Resources

HPV Vaccine

Overview

Human papilloma virus (HPV) widely infects humans and can cause cervical cancer and other malignant tumors, seriously endangering human health. In 2006, Merck launched the first HPV vaccine, making cervical cancer and condyloma caused by HPV preventable. Glaxosmithkline's Cervarix, Merck's Gardasil and Gardasil 9 are on the market. The HPV vaccine is effective in preventing HPV infection and the disease it causes, and the World Health Organization (WHO) is implementing a global plan to eliminate cervical cancer. However, how to evaluate vaccines scientifically, especially their clinical immunogenicity, is very important.

Immunological mechanisms of HPV

The natural and adaptive immunity of the body's immune system controls and removes infected HPV, so most infections are self-limiting. Studies have shown that more than 90% of infected people will clear the virus within 3 years1. However, HPV has multiple mechanisms to escape the immune system so that a small number of people remain infected.

HPV infection will produce antibodies against L1, L2 and E6 and other proteins, among which L1 antibodies account for the largest proportion, but mostly type specific antibodies, it is difficult to produce type cross immune protection. The antibody molecular types of HPV are mainly IgG and IgA, among which IgA is divided into serum type and secreting type. In the early stage, IgA was thought to be more useful in mucous membrane than IgG. However, later clinical trials proved that IgG plays a major role in anti-HPV infection2-3. L2 plays an important role in HPV immune escape, mainly by preventing the increase of immune-related signal molecules on the surface of Langerhans cells and inhibiting their ability to activate T cells4. In addition, certain types of E6 and E7 can down-regulate type I IFN expression by interfering with cell cycle5.

Prophylactic vaccine

The immune response caused by natural infection is weak and it is difficult to produce antibodies with enough titer to neutralize the invading virus. Inoculation of preventive vaccine can stimulate the body to produce a high titer of neutralizing antibodies, clear the invasion of the virus. Clinical results show that neutralizing antibodies produced by HPV vaccine can penetrate into the blood vessel wall to reach the infection site and bind the virus to make it lose the ability to infect cells6. Both L1 and L2 are good candidate proteins for preventive vaccine.

Gardasil®4, developed by Merck in the United States in 2006, was the first HPV vaccine to be marketed in the world. The vaccine contains HPV 6, 11, 16 and 18 strains expressed by Saccharomyces cerevisiae. Vaccines made by adsorption to AAHS can prevent about 90% of condyloma acuminatum and about 70% of cervical cancer.

Cervarix®, developed by GlaxoSmithKline (GSK) of the UK, was approved by the European Union in September 2007 and the US FDA in October 2009. The vaccine, which contains baculovirus-insect cells expressing HPV 16 and 18 VLP components, does not contain low-risk HPV and therefore does not protect against condyloma acuminatum, but it also protects about 70 percent of cervical and uterine cancers. The vaccine uses AS04, an adjuvant developed by GSK.

Gardasil®9, developed by Merck, was approved in the United States in December 2014. The vaccine, also expressed in Saccharomyces cerevisiae, contains nine VLP components of HPV 6, 11, 16, 18, 31, 33, 45, 52 and 58, and can prevent approximately 90% of cervical, vaginal, vulvar and anal cancers caused by seven high-risk HPV types, and approximately 90% of condyloma acuminatum caused by low-risk HPV 6 and 11.

Methods for immunogenicity evaluation

Three main types of assays or immunogenicity evaluation

Fig. 1 Three main types of assays or immunogenicity evaluation12

  • Competitive immunoassay
  • Merck's competitive immunoassay Luminex (cLIA) detects antibodies in serum samples that compete with specific neutralizing monoclonal antibodies7-8. CLIA detects type specific neutralizing monoclonal antibodies and conformation intact VLP that need to be labeled. Antibodies in serum bind to VLP and inhibit the binding of labeled monoclonal antibodies, thus reducing the signal value of detection. By labeling different types of specific monoclonal antibodies and combining Luminex detection, the synchronous detection of different types of antibodies can be realized, thus improving the detection flux of the method. This method was used to evaluate the clinical immunogenicity of Gardasil® and Gardasil®9 of Merck.

    cLIA can detect all classes of antibodies (IgG, IgM, IgA, etc.), but only for a single epitope. Therefore, the method has good specificity, but may underestimate the level of functional antibodies in the sample.

