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

#### **10.1. Genetic vaccination**

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‐

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

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

Both current commercial vaccines show excellent safety and efficacy profiles and there seems to be little room for improvement in either aspect when addressing the HPV type-specific protection. Some countries are considering or are in the process of implementing a two dose regiment, driven by the intention to minimize costs [74, 75]. Such deliberations would benefit from higher immunogenicity of the VLP vaccine, which could possibly be achieved by using stronger adjuvant systems. But naturally, it seems unlikely that Merck or GSK would find a sufficient economical motivation to move along this road. What's more, there is only a limited repertoire of adjuvants that can be used in prophylaxis for a young target population.

Both Merck and GSK are probably not highly motivated in developing second generation HPV vaccines that would compete with their blockbusters. An exception is the nonavalent HPV VLP vaccine that is currently evaluated by Merck in clinical trials. A number of pre-clinical studies focused on the development of L1-based vaccines that overcome one or more of the limitations discussed above. These second generation approaches addressed delivery (e.g. oral), production systems (plant, E. coli), stability (e.g. capsomeres) and extension to thera‐ peutic applications (chimeric L1 proteins) [76-78]. In light of the fact that the current VLP vaccines are inducing a limited degree of cross-protection, for which the nature is not yet known, one could envision modifying the L1 protein so as to extend the breadth of protection,

As indicated above, the protective range of Cervarix® and Gardasil® is mainly limited to the vaccine type papillomaviruses. In their clinical trial GSK could show that immunization with

nately, however, this benefit was not observed in clinical trials to date.

addressed in more detail below.

**9. Second generation vaccines targeting L1**

but to our knowledge, this strategy is currently not pursued.

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]. Currently, no clinical testing involving human subjects is being performed with naked DNA or with a genetic vector. For one, DNA immunization has not found its way to human immune prophylaxis to date. The main reason is the much lower efficacy of DNA vaccines in primates compared to the murine system. Further, there are concerns about the safety of DNA vaccines in general. Although these concerns are of theoretical nature only, they still pose a major hurdle for application in routine vaccine prophylaxis.

responses against the L1 antigen, although the authors could demonstrate *in vitr*o neutraliza‐

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The current HPV vaccines are produced either in yeast (Gardasil®) or insect cells infected with recombinant baculoviruses (Cervarix®). It is not disclosed by the vaccine manufacturers what the production costs per dose really are, but insect cells present a rather complex platform and yeast cells provide challenges in the extraction procedures. In the early phases of HPV VLP technology, several labs worked on expressing L1 in *E. coli* but only recently has it been possible to produce properly folded L1 in this system. It was Chen et al. who showed in 2001 that Nterminally modified L1 protein of HPV 11 and 16 can be expressed in *E. coli* and purified in the form of native pentamers (capsomers) [112]. Yuan and colleagues reported that two doses of 400 ng of a GST-L1 fusion protein, assembled in capsomere-like structures protected dogs from a challenge with COPV. HPV 16 L1 pentamers share essential conformational epitopes with VLPs [113, 114]. L1 pentamers are less immunogenic compared to VLPs but use of appropriate adjuvant systems (e.g. ASO4) can largely compensate for this [113]. In addition to being produced cost-effectively in *E. coli*, L1 pentamers are also more stable than VLPs making an intact cold-chain in vaccine distribution obsolete. Although clinical trials are in preparation, efficacy of L1 pentamers has not yet been assessed in human subjects. However, Stahl-Hennig could show capsomeres adjuvanted with synthetic double stranded RNA, either poly ICLC or poly IC induced strong anti-L1 antibody and T-helper responses in rhesus macaques [115]. In a number of studies the production of L1 antigens in transgenic plants has been evaluated. Earlier studies showed that the surface antigen of hepatitis B virus can be expressed and assembled in transgenic plants [116]. Importantly, oral delivery of unprocessed plant material induced HBsAg specific immunity in mice and healthy volunteers [117]. This report ignited the idea that vaccine antigens can be produced with the aid of transgenic plant technology. The great advantage of plants is the simplicity by which vast quantities of biomass can be produced with all required technology already in place. Bypassing the requirement for antigen extraction and purification would allow to meet the worlds growing, yet unmet, demand for cheap vaccines. In this light, production of L1 in plants was initiated, [118-128], and immuno‐ genicity after either oral or systemic delivery was confirmed. Yield of L1, which initially posed a major problem, improved significantly to more than 10% of the total soluble protein [125]. Today's consensus on antigen production in plants stresses standardized extraction and purification to ensure antigens with defined properties and limited inter-batch variability will be an essential criteria. Also, much of the L1 antigen in the plant tissue is incorrectly folded and hence has only little immunogenicity. Overall, there are strong resentments by regulatory agencies and vaccine manufacturers on introducing poorly standardize-able oral vaccines

