**2. HPV vaccines — Early studies in animal models**

The first observations in respect to therapeutic or prophylactic vaccination against papillomaviruses (PV) were made using models of experimental induction of warts in rabbits and humans. In heroic and bold self-experimentation Findlay inoculated himself with wart extracts and noted that he became 'immune' to wart induction. Similarly, Grigg and Wilhelm noted patterns for the appearance of skin warts in school children and attributed their findings to a possible 'resistance' of some individuals [5]. In the first half of the last century a number of efforts were undertaken to treat skin and genital warts by the injection of autologous and heterologous wart extracts; some of these attempts were seemingly met with success [6].

A systematic development of prophylactic papillomavirus vaccines proved difficult without a virus that can be replicated in culture, suitable animal models, and markers for protection. Still, a number of prophylactic vaccine approaches were performed either by the use of formalin-fixed wart extracts or by inactivated purified viruses e.g. in dogs, rabbits, cattle and horses (for review see: [7]). By passive transfer Chambers et al. demonstrated that antibodies confer protection against induction of oral papillomas [8]. One of the first *in vitro* assays that allowed detection of virus-neutralizing antibodies, the so-called focus-formation assay, was based on transformation of mouse fibroblasts [9]. Initially, this assay was limited to the use of BPV but was later extended to HPV types, by encapsidating the BPV genome in an HPV capsid. Inhibition of virion induced agglutination (HI assay) of mouse erythrocytes by capsid-specific antibodies was employed as a simple surrogate assay before the development of functional reporter-based neutralization assays [10]. The HI assay has intrinsic limitations as it, first, only detects L1-specific antibodies that prevent binding of particles to the cell surface and, second, the nature of the interaction of PV virions with mouse erythrocytes is not well defined. On a different note, it should be mentioned that Kreider and colleagues were the first to develop a functional neutralization assay for HPV 11 by implanting human tissue under the renal capsule of nude mice and subsequently monitoring HPV induced lesions [11]. Because of the complex technique this assay was established only in very few laboratories.

**Figure 1.** Pseudovirion-based neutralization assay (PBNA). Gaussia = Gaussia luciferase; GFP: green fluorescent pro‐ tein; SV40 ORI = SV40 origin for replication. Pseudovirions (PSV) encapsidating a Gaussia luciferase reporter gene were produced in mammalian cells and used for infection of HeLa cells. The levels of secreted Gaussia (light blue arrows) can be quantified by a luminescence assay. The presence of neutralizing antibodies (dark blue) abrogates PSV infec‐

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PV pseudovirions have also been used in a cervicovaginal mouse model for the detection of neutralizing antibodies. In this model, the female mouse genital epithelium is infected with pseudovirions carrying a firefly luciferase gene and luciferase activity is monitored by *in vivo* imaging. Compared to the *in vitro* PBNA, the mouse model shows increased sensitivity

Many years of research showing that anti-L1 antibodies protect against HPV infection and L1 can assemble into particles called virus-like particles culminated and [14] triggered the

Two commercially available prophylactic HPV vaccines, Cervarix® (GSK) and Gardasil® (Merck) have been licensed in over 100 countries. Both are composed of the L1 major capsid proteinassembledintonon-infectiousandhighlyimmunogenicvirus-like-particles (VLPs)[16].

tion and the subsequent secretion of Gaussia.

**3. Current HPV vaccines**

for the detection of L1 but moreover, of L2 antibodies [13].

development of the current HPV vaccines [15].

**3.1. The two commercial HPV vaccines — Similarities and differences**

In recent years, the so called pseudovirion-based neutralization assays (PBNAs) have been regarded as the gold standard for the detection of neutralizing antibodies against PVs [12]. These assays have in common that a plasmid encoding a reporter gene (such as secreted alkaline phosphatase, luciferases, fluorescent proteins) is encapsidated in mammalian cells by expression of codon-optimized L1 and L2 genes (Fig. 1). These pseudoviruses can be purified e.g. by gradient centrifugation and used to infect cells *in vitro* and *in vivo*. Presence of neutral‐ izing antibodies will prevent infection and thus reporter gene expression. The assay is tedious and does not readily allow for screening of large serum sample collections e.g. for the moni‐ toring of clinical vaccine trials. Recently, we have developed a modified, high-throughput PBNA that allows automated and reproducible detection of neutralizing antibodies (Sehr et al. in preparation).

Human Papillomavirus Prophylactic Vaccines and Alternative Strategies for Prevention http://dx.doi.org/10.5772/55852 151

**Figure 1.** Pseudovirion-based neutralization assay (PBNA). Gaussia = Gaussia luciferase; GFP: green fluorescent pro‐ tein; SV40 ORI = SV40 origin for replication. Pseudovirions (PSV) encapsidating a Gaussia luciferase reporter gene were produced in mammalian cells and used for infection of HeLa cells. The levels of secreted Gaussia (light blue arrows) can be quantified by a luminescence assay. The presence of neutralizing antibodies (dark blue) abrogates PSV infec‐ tion and the subsequent secretion of Gaussia.

PV pseudovirions have also been used in a cervicovaginal mouse model for the detection of neutralizing antibodies. In this model, the female mouse genital epithelium is infected with pseudovirions carrying a firefly luciferase gene and luciferase activity is monitored by *in vivo* imaging. Compared to the *in vitro* PBNA, the mouse model shows increased sensitivity for the detection of L1 but moreover, of L2 antibodies [13].