  • VLP binding antibody detection
  • The assay can detect VLP-bound IgG in samples, and other types of antibodies, such as IgA or different IgG subclasses, can also be detected by altering the secondary antibody. This method can recognize both conformational epitopes and non-conformational epitopes, and the detected antibodies include both neutralizing and non-neutralizing antibodies.

    • Virus like particle-enzyme linked immunosorbent assay (VLP-ELISA): VLPs are bound to a solid surface (beads or wells) and antibodies in the sample bind directly. Bound antibody is detected with labeled secondary antibodies directed against a specific Ig class, usually anti-IgG. The label can be an enzyme, an affinity label or a fluorescent molecule. L1 VLP ELISAs for type-specific IgGs have been used by GlaxoSmithKline and several research labs as the main assay in a number of immunogenicity trials12.
    • IgG Luminex immunoassay (IgG LIA): The coated antigen and serum standard used in this method were the same as HPV-9 cLIA, and the magnitude of the serum standard was the same as HPV-9 cLIA. Mouse anti-human IgG was used as the secondary antibody to detect VLP-bound IgG.
  • Pseudovirion-based neutralization assay (PBNA)
  • The neutralization test is considered the “gold standard” for detecting protective antibodies and is recommended by the WHO as a reference method for evaluating vaccine-induced protective antibodies. PBNA can detect all neutralizing antibodies (such as IgM, IgA, IgG) in vitro9.

    • PBNA based on Green Fluorescent Protein (GFP) and Secreted Alkaline Phosphatase (SEAP): Pseudovirus can infect target cells (293TT or 293FT cells). After infection, the reporter gene plasmid is transported into the nucleus to express the corresponding protein. The number of cells expressing the reporter gene or the signal value of the reporter protein is proportional to the number of pseudovirus infection. If neutralizing antibodies or serum samples containing neutralizing antibodies are added in the infection process, neutralizing antibodies can block the pseudovirus from infecting cells, and the level of neutralizing antibodies in the detected samples can be calculated by detecting the signal value of reporting proteins.
    • PBNA based on Gaussia luciferase (Gluc): Gluc is a small molecule luciferase derived from marine organisms, which has a low background in mammalian cells (100 times lower than SEAP) and can be secreted into the cell supernatant after expression. Its detection procedure is simple, without repeated incubation at different temperatures. The detection time (5 min/ 96-well plate) was significantly shorter than that of SEAP (60 min/ 96-well plate).
    • Polychromatic PBNA based on fluorescent protein: In this method, different types of HPV structural gene plasmids and fluorescent protein expression plasmids of different colors were co-transfected into eukaryotic cells to prepare different types of pseudoviruses containing different fluorescent protein reporter genes. To establish the method, the first step is to select the fluorescent protein, which does not interfere with each other under a certain combination of excitation light and filter.

Neutralization test is a detection method based on cell culture. Compared with binding test, the original method has a higher degree of variation, and the operation is relatively cumbersome, which makes it difficult to conduct high-throughput detection. With the development of detection methods, the detection flux of neutralizing antibody detection method is also gradually improving, especially the 384 well plate automatic detection method based on Gluc10 and the three-color pseudovirus binding immune spot counting method11, which greatly improves the detection flux while reducing the sample dosage. It lays a foundation for the application of this method in clinical trials.

References:

  1. Maglennon, GA.; et al. Immunosuppression facilitates the reactivation of latent papillomavirus infections. J Virol. 2014 Jan;88(1):710-6.
  2. Fife, KH.; et al. Dose-ranging studies of the safety and immunogenicity of human papillomavirus Type 11 and Type 16 virus-like particle candidate vaccines in young healthy women. Vaccine. 2004 Jul 29;22(21-22):2943-52.
  3. Lowe, RS.; et al. Human papillomavirus type 11 (HPV-11) neutralizing antibodies in the serum and genital mucosal secretions of African green monkeys immunized with HPV-11 virus-like particles expressed in yeast. J Infect Dis. 1997 Nov;176(5):1141-5.
  4. Fahey, LM.; et al. A major role for the minor capsid protein of human papillomavirus type 16 in immune escape. J Immunol. 2009 Nov 15;183(10):6151-6.
  5. Murty, VV.; et al. Chromosomal aberrations & sister chromatid exchanges in relation to herpes simplex virus antibody in women with cervical dysplasia. Indian J Med Res.1986 Jan; 83:27-32.
  6. Nardelli-Haefliger, D.; et al. Specific antibody levels at the cervix during the menstrual cycle of women vaccinated with human papillomavirus 16 virus-like particles. J Natl Cancer Inst. 2003 Aug 6;95(15):1128-37.
  7. Roberts, C.; et al. Development of a human papillomavirus competitive luminex immunoassay for 9 HPV types[J]. Hum Vaccin Immunother. 2014, 10(8): 2168-2174.
  8. Dias, D.; et al. Optimization and validation of a multiplexed luminex assay to quantify antibodies to neutralizing epitopes on human papillomaviruses 6, 11, 16, and 18[J]. Clin Diagn Lab Immunol. 2005, 12(8): 959-969.
  9. FrazerI, H. Measuring serum antibody to human papillomavirus following infection or vaccination[J]. Gynecol Oncol. 2010, 118(1Suppl): S8-11.
  10. Sehr, P.; et al. High-throughput pseudovirion-based neutralization assay for analysis of natural and vaccine-induced antibodies against human papillomaviruses[J]. PLoS One. 2013, 8(10): e75677.
  11. Nie, J.; et al. Development of a triple-color pseudovirion-based assay to detect neutralizing antibodies against human papillomavirus[J]. Viruses. 2016, 8(4): 107.
  12. Pinto, LA.; et al. Immunogenicity of HPV prophylactic vaccines: Serology assays and their use in HPV vaccine evaluation and development. Vaccine. 2018 Aug 6;36(32 Pt A):4792-4799.

Reagents Solutions

HPV L1 Antibodies for Neutralization and Vaccine Development

Creative Diagnostics has launched the most complete HPV L1 antibody products, covering HPV 6/11/16/18/31/33/45/52/58, including polyclonal, monoclonal antibody and monoclonal antibody screening set, mainly used for ELISA, Western Blot, neutralization and in vitro vaccine in vitro efficacy testing. View More

Neutralizing monoclonal antibody sets:

Monoclonal antibody set Clone number-IC50 (μg/ml) for Neut:
Anti-HPV 6 L1 monoclonal antibody (set) 1E5-0.0092, 39G7-0.0085, 39G2-0.00081, 42G5-0.00076, 44B11-0.00008, 43C7-0.0033
Anti-HPV 11 L1 monoclonal antibody (set) 34C9-4.27, 35C1-15.87, 35H5-743, 34E5-43
Anti-HPV 16 L1 monoclonal antibody (set) 2A1-0.00033, 3D5-0.0021, 4G12-0.0020, 5A6-0.0024, 6C7-0.0020, 7B9-0.00027
Anti-HPV 18 L1 monoclonal antibody (set) 1B1-97, 3A2-0.00030, 3A4-1.46, 4H1-0.76, 4H5-1.31, 7H8-0.0025
Anti-HPV 31 L1 monoclonal antibody (set) 19C2-0.0061, 19B6-0.12
Anti-HPV 33 L1 monoclonal antibody (set) 4C2, 4F1, 4G4, 4H4, 6C11, 6D2
Anti-HPV 45 L1 monoclonal antibody (set) 30H6-0.54, 35B12-1.70, 35H9-0.49, 40H1-0.15, 46G5-0.028, 48B1-0.14
Anti-HPV 52 L1 monoclonal antibody (set) 36B9-0.00056, 36E12-0.00061, 40C2-0.0035, 41C3-0.0011, 41D1-0.00015, 42A2-0.00040
Anti-HPV 58 L1 monoclonal antibody (set) 1D2-0.015, 2E11-0.021, 2F11-0.014, 2F7-0.020, 2F9-0.10, 2G7-0.015

*The antibodies are provided as screening sets (6 different clones per set), each clone is also available separately.

