Background
Human papillomaviruses (HPVs) are a group of non-enveloped viruses with a double-stranded DNA [
1,
2], which mainly infect epithelial cells and can further induce a variety of benign and malignant hyperplasia [
3]. The high-risk, oncogenic HPV types are related to invasive cervical cancer and other genital carcinomas; whereas the low-risk HPVs cause anogenital warts [
4,
5]. The high-risk types include HPV types 16, 18, 31, 33, 35, 39, 45, 91, 52, 56, 58, 59, 68, 69, 73, and 82, among which HPV type 16 (HPV16) and HPV type 18 (HPV18) together are responsible for approximately 70% of cervical cancer cases [
6]. Because the diseases caused by HPV infections are a serious harm to human health, the development of safe and effective preventive or therapeutic vaccines is of great significance [
7].
There is an early-gene and a late-gene region within the genome of HPV virus. The early genes E1-E7 play roles in viral life cycle and pathogenesis, whereas the late genes L1 and L2 code for the viral capsid proteins L1 and L2 respectively [
8‐
11]. L1 is the major capsid protein, and 72 copies of L1 pentamers make up the viral envelope; L2, the minor capsid protein, sits in the center of the L1 pentamers [
8,
11,
12]. Numerous studies have confirmed that HPV-L1 protein is the major target protein for HPV vaccine: without HPV-L2 protein, the HPV-L1 protein expressed in vitro can self-assemble into virus-like particles (VLPs) that mimic the structure of the native virus [
13,
14]. The L1-VLPs retain the vast majority neutralizing epitopes of the natural virus, and can induce high titers of neutralizing antibodies [
15,
16]. Therefore, L1-VLPs of different types of HPV are the major component of the HPV vaccines currently available or during development.
Three prophylactic vaccines against HPV are currently available on the market. Cervarix™ (GSK) is a bivalent vaccine designed to prevent high-risk HPV type 16 and 18 infections [
17], which has recently been approved by China FDA. Gardasil
® (Merck) — a quadrivalent vaccine — targets two oncogenic types (HPV16 and HPV18) and two types that cause genital warts (HPV6 and HPV11) [
1,
18]. Merck has developed a nonavalent vaccine targeting five additional high-risk HPV types (HPV 31/33/45/52/58) to the HPV types 6/11/16/18 contained in Gardasil
® [
17], and was licensed by U.S. FDA in December 2014 [
18]. The nonavalent HPV vaccine Gardasil
® 9 appears to be safe and effective in preventing persistent infection and precancerous lesions associated with HPV types 16/18/31/33/45/52/58, as well as genital warts related to HPV types 6 and 11 [
19].
It is crucial to monitor the quality of products of every step during the development and production of vaccines. For HPV vaccines, tests for the purpose of quality control include HPV type identification experiment, determination of antigen content, in vitro and in vivo potency assays. These tests utilize antibody binding and competition, making quantifications and comparisons of samples easy to be completed and adapted to other platforms [
4,
20,
21]. The antibodies used in such tests are often HPV type-specific, and need to reflect the biological properties of the original potency and neutralization assays [
20,
22].
During our research and development of a nonavalent HPV vaccine (valence include 6/11/16/18/31/33/45/52/58, same as Gardasil® 9 by Merck), we produced hybridoma cell lines that produce type-specific HPV18 neutralizing mAbs, and assembled an ELISA detection kit using two of the mAbs. This kit, which can rapidly identify and quantify HPV18 L1-VLPs in HPV vaccines, is crucial for vaccine quality control. More importantly, this kit can be used in preliminary clinical analysis of HPV18 infections, and the mAbs can be applied in the development of more sophisticated immunohistochemistry systems.
Discussion
In general, when developing and selecting mAbs for a detection kit of HPV18, we seek to satisfy four criteria. First, the sequence of each mAb must be a specific sequence in the kit. Second, the mAbs should have good binding affinity towards the antigen and would not rapidly dissociate [
20]. Third, two mAbs used in the same assay should be able to quickly and accurately identify the antigen than other mAbs. Last but not least, the mAbs should represent critical quality attributes of the kit, such as linearity, repeatability and affinity.
In this study, two mAbs (1B1 and 4C2) were screened from 10 mAbs expressed by hybridoma cells stably expressing type-specific neutralizing antibodies against HPV18 L1-VLPs. A few features of mAbs 1B1 and 4C2, including antibody isotype, HPV type specificity, conformational epitopes, neutralization ability, and sequences were characterized. Both 1B1 and 4C2 had good specificity towards native HPV18 L1-VLPs and showed no cross-reactivity to the L1-VLPs of the other eight HPV types tested. Compared with antibodies described in similar studies [
7,
15] that have cross-neutralizing activities towards other types of HPVs, our mAbs are HPV18 type-specific and recognize conformational epitopes on HPV18 L1-VLPs.
Amongst all the HPV vaccines currently available, the nonavalent Gardasil® 9 has the widest protection range. Our mAbs, which were demonstrated to have high titers only towards HPV18 L1-VLPs within the 9 HPV types analyzed (valences same as those of Gardasil® 9), can be used in the specific detection of HPV18 VLPs in HPV vaccines with up to 9 valences or compounds that have HPV18 component. Furthermore, they can be applied to the detection of HPV18 infections in clinic.
This pair of mAbs was subsequently used to develop an HPV18 Detection Kit. One mAb coated on the ELISA plate would capture the L1-VLPs, whereas the other mAb with conjugated HRP was used for detection. Such sandwich ELISA approach uses two mAbs to detect and quantify HPV18, allowing the detection of not only HPV18 L1 pentamers, but also HPV18 L1-VLPs. The detection limit of the kit was 0.l μg/ml, with a linear range of 0.l – 10 μg/ml. Assays have also confirmed the kit had high specificity and sensitivity only towards native HPV18 L1-VLPs. This Detection Kit has been applied in our routine quality control tests of HPV vaccine productions, and it was also used by other organizations in relevant analyses.
This Detection Kit was designed primarily for the quality control throughout HPV vaccine production as well as preliminary detection of HPV types. We have taken this research further by developing a more sophisticated IHC system using these mAbs, aiming for a more accurate and convenient detection of HPV18 infections in tissue samples (to be reported elsewhere).
Conclusions
In summary, high-affinity and strongly neutralizing antibodies were generated, characterized, and used to develop a sandwich ELISA kit that could identify and quantify HPV18 type-specific L1-VLP antigens within the nonavalent HPV vaccine range (HPV6/11/16/18/31/33/45/52/58). With the two mAbs we produced, the Detection Kit could detect the presence of HPV18 L1 pentamer or HPV18 L1-VLPs from up to nine different types of HPVs, thus could be widely used in quality control for clinical testing and vaccine manufacturers. This study has also provided us a guideline for the rational design of high-affinity monoclonal antibodies that could be applied to the diagnosis and treatment of HPV18 infections.
Acknowledgements
We sincerely thank Professor Xiaojiang Chen at the University of Southern California for editorial assistance.