**13. Approaches to measure induction of neutralizing antibodies**

A nowadays routine assay is the pseudovirion-based neutralization (PBNA) assay developed byBucket al.thatmeasures transductionefficiencyofPVcapsids encapsidatinga reportergene. In the presence of L1 or L2 neutralizing antibodies or compounds that interfere with virus infection such as carrageenan (see below), transduction of cells is inhibited. This assay is considered the gold-standard for *in vitro* assays and (theoretically) measures any antibody that prevents binding, uptake, uncoating and trafficking of viruses. Although the PBNA has been routinely used for the detection of L2-directed neutralization, recently, Day et al. described a modified *in vitro* neutralization assay with increased sensitivity for L2- (and L1-) specific neutralizing antibodies [144]. In this assay, the virus is treated with exogenous furin conver‐ taseafterinducingaconformationalchange.FurinhasbeenshowntobeessentialforPVinfection and the L2 proteins have a conserved cleavage site at their N-terminus. Cleavage of L2 is a prerequisiteforthebindingofantibodiestothemajorcross-neutralizingepitope17-36.Typically, the L2-specific titers in the L2-PBNA are at 10-100 fold higher compared to the standard PBNA.

As described above, early vaccination experiments have been carried out in rabbits and cows, followed by challenge with the corresponding virus, CRPV or BPV. Readout was induction of papillomas. The CPRV model was extended for the use of HPV by 'pseudotyping', i.e. encapsidating CRPV genomes into HPV 16 capsids. By this, rabbits can serve as an *in vivo* model for testing HPV vaccine antigens. Protection against oral papillomas in dogs infected with the canine oral papillomavirus was an essential milestone to demonstrate that VLPs can induce sterilizing immunity against PV infection. Also, by passive transfer it could be shown that antibodies are sufficient for protection.

However, despite the highly valuable contribution of BPV, CRPV, and COPV models, only a few laboratories around the world had the available means and resources to establish them for routine use.

The laboratory of John Schiller developed a mouse model for PV infection that can find widespread routine application more easily [13]. In this model, the genital mucosa is infected with pseudovirions encapsidating a luciferase reporter gene. Infection can be quantified by *in vivo* imaging. For efficient infection, microtraumata are induced into the mucosal epithelium, either mechanically or chemically. Vaccine antigens can be analyzed directly, i.e. by immu‐ nizing the mice before performing the challenge or indirectly by a passive transfer of antibodies from immunized animals or even humans. This model has later been translated to macaques. In one interesting study it was demonstrated that cytology specimen collection carried out in the macaques, as performed in routine pap screening in women, increases the likelihood of infection by papillomaviruses [145], which, in return, can be prevented by the use of carra‐ geenan in the lubricant which is used in the pelvic exam.

Interestingly and similar to the L2-PBNA, the *in vivo* challenge model shows increased sensitivity compared to the standard PBNA. In fact, we have learned from these assays that extremely low amounts of L2-specific antibodies, which were not detected by the standard PBNA, are sufficient for protection *in vivo* in mice. It is not clear, whether this is due to the same mechanisms, e.g. better access of the L2 neutralizing epitopes. Further, it should be noted that it is not certain whether the increased sensitive of the L2-PBNA or the *in vivo* challenge model correlate with protection *in vivo* in humans.

The existing animal models are unlikely to make functional *in vitro* assays obsolete. First, they are not suited for analyzing large sets of samples and also, it is difficult to produce quantitative estimates of protection as they allow only very limited titration of sera.