Polyclonal antibodies For WB

Recombinant HPV L1 VLP

Creative Diagnostics has established a global-leading virus-like particles (VLPs) manufacture platform using E. coli cell system. With years of exploration, our scientists have successfully obtained various highly purified HPV VLPs. Our products have significantly contributed to the HPV vaccine related research. View More

Catalog# Product Description Expression System Application
DAGF-227 Recombinant HPV type 6 L1 protein (VLP) E. Coli ELISA, Antibody Detection
DAGF-228 Recombinant HPV type 11 L1 protein (VLP) E. Coli ELISA, Antibody Detection
DAGF-229 Recombinant HPV type 16 L1 protein (VLP) E. Coli ELISA, Antibody Detection
DAGF-230 Recombinant HPV type 18 L1 protein (VLP) E. Coli ELISA, Antibody Detection
DAGF-231 Recombinant HPV type 31 L1 protein (VLP) E. Coli ELISA, Antibody Detection
DAGF-232 Recombinant HPV type 33 L1 protein (VLP) E. Coli ELISA, Antibody Detection
DAGF-233 Recombinant HPV type 45 L1 protein (VLP) E. Coli ELISA, Antibody Detection
DAGF-234 Recombinant HPV type 52 L1 protein (VLP) E. Coli ELISA, Antibody Detection
DAGF-235 Recombinant HPV type 58 L1 protein (VLP) E. Coli ELISA, Antibody Detection
DAGC142 Recombinant HPV type 35 L1 protein (VLP) E. Coli ELISA, Antibody Detection
DAGC143 Recombinant HPV type 39 L1 protein (VLP) E. Coli ELISA, Antibody Detection
DAGC144 Recombinant HPV type 51 L1 protein (VLP) E. Coli ELISA, Antibody Detection
DAGC145 Recombinant HPV type 56 L1 protein (VLP) E. Coli ELISA, Antibody Detection
DAGC146 Recombinant HPV type 59 L1 protein (VLP) E. Coli ELISA, Antibody Detection
DAGC147 Recombinant HPV type 68 L1 protein (VLP) E. Coli ELISA, Antibody Detection

HPV Pseudovirus

The CD Pseudotyped GFP HPV production is based on the transfection of a 293 cell line, 293FT, engineered to express high levels of SV40 large T antigen. The cells are co-transfected with codon-modified papillomavirus capsid genes, L1 and L2, together with a pseudogenome plasmid containing the SV40 origin of replication. Pseudovirus (PsV) encapsidating a GFP reporter plasmid can be used to develop a high-throughput in vitro neutralization assay in a 96-well plate format. View More

Cat. No. Product Name Reporter Cell Line
PSVG-HPV6 Pseudotyped GFP HPV6 GFP HEK293 FT
PSVG-HPV11 Pseudotyped GFP HPV11 GFP HEK293 FT
PSVG-HPV16 Pseudotyped GFP HPV16 GFP HEK293 FT
PSVG-HPV18 Pseudotyped GFP HPV18 GFP HEK293 FT
PSVG-HPV31 Pseudotyped GFP HPV31 GFP HEK293 FT
PSVG-HPV33 Pseudotyped GFP HPV33 GFP HEK293 FT
PSVG-HPV35 Pseudotyped GFP HPV35 GFP HEK293 FT
PSVG-HPV39 Pseudotyped GFP HPV39 GFP HEK293 FT
PSVG-HPV45 Pseudotyped GFP HPV45 GFP HEK293 FT
PSVG-HPV51 Pseudotyped GFP HPV51 GFP HEK293 FT
PSVG-HPV52 Pseudotyped GFP HPV52 GFP HEK293 FT
PSVG-HPV56 Pseudotyped GFP HPV56 GFP HEK293 FT
PSVG-HPV58 Pseudotyped GFP HPV58 GFP HEK293 FT
PSVG-HPV59 Pseudotyped GFP HPV59 GFP HEK293 FT
PSVG-HPV68 Pseudotyped GFP HPV68 GFP HEK293 FT

Online Inquiry

Name:
Phone: *
E-mail Address: *
Technology Interest:
Type of Organization:
Service & Products Interested: *
Project Description:

Resources

OUR PROMISE TO YOU Guaranteed product quality expert customer support

Inquiry Basket