**8. Is there room and need for second generation vaccines?**

As outlined above, the two commercial vaccines induce long lasting high titer, protective antibody responses against the HPV types included in the vaccines. The efficacy of preventing vaccine type induced lesions can reach up to 100%. This success is based on the exceptional immunogenicity of HPV virus-like-particles and the current vaccination programs will surely have significant impact on reducing the HPV associated cancer burden in the near future. Still, there are several shortcomings of the commercial vaccines which include costly production, need for invasive administration, low stability requiring intact cold chains in vaccine delivery and a narrow range of protection limited mainly to vaccine type papillomaviruses. Further, studies have shown that vaccination with the commercial vaccines has no impact on the progression of pre-existing lesions, i.e. neither Gardasil® nor Cervarix® seem to have a therapeutic effect [56]. Although basically all vaccines used in routine medicine are of pro‐ phylactic nature, this was not necessarily expected to be the case for the HPV VLP vaccines. In a number of preclinical studies it was demonstrated that vaccination of mice with VLPs induces strong cytotoxic T-cell responses against the L1 antigen and in case of L1-E7 chimeric particles also against the E7 portion [70-73]. The response had strong anti-tumorigenic properties in different tumor challenge models. Therefore, there was reason to hope for a vaccination benefit for humans already infected with the corresponding HPV type. Unfortu‐ nately, however, this benefit was not observed in clinical trials to date.

Cervarix® induces cross-protection against additional types such as HPV 31, 33 and 45 and Gardasil® induces protection against HPV 31, albeit at lower efficacy. As a consequence, in 2010 the European Medicines Agency has approved the amendment of the license of Cervarix® in prevention of HPV 31, 33 and 45 induced lesions. The molecular mechanisms for the enhanced cross-protection of Cervarix® in comparison to Gardasil® is not fully understood. One explanation could be the fact that Cervarix® is inducing higher titers against HPV 16 and HPV 18, possibly due to the stronger adjuvants used in the formulation of Cervarix®. Another explanation could be structural differences of the VLPs contained in the two vaccines.

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However, despite this extended cross-protection observed for Cervarix® about 20% of cervical cancer cases remain uncovered by the vaccine. To breach this gap, Merck MSD is currently evaluating a nonavalent HPV VLP vaccine in phase III clinical trials. In addition to the nononcogenic HPVs 6 and 11, this vaccine includes VLPs of HPV types 16, 18, 31, 33, 45, 52 and 58 and theoretically would reach close to 88% efficacy. It remains to be determined whether this cocktail of nine different VLPs is able to induce prolonged protective responses against the corresponding HPV types or if due to interference this may not be possible. Further, because of increasing vaccine complexity this strategy will be limited due to rising costs in production. Also, it will be difficult to prove vaccine efficacy in preventing cervical dysplasia induced by rather rare HPV types, such as HPV 52 and HPV 58, if neutralizing antibodies or at least prevention of infection by these types are not accepted as surrogate markers by the

There are more than 200 different papillomaviruses infecting vertebrates. Among them are roughly 50 types for which there is a theoretical interest of implementing prophylaxis and these include oncogenic HPVs, skin type HPVs relevant in immune compromised patients, bovine PV infecting cattle [79] and horses and PV viruses infecting pets. It has been shown in a number of studies that genetic vaccination with codon-adapted L1 genes leads to the induction of high titer neutralizing antibodies. Vaccination has been performed by intramus‐ cular needle injection or by the use of a gene gun [80-90]. We observed particularly strong neutralizing antibody responses when administering codon-modified L1 genes using a tattooing device [84, 91]. In addition to delivering the expression constructs to muscle and/or antigen presenting cells, tattooing induces a certain degree of local tissue damage which might serve as a danger signal [92, 93]. The great advantage of immunization with naked DNA is the ease of constructing and producing the vaccine vectors for many different L1 antigens since standardized procedures can be applied. Also, DNA is a very stable molecule making the need for intact cold chains in vaccine distribution obsolete. In addition, it has been shown that cocktails of different L1 expression constructs can be applied to mount a broad range of protection, although some kind of interference between different L1s has been observed [94].

**10. Examples for second generation L1-based vaccines**

licensing agencies.

**10.1. Genetic vaccination**

To overcome at least some of the limitations of the commercial vaccines a number of different approaches to develop a second generation PV vaccine are followed, some of which will be addressed in more detail below.
