21.  Sankaranarayanan, R., Prabhu, P., Pawlita, M., Gheit, T., Bhatla, N., Muwonge, R., Nene, B., Esmy, P., Joshi, S., Poli, U., Jivarajani, P., Verma, Y., Zomawia, E., Siddiqi, M., Shastri, S., Jayant, K., Malvi, S., Lucas, E., Michel, A., Butt, J., Vijayamma, J., Sankaran, S., Kannan, T., Varghese, R., Divate, U., Thomas, S., Joshi, G., Willhauck-Fleckenstein, M., Waterboer, T., Müller, M., Sehr, P., Hingmire, S., Kriplani, A., Mishra, G., Pimple, S., Jadhav, R., Sauvaget, C., Tommasino, M. and Pillai, M. (2016). Immunogenicity and HPV infection after one, two, and three doses of quadrivalent HPV vaccine in girls in India: a multicentre prospective cohort study. The Lancet Oncology, 17(1), pp.67-77.

                         22.  Bhatla, N., Nene, B., Joshi, S., Esmy, P., Poli, U., Joshi, G., Verma, Y., Zomawia, E., Pimple, S., Prabhu, P., Basu, P., Muwonge, R., Hingmire, S., Sauvaget, C., Lucas, E., Pawlita, M., Gheit, T., Jayant, K., Malvi, S., Siddiqi, M., Michel, A., Butt, J., Sankaran, S., Kannan, T., Varghese, R., Divate, U., Willhauck-Fleckenstein, M., Waterboer, T., Müller, M., Sehr, P., Kriplani, A., Mishra, G., Jadhav, R., Thorat, R., Tommasino, M., Pillai, M. and Sankaranarayanan, R. (2018). Are two doses of human papillomavirus vaccine sufficient for girls aged 15–18 years? Results from a cohort study in India. Papillomavirus Research, 5, pp.163-171.

                         23.  Schmitt, M., Dondog, B., Waterboer, T., Pawlita, M., Tommasino, M. and Gheit, T. (2009). Abundance of Multiple High-Risk Human Papillomavirus (HPV) Infections Found in Cervical Cells Analyzed by Use of an Ultrasensitive HPV Genotyping Assay. Journal of Clinical Microbiology, 48(1), pp.143-149.

                         24.  Schmitt, M., Bravo, I., Snijders, P., Gissmann, L., Pawlita, M. and Waterboer, T. (2006). Bead-Based Multiplex Genotyping of Human Papillomaviruses. Journal of Clinical Microbiology, 44(2), pp.504-512.

                        ACKNOWLEDGEMENTS

                        I deeply thank my advisor Prof. M. Radhakrishna Pillai, Director, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram for selecting and providing me the opportunity to carry out the internship.

                        I express my deep sense of gratitude to Dr Devasena Anantharaman, Scientist E II, RAjiv Gandhi Centre for Biotechnology, Thiruvananthapuram for her endeavour approach and supervision throughout my period of training.

                        I am extremely thankful and pay my gratitude to PhD scholar Ms Aparana G J for her valuable guidance and support throughout the period of my training.

                        I am also thankful to scholars Ms Ahalya S, Mr Vivek Ashok Kumar, Post-doctoral fellows and to the lab technicians of HPV Lab for their support during this period.

                        APPENDICES

                        1.     Hybridization protocol

                        Reagents:

                        ·       DNase, RNase free distilled water (Qiagen, Germany)

                        ·       Bio-Rad non-magnetic beads

                        ·       Hybridization buffer:0.15M TMAC, 75mM Tris-HCl (pH 8), 6mM EDTA (pH 8), 1.5g/l sarkosyl

                        ·       TE buffer (10 mM Tris-HCl, 1mM EDTA, pH 8)

                        ·       Wash buffer (WB) 1: 0.02% Tween, PBS pH 7.4

                        ·       Wash buffer 2:0.1% SDS

                        ·       Streptavidin-R-phycoerythrin (Molecular Probes, OR)

                        ·       DNase, RNase, Protease free, TE buffer pH 8.0

                        ·       Ice Controls

                        Each hybridization experiment should include several controls:

                        ·       negative water hybridization control (indicates individual bead background )

                        ·       positive hybridization control (indicates positive performance of the hybridization), i.e. either biotinylated antisense oligonucleotides or a mixture of characterized PCR products.

                        ·       water PCR (indicates contaminations of the PCR mix during pipetting and unspecific hybridization of biotinylated primers)

                        ·       master-mix PCR (indicates contaminations of PCR components)

                        ·       positive PCR controls

                        ·       Switch on the thermomixer prior to use. Set the temperature at 41°C.

                        ·       Switch on the thermal cycler and set to 95°C.

                        ·       Maintain ice on a tray beside the thermal cycler.

                        ·       Thaw the PCR products on another ice tray.

