Development of Antibodies against HPV-6 and HPV-11 for the Study of Laryngeal Papilloma
Abstract
:1. Introduction
2. Materials and Methods
2.1. Subjects
2.2. Detection of HPV DNA
2.3. Measurement of Viral DNA Load and mRNA Expression in HPV-11-Infected Papilloma by Quantitative Real-Time PCR
2.4. ISH with HPV DNA Probes
2.5. RNA-ISH with HPV-11 E6, E2, E4, and E5b Digoxigenin RNA Probes
2.6. Generation of an Anti-HPV-11 E1^E4 Polyclonal Antibody
2.6.1. Preparation of the Target Antigen
2.6.2. Preparation of the Polyclonal Antibody against HPV-11 E1^E4
2.7. Western Blotting
2.8. Immunohistochemistry Using the Newly Developed Anti-HPV-11 E1^E4 Antibody and Anti-HPV-6 E1^E4 Antibody
2.9. Statistical Analysis
2.10. Ethics Approval and Informed Consent
3. Results
3.1. HPV-11 DNA Distribution, Subtypes, and Viral Load
3.2. Expression of HPV-11 mRNAs
3.2.1. Expression Levels of the Nine Viral mRNAs
3.2.2. mRNA Expression in LP Detected by RNA-ISH
3.3. Western Blot Analysis Using the Anti-HPV-11 E1^E4 Antibody
3.4. Immunohistochemistry Using the Anti-HPV-11 and Anti-HPV-6 E1^E4 Antibodies
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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All Cases (n = 66) | Anti-HPV-6 E4 IHC | Anti-HPV-11 E4 IHC | |||
---|---|---|---|---|---|
Positive | Negative | Positive | Negative | ||
Laryngeal papilloma | |||||
HPV-6-positive | 20 | 16 | 4 | 0 | 20 |
HPV-11-positive | 3 | 0 | 3 | 3 | 0 |
HPV-negative | 5 | 0 | 5 | 0 | 5 |
Nasoseptal exophytic papilloma | |||||
HPV-6-positive | 1 | 0 | 1 | 0 | 1 |
HPV-11-positive | 1 | 0 | 1 | 1 | 0 |
HPV-negative | 2 | 0 | 2 | 0 | 2 |
Paranasal exophytic papilloma (HPV-negative) | 1 | 0 | 1 | 0 | 1 |
Nasopharyngeal papilloma (HPV-11-positive) | 1 | 0 | 1 | 1 | 0 |
Oropharyngeal papilloma (HPV-negative) | 1 | 0 | 1 | 0 | 1 |
Inverted papilloma with SCC (HPV-18-positive) | 1 | 0 | 1 | 0 | 1 |
Normal vocal cord (HPV-negative) | 5 | 0 | 5 | 0 | 5 |
Chronic tonsillitis (HPV-negative) | 5 | 0 | 5 | 0 | 5 |
Laryngeal cancer | |||||
High-risk HPV-positive 1 | 5 | 0 | 5 | 0 | 5 |
HPV-negative | 5 | 0 | 5 | 0 | 5 |
Oropharyngeal cancer | |||||
High-risk HPV-infected (HPV-16, 33, 35, 56, 67) | 5 | 0 | 5 | 0 | 5 |
Tongue cancer (HPV-negative) | 5 | 0 | 5 | 0 | 5 |
Clinicopathological Characteristics | DNA Viral Load | HPV mRNA Expression/β-Actin | ||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Case | Age (Years) | Sex | Surgery | Number of Tumors | Subsite | Derkay Score | (Copies/ ng DNA) | E6 | E7 | E1 | E2 | E4 | E5a | E5b | L2 | L1 |
1 | 44 | M | 1st | multiple | Bil TVC | 5 | 64,192 | 0.11588 | 0.10541 | 0.23529 | 0.39033 | 3.95554 | 0.98977 | 2.33520 | 0.03630 | 0.02664 |
