Pseudotyped Reporter VirusesInquiry Basket
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Immunogenicity assessments in vaccine trials have focused on measurements of antibody responses to the vaccine. A series of functional assays have been generated to measure antibody-mediated neutralization for papillomaviruses. Some of the functional assays include challenges of animals that are so complex that they cannot be applied for routine testing. The current "gold standard" for measuring neutralizing anti-HPV antibodies is the pseudovirion-based neutralization assay (PBNA). Instead of using authentic viruses, pseudovirions with an encapsidated reporter can be used to avoid high risks and provide more direct measurements for antibodies.
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.
![Adapted from: Schiller J T, Müller M. Next generation prophylactic human papillomavirus vaccines[J]. The Lancet Oncology, 2015, 16(5): e217-e225.](/upload/image/Pseudotyped-GFP-HPV-1.png)
Adapted from: Schiller J T, Müller M. Next generation prophylactic human papillomavirus vaccines[J]. The Lancet Oncology, 2015, 16(5): e217-e225.
Human papillomaviruses (HPVs) compose a group of more than 100 genotypes, which can result in cervical cancer and its precursor lesions. Currently, 12 types of HPV (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59) have been classified as carcinogenic types to humans and an additional 8 types are classified as possibly carcinogenic types to human. We can provide up to 15 different types of HPV pseudovirus, which include all 12 carcinogenic HPV types.
HPVs are double-stranded non-enveloped DNA viruses that infect keratinocytes of stratified squamous epithelia. There are roughly 8,000 base pairs inside the HPV genome to encode 8 essential proteins for completion of the viral life cycle.
L1 (major) and L2 (minor) capsid proteins, encoded by two late genes, can form an icosahedral lattice around the HPV genome. Over-expression of L1 and L2 proteins are needed for the self-assembly of viral particles. While L1 protein alone is able to form native capsids, the DNA encapsidation process does require L2 proteins. Codon optimization of L1 and L2 DNA sequences were found to increase protein expression by about 100-fold, enabling the production of high titer stocks.
| Cat. No. | Product Name | Reporter | Cell Line | |
| PSVG-HPV6 | Pseudotyped GFP HPV6 | GFP | HEK293 FT | Inquiry |
| PSVG-HPV11 | Pseudotyped GFP HPV11 | GFP | HEK293 FT | Inquiry |
| PSVG-HPV16 | Pseudotyped GFP HPV16 | GFP | HEK293 FT | Inquiry |
| PSVG-HPV18 | Pseudotyped GFP HPV18 | GFP | HEK293 FT | Inquiry |
| PSVG-HPV31 | Pseudotyped GFP HPV31 | GFP | HEK293 FT | Inquiry |
| PSVG-HPV33 | Pseudotyped GFP HPV33 | GFP | HEK293 FT | Inquiry |
| PSVG-HPV35 | Pseudotyped GFP HPV35 | GFP | HEK293 FT | Inquiry |
| PSVG-HPV39 | Pseudotyped GFP HPV39 | GFP | HEK293 FT | Inquiry |
| PSVG-HPV45 | Pseudotyped GFP HPV45 | GFP | HEK293 FT | Inquiry |
| PSVG-HPV51 | Pseudotyped GFP HPV51 | GFP | HEK293 FT | Inquiry |
| PSVG-HPV52 | Pseudotyped GFP HPV52 | GFP | HEK293 FT | Inquiry |
| PSVG-HPV56 | Pseudotyped GFP HPV56 | GFP | HEK293 FT | Inquiry |
| PSVG-HPV58 | Pseudotyped GFP HPV58 | GFP | HEK293 FT | Inquiry |
| PSVG-HPV59 | Pseudotyped GFP HPV59 | GFP | HEK293 FT | Inquiry |
| PSVG-HPV68 | Pseudotyped GFP HPV68 | GFP | HEK293 FT | Inquiry |
The HPV pseudotyped virus neutralization test begins with serial dilutions of the samples to be tested, which are then mixed with a certain amount of pseudotyped virus. After that, the target cells are incubated for 24 h, and the amount of pseudotyped virus entering the target cells is calculated by detecting the expression of GFP/RFP, to obtain the neutralizing antibody content of the sample. The cell control (CC) with only cells and the virus control (VC) with virus and cells are set up in each plate. The half maximal effective concentration (EC50) is calculated for the tested samples.
Set up pseudovirus dilutions: for 96 well plate, serially dilute the HPV pseudovirus to a final volume of 100 µL in cell culture media per well.
Infect cells: For a 96 well plate, resuspend cells at a density of 3 x 105 cells/mL. Add 100 µL of the cell suspension (3 x 104 cells) to each well containing the pseudovirus and mix gently by pipetting 1-2 times. Place plate in a 5% CO2, 37°C humidified incubator for 24-48 hours.
Quantification: Acquire GFP signal (ex488/em530) using fluorescent microscopy, flow cytometry, or a high-content imager.
Infection of 293FT cells with Pseudotyped GFP HPV
Infection of 293FT cells with Pseudotyped RFP HPV
Q: How high is the virus titer of this product?
A: Typically 1000 TU/mL. The recommended amount of pseudovirus per well can be found in the Certificate of Analysis for each lot of pseudovirus but should be determined empirically based on your needs.
Q: Can the virus stock thawed and frozen for several times, without loosing efficiency?
A: For our pseudovirus, freezing at -80°C in small aliquots is the optimal storage condition. Aliquots should NOT be frozen and thawed more than once.
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