                        ·       Prepare the bead mix as per the standardised lab protocol. The bead mix will contain 21 types of bead sorts (one per HPV type) coupled to HPV type-specific target probe sequences.

                        ·       Vortex the coupled bead mix thoroughly and transfer 4ml of the coupled beads on to a trough.

                        ·       Transfer 40µl of the coupled beads to the 96 well PCR plate using a multi-channel pipette.

                        ·       Quick spin the PCR products and transfer 10µl of each amplified sample using a multichannel pipette in a predetermined sequence by mixing to its respective position in the 96 well reaction plate.

                        ·       Maintain the corresponding positions in the 96 well reaction plates for controls.

                        ·       Cover the reaction plate with sealing foil and place within the thermal cycler for a pre-programmed run of 95°C for 15 minutes.

                        ·       Once this is completed, quickly transfer the reaction plate on ice for 1 minute and place in a pre-set thermomixer at 41°C, 750rpm for 30 minutes.

                        ·       Equilibrate the wash plate with wash buffer by wetting the filter plate with 100µl wash buffer (1X PBS+ Tween 20). The wash plate is a 96 well plate with nitrocellulose filter bottom. Allow this to rest until hybridization is completed.

                        ·       Meanwhile, prepare 5ml of staining buffer by adding 2M TMAC + StrepE (1:1600 dilution). Ensure the vial is covered with aluminium foil.

                        ·       At the 28th minute of hybridization incubation, drain the liquid in the filter plate from the base using a vacuum pump. The vaccum should be applied approximately for 30 seconds at 100mmhg pressure.

                        ·       After 30 minutes of hybridization incubation, transfer the full volume of the hybridized product to the equilibrated filter plate.

                        ·       Drain the liquid from filter plate using a vacuum pump at 100mmhg.

                        ·       Wash once by adding 100ul wash buffer. Drain liquid by vacuum filtration (same as in step16) to remove any un-hybridized DNA.

                        ·       Place the filter plate over several sheets of dry blotting paper. Excess liquid in the filter plate is drained by vigorous hammering action applied on top of the covered plate.

                        ·       Add 50µl of staining buffer using a multi-channel pipette to each well and incubate in dark for 30 minutes under agitation.

                        ·       After 30 minutes, remove liquid by vacuum filtration.

                        ·       Add 100 µl wash buffer to each well and drain the plate by vacuum filtration. Repeat this step twice.

                        ·       Repeat step 18

                        ·       Add 100µl wash buffer to each well of the filter plate and incubate for 10 minutes in a horizontal shaker

                        ·       Read the plate in the Bioplex-200 machine located in the third floor.

                        ·       Of the two lasers in the analyser, one identifies the bead sort by the internal bead fluorescence and the other one quantifies the reporter fluorescence.

                        ·       The output was expressed as Median Flores2cence Intensity (MFI) value of 100 beads per sort.

                        2.     CSHL Protocol for DNA isolation by salt precipitation method

                        Reagents required:

                        ·       Cell lysis buffer

                        10mM Tris-Cl (pH 8.0)

                        1mM EDTA (pH 8.0)

                        0.1% (w/v) SDS

                        ·       EtOH

                        ·       Isopropanol

                        ·       5M Potassium Acetate solution

                        60 ml of 5M Potassium acetate+11.5 ml of glacial acetic acid +28.5 ml of d.H2O – resulting solution has 3M concentration with respect to potassium ion concentration and 5M concentration with respect to acetate ion concentration.

                        ·       The cultured cells are pelleted to which 600 µl of ice-cold lysis buffer and 3µl of Proteinase-K is added and incubated at 550C for 3 hours.

                        ·       After incubation the tubes were cooled and around 3 µl of RNase is added and incubated at 370C for15-60 minutes. Cool and add 200µl of 5M potassium acetate.

                        ·       The tubes are the vortexed foe 20 seconds and then centrifuged at 13000rpm for 3 minutes at 40C.

                        ·       The supernatant obtained is collected in fresh tubes to which 600 µl of chilled isopropanol is added and mixed well.

                        ·       Centrifugation is performed at 13000rpm for 1 min at room temperature and the supernatant obtained is discarded.

                        ·       Around 600 µl of chilled 70% EtOH is added to the pellet and the tubes are inverted vigorously for 1 minute. Centrifuge at 13000rpm for a minute at room temperature. Air dry the pellet and re-dissolve in 100 µl of TE.

                        3. Overall observations of HPV detection from cervical samples

                        Sample nature Number Positivity for any HPV type HPV positivity (for vaccinated types-6, 11, 16, 18) HPV positivity (for related types-31, 33, 45, 52, 58) Other HPV types found positive
                        Unvaccinated 15 14 (93.333%) 9 (60%) - 5 (33.333%)
                        Vaccinated 15 1 (6.667%) 1 (6.667%) - -
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