**12. L2: Candidate for a potential pan HPV vaccine?**

At the time when PV VLP technology started to have its major impact on papillomavirus research and vaccine development, the group of Saveria Campo in Glasgow reported that vaccination of cattle with a bacterially produced minor capsid protein L2 induced protection against challenge with infectious BPV 4 virus [129]. The authors identified epitopes located in a region of L2 encompassing amino acids 131-151 of BPV 4. Although the report describes these epitopes as B-cell epitopes, no neutralization assay could be performed at the time and hence an involvement of cellular immunity could not be ruled out. Also, the antigens that were either GST-L2 fusion proteins or conjugated peptides were of rather poor immunogenicity. As the field was moving towards VLP vaccines that induce very strong protective effects, L2 was not given further thought as a vaccine antigen at the time.

Later, Richard Roden and his colleagues investigated in detail the suitability of L2 as a vaccine antigen. They observed that L2 antigens purified from *E. coli* induced cross-neutralizing antibodies as assessed by the focus formation assay developed by the investigators [130]. Subsequently, they mapped a cross-neutralizing epitope to a region spanning amino acids 1-88, which was later pin-pointed to amino acids 17-36 [131-133]. Interestingly, human sera from a therapeutic vaccine study using a L2-E6-E7 fusion protein produced in *E. coli* (TA-CIN; [134]) came back positive for neutralizing activity [132].

The presence of neutralizing and cross-neutralizing epitopes in the N-terminus of L2 was reported and confirmed by others. Kondo and colleagues mapped several regions in the L2 protein between amino acids 1-140 [135]. Some of the neutralizing epitopes were later con‐ firmed by others, however it seems clear today that only one epitope, comprising amino acids 17-36, consistently elicited cross-protection [136-138].

After identifying the target region in the L2 protein, the major challenge in developing L2 as a vaccine antigen was posed by L2's low immunogenicity compared to L1. No or very little neutralizing activity is induced when fragments or peptides of L2 are used as antigens [129, 137]. Further, VLPs composed of L1 and L2 do not induce measurable anti-L2 responses. Because of this, a number of strategies were pursued with the goal of increasing immunoge‐ nicity of the L2 cross-neutralizing epitope.

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 approach is expected to enter a clinical phase in 2013.

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,

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

At the time when PV VLP technology started to have its major impact on papillomavirus research and vaccine development, the group of Saveria Campo in Glasgow reported that vaccination of cattle with a bacterially produced minor capsid protein L2 induced protection against challenge with infectious BPV 4 virus [129]. The authors identified epitopes located in a region of L2 encompassing amino acids 131-151 of BPV 4. Although the report describes these epitopes as B-cell epitopes, no neutralization assay could be performed at the time and hence an involvement of cellular immunity could not be ruled out. Also, the antigens that were either GST-L2 fusion proteins or conjugated peptides were of rather poor immunogenicity. As the field was moving towards VLP vaccines that induce very strong protective effects, L2 was not

Later, Richard Roden and his colleagues investigated in detail the suitability of L2 as a vaccine antigen. They observed that L2 antigens purified from *E. coli* induced cross-neutralizing antibodies as assessed by the focus formation assay developed by the investigators [130]. Subsequently, they mapped a cross-neutralizing epitope to a region spanning amino acids 1-88, which was later pin-pointed to amino acids 17-36 [131-133]. Interestingly, human sera from a therapeutic vaccine study using a L2-E6-E7 fusion protein produced in *E. coli* (TA-CIN;

The presence of neutralizing and cross-neutralizing epitopes in the N-terminus of L2 was reported and confirmed by others. Kondo and colleagues mapped several regions in the L2 protein between amino acids 1-140 [135]. Some of the neutralizing epitopes were later con‐ firmed by others, however it seems clear today that only one epitope, comprising amino acids

After identifying the target region in the L2 protein, the major challenge in developing L2 as a vaccine antigen was posed by L2's low immunogenicity compared to L1. No or very little neutralizing activity is induced when fragments or peptides of L2 are used as antigens [129, 137]. Further, VLPs composed of L1 and L2 do not induce measurable anti-L2 responses. Because of this, a number of strategies were pursued with the goal of increasing immunoge‐

making major investments for manufacturers less attractive.

**12. L2: Candidate for a potential pan HPV vaccine?**

given further thought as a vaccine antigen at the time.

[134]) came back positive for neutralizing activity [132].

17-36, consistently elicited cross-protection [136-138].

nicity of the L2 cross-neutralizing epitope.

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 clear how robust the anti-L2 responses were.

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

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 due to the low immunogenicity of L2 an appropriate scaffold and/or adjuvant system is required. Still, there are several issues to be addressed. First, no systematic comparison of the different strategies of L2 epitope presentation has been carried out. No consensus has been reached as to which parameters for L2 vaccination would be an indicator for vaccine efficacy or would present a correlate for protection *in vivo*. Currently, there are a number of different assays to determine L2-directed humoral immune responses. Although anti-L2 antibodies can be readily measured by ELISA assays, this does not provide a meaningful result, as many antibodies recognizing the neutralizing epitopes seem to be non-functional. Typically, ELISA titers are orders of magnitude higher compared to titers obtained in functional neutralization assays.

either mechanically or chemically. Vaccine antigens can be analyzed directly, i.e. by immu‐ nizing the mice before performing the challenge or indirectly by a passive transfer of antibodies from immunized animals or even humans. This model has later been translated to macaques. In one interesting study it was demonstrated that cytology specimen collection carried out in the macaques, as performed in routine pap screening in women, increases the likelihood of infection by papillomaviruses [145], which, in return, can be prevented by the use of carra‐

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Interestingly and similar to the L2-PBNA, the *in vivo* challenge model shows increased sensitivity compared to the standard PBNA. In fact, we have learned from these assays that extremely low amounts of L2-specific antibodies, which were not detected by the standard PBNA, are sufficient for protection *in vivo* in mice. It is not clear, whether this is due to the same mechanisms, e.g. better access of the L2 neutralizing epitopes. Further, it should be noted that it is not certain whether the increased sensitive of the L2-PBNA or the *in vivo* challenge

The existing animal models are unlikely to make functional *in vitro* assays obsolete. First, they are not suited for analyzing large sets of samples and also, it is difficult