HPV Vaccine

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 infection^2-3. 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.

Methods for 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 antibodies^7-8. 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.