2 | 44 | M | 1st | multiple | Bil TVC | 7 | 226,916 | 0.38194 | 1.95009 | 6.36798 | 10.67637 | 56.84702 | 16.06985 | 39.08463 | 0.32797 | 0.11461 |
3 | 5 | M | 13th | multiple | E | 5 | 84 | 0.00316 | 0.06096 | 0.01683 | 0.02952 | 0.43326 | 0.20933 | 0.54957 | 0.00101 | 0.00059 |
5 | multiple | PW | 3 | 642,111 | 0.02153 | 0.04717 | 0.04567 | 0.09687 | 0.87938 | 0.20339 | 0.49420 | 0.00542 | 0.00482 | |||
5 | 14th | multiple | Bil TVC | 4 | 230,366 | 0.03669 | 0.03956 | 0.08475 | 0.13412 | 1.45394 | 0.36106 | 0.85643 | 0.00581 | 0.00167 | ||
6 | 15th | multiple | Bil TVC | 5 | 33,153 | 0.02311 | 0.55589 | 0.03616 | 0.12762 | 0.83358 | 0.89123 | 5.44958 | 0.00210 | 0.00170 | ||
7 | 16th | multiple | E | 2 | 15,711 | NA | NA | NA | NA | NA | NA | NA | NA | NA | ||
7 | multiple | Bil TVC | 4 | 601,070 | 0.04394 | 0.20269 | 0.03255 | 0.15081 | 1.16959 | 0.80580 | 1.18013 | 0.00918 | 0.00594 | |||
7 | multiple | PW | 2 | 71,468 | 0.18674 | 0.62598 | 0.11146 | 0.35295 | 4.58308 | 4.86911 | 5.82166 | 0.04036 | 0.06345 | |||
4 | 44 | F | 1st | multiple | NP | - | 11,346 | 0.17928 | 0.67253 | 0.08880 | 0.41194 | 3.45012 | 3.11286 | 4.59908 | 0.00536 | 0.00490 |
5 | 63 | M | 1st | multiple | NS | - | 246,077 | 0.17783 | 0.77629 | 0.47749 | 0.41261 | 4.80617 | 1.05967 | 2.48394 | 0.13504 | 0.10001 |
HPV Status of Specimen | No. of Specimens | Anti-HPV-6 E4 IHC | Anti-HPV-11 E4 IHC | ||
---|---|---|---|---|---|
Positive | Negative | Positive | Negative | ||
HPV-6-positive | 21 | 16 | 5 | 0 | 21 |
HPV-11-positive | 5 | 0 | 5 | 5 | 0 |
High-risk HPV-positive | 11 | 0 | 11 | 0 | 11 |
HPV-negative | 29 | 0 | 29 | 0 | 29 |
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Ikegami, T.; Kise, N.; Kinjyo, H.; Kondo, S.; Suzuki, M.; Tsukahara, N.; Murakami, A.; Kiyuna, A.; Agena, S.; Tanaka, K.; Hasegawa, N.; Kawakami, J.; Ganaha, A.; Maeda, H.; Hirakawa, H. Development of Antibodies against HPV-6 and HPV-11 for the Study of Laryngeal Papilloma. Viruses 2021, 13, 2024. https://doi.org/10.3390/v13102024
Ikegami T, Kise N, Kinjyo H, Kondo S, Suzuki M, Tsukahara N, Murakami A, Kiyuna A, Agena S, Tanaka K, Hasegawa N, Kawakami J, Ganaha A, Maeda H, Hirakawa H. Development of Antibodies against HPV-6 and HPV-11 for the Study of Laryngeal Papilloma. Viruses. 2021; 13(10):2024. https://doi.org/10.3390/v13102024
Chicago/Turabian StyleIkegami, Taro, Norimoto Kise, Hidetoshi Kinjyo, Shunsuke Kondo, Mikio Suzuki, Narutoshi Tsukahara, Akikazu Murakami, Asanori Kiyuna, Shinya Agena, Katsunori Tanaka, Narumi Hasegawa, Junko Kawakami, Akira Ganaha, Hiroyuki Maeda, and Hitoshi Hirakawa. 2021. "Development of Antibodies against HPV-6 and HPV-11 for the Study of Laryngeal Papilloma" Viruses 13, no. 10: 2024. https://doi.org/10.3390/v13102024