**11. Alternative production systems**

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

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

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

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

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

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

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

recombinant adenoviruses have not yet been tested in the COPV challenge model.

it might not be possible to generate responses against L1 proteins of multiple PVs.

for application in routine vaccine prophylaxis.

of the induction of cellular immune responses against L1.

humans.

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 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 commercial vaccines. The main challenge seems to be the need for demonstrating noninferiority. Licensing of Gardasil® and Cervarix® has been a mammoth task, involving tens of thousands participants in clinical trials. It is very unlikely that such evaluation can be reproduced with a vaccine approach that presents only an incremental improvement in one of the other shortcomings of Gardasil® and Cervarix®. Other equally important issues are safety and simplicity of second generation vaccines, especially in light of the target popula‐ tion's young age. Lastly, intellectual property is an important factor in vaccine development. While the tight patent situation on L1 VLP technology might eventually be less stringent in the coming years, this will also leave novel developments without sufficient protection, making major investments for manufacturers less attractive.

Alphs et al. observed a strong increase in immunogenicity of the 17-36 epitope when conju‐ gating the L2 peptide to a synthetic lipopeptide (TLR2 agonist) and a broadly acting T-helper epitope [139]. This antigen induced rather high neutralizing titers against HPV 16 while responses against other high-risk HPVs including HPV 18 or HPV 45 were 1-2 orders of magnitude lower. Still, this fully synthetic L2 vaccine provided an elegant basis for the development of a L2 vaccine. Jagu et al. reported that a concatenated L2 fusion protein, consisting of the amino acids 11-88 of five different HPV types induced strong neutralization and cross-neutralization and was superior compared to monotypic HPV 16 L2 antigen. This

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Displaying the 17-36 epitope on bacteriophage PP7 capsids was shown to be an attractive alternative approach in generating a functional L2-based vaccine [140, 141]. VLPs of bacterio‐ phage PP7 can be produced in large quantities and are tolerant for the insertion of heterologous peptides. Immunization of mice leads to high titers of ELISA reactive L2-specific antibodies. Cross-protective neutralization of HPV pseudovirions was shown in an *in vivo* challenge model. The authors did not titrate the sera in an *in vitro* neutralization assay and thus it is not

A 'natural' scaffold for the presentation of L2 epitopes would be to insert the cross-neutralizing epitope into L1 loops located on the VLP surface. This would provide for a highly repetitive presentation of the L2 region. Schellenbacher et al. pursued this approach and tested various peptide insertions into the BPV1 and HPV 16 L1 protein [142]. Such insertions often interfere with proper assembly of the L1 into higher ordered structures but the authors were able to produce and purify a number of L1-L2 chimeric particles. They demonstrated that the CVLPs still induced L1-specific neutralization, indicating mostly correct conformation of the L1 protein. More importantly, chimeric particles carrying the 17-36 epitope of HPV 16 L2 induced neutralizing antibody responses in rabbits against HPV 5, 11, 16, 18, 45, 52, 58 pseudovirions

Recently, we have developed a strategy to boost the immunogenicity of the L2 cross-neutral‐ izing epitope by using bacterial thioredoxin (*Trx*) as a carrier [137]. Due to its rigid structure, this small, 109 amino acid long protein can constrain rather large multi-peptide insertions of heterologous antigens without compromising carrier structure. Previously, presenting an amyloid-ß peptide in context of an *E. coli Trx* scaffold allowed induction of Aß immune responses in a mouse model for Alzheimer [143]. When we inserted the HPV 16 L2 crossneutralizing epitope (aa 20-38 corresponding to 17-36 described by Roden et al.) we achieved a boost in immunogenicity by several orders of magnitude, compared to the peptide linked to keyhole limpet hemocyanin. Further, multimerization of the L2 epitope in the *Trx* led to further increase in induction of neutralizing antibodies. While we also confirmed the existence of other regions in the L2 N-terminus as targets for neutralizing antibodies, we only found crossneutralization for the 20-38 epitope [136]. We also found that a subset of antibodies reactive against the different L2 epitopes fail to neutralize HPV pseudovirions *in vitro* and this might

be due to steric hindrance of L2 epitope recognition in the context of virus capsids.

Ultimately, there is convincing evidence that the L2 protein of HPV contains a number of neutralizing epitopes and importantly one major cross-neutralizing epitope. It is also clear that

approach is expected to enter a clinical phase in 2013.

clear how robust the anti-L2 responses were.

with titers ranging from 1:100 to 1:10,000.