tion activity of the rabbit sera.

**11. Alternative production systems**

originating from partially processed plant material.

In summary, there are tremendous hurdles that novel second generation vaccines based on the L1 antigen must be overcome starting with facing and competing with the two existing

The ease of targeting multiple L1 antigens has also been a motivation to evaluate viral vector based genetic approaches. Different viral vectors have been used and these include vaccinia virus, vesicular stomatitis virus, and adenoviruses [95-98]. High titer neutralizing antibody responses were induced in vaccinated mice. Additionally, in some of the studies strong cellular immune responses against the L1 antigen could be demonstrated. Using the cottontail rabbit papillomavirus model, it was shown that single intranasal administration of recombinant vaccinia virus [99] or vesicular stomatitis virus (VSV) [96, 97] induces anti-L1 antibodies and protections against CRPV challenge, although the latter could also have been a consequence of the induction of cellular immune responses against L1.

Berg et al. ([98] [100] produced correctly folded canine oral PV VLPs using recombinant adenoviruses. Immunization of mice led to high titer neutralizing antibody responses, but the recombinant adenoviruses have not yet been tested in the COPV challenge model.

When considering administration, the use of complex virus systems including vaccinia virus, VSV and adenoviruses faces significant safety issues. Moreover, most vaccinations will likely be limited to single administration due to the strong responses against the vectors. In this light it might not be possible to generate responses against L1 proteins of multiple PVs.

Adeno-associated virus (AAV) vectors combine the simplicity of naked DNA with the efficacy of viral vector gene delivery. AAV vectors are extremely stable and can be lyophilized without compromising their transduction activity. Also, these viral vectors do not encode for viral gene products. We have used AAV vectors for intranasal and systemic delivery of the L1 gene. Single doses of AAV-L1 induce long lasting (>1 yr) neutralizing antibody responses in mice. The intranasal application also induced mucosal antibodies and cellular immunity. Non-adjuvant‐ ed intranasal application in macaques with recombinant AAV9 vectors also induced immunity against the encoded L1 antigen [101-104]. Liu and colleagues reported on the co-administration of AAV-L1 vectors together with a recombinant adenoviruses encoding for granulocyte macrophage colony-stimulating factor [105]*.* This strategy yielded higher neutralizing titers compared to VLP immunization but might prove difficult in translating into application in humans.

In addition to viral vectors, L1 has also been delivered by live prokaryotic vectors such as Salmonella enterica Typhii [106-109] and recombinant Bacille Calmette-Guerin (rBCG) [110, 111]. Nardelli-Haefliger was the first to demonstrate that live L1-recombinant bacteria (S. typhii) induced mucosal and systemic antibody responses in mice. In another study, Govan et al. showed that rabbits vaccinated with rBCG encoding the CRPV L1 protein are protected against viral challenge [110]. This protection might, however, in part be due to cellular immune responses against the L1 antigen, although the authors could demonstrate *in vitr*o neutraliza‐ tion activity of the rabbit sera.
