**3. Current HPV vaccines**

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

technique this assay was established only in very few laboratories.

seemingly met with success [6].

al. in preparation).

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

150 Human Papillomavirus and Related Diseases – From Bench to Bedside A Diagnostic and Preventive Perspective

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

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

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

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 development of the current HPV vaccines [15].

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]. Cervarix® is a bivalent vaccine containing VLPs from the two most prevalent high-risk HPV types 16 and 18. The VLPs are produced in insect cells and formulated with the adjuvant system AS04 (composed of aluminium hydroxyphosphate sulfate combined with MPL- 3-O-deacyl-4' monophosphoryl lipid A) [17]. Gardasil® is a quadrivalent vaccine that in addition to HPV16 and HPV18 VLPs also contains HPV6 and HPV11 VLPs. These two low-risk types are respon‐ sible for nearly 90% of the genital warts. The VLPs in Gardasil® are produced in a yeast system and adjuvanted with aluminium hydroxiphosphate sulfate salt [18] (Table 1).

The most common adverse effects for both vaccines are pain, reddening and swelling at the site of the injection as well as syncope, fatigue, nausea, dizziness and migraine. No severe side effects including auto-immune response abortion or abnormal pregnancy were observed with increased frequency after vaccination with Cervarix® or Gardasil® when compared to the

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**Symptom Cervarix® Gardasil®** Pain 92.9 [90.4, 95.0] 71.6 [67.5, 75.4] Redness 44.3 [40.0, 48.6] 25.6 [21.9, 29.5] Swelling 36.5 [32.3, 40.7] 21.8 [18.3, 25.5] Fatigue 49.8 [45.5, 54.2] 39.8 [35.6, 44.1] Headache 47.5 [43.2, 51.9] 41.9 [37.6, 46.3] Fever ≥ 39.0 °C 0.4 [0.0, 1.4] 0.0 [0.0, 0.7]

**Table 2.** Percentage of women reporting symptoms at least once within seven days after any vaccine dose (total

As HPV infection is limited to basal epithelial cells, the virus is normally "hiding" from circulating immune cells during initial stages of infection, limiting the host's immune respons‐ es. Additionally, to evade the host's immune system and achieve persistent infection, HPV has developed several mechanisms to down-regulate host immunity [27, 28]. The virus's success in evading the immune system is corroborated by the finding that of the women infected with HPV, only 50% develop anti-HPV antibodies (mainly anti-L1). Whether these antibodies can

The mechanisms of immunity induced by the HPV vaccines are not fully understood but it seems that humoral immunity (virus-specific neutralizing immunoglobulin G antibodies) plays an important role. Passive transfer of immune serum in pre-clinical animal models, for example, have demonstrated that L1 virus-specific antibodies are sufficient to prevent

Cervarix® and Gardasil® induce production of high levels of anti-L1 antibodies that reach their peak seven months after the administration of the third dose. The level of antibodies gradually decreases over time but even after several years the titers remain higher than in

control groups [24-26]

Modified from [23]

**5. Immunity**

vaccinated cohort) – Einstein et al., 2009 study [23]

**5.1. Immunity of natural HPV infection**

papillomavirus infection [14, 29, 30].

naturally infected women.

protect against re-incident infection remains unclear.

**5.2. Vaccine induced immunity and duration of protection**


**Table 1.** Comparison of the two prophylactic HPV vaccines, Gardasil® and Cervarix®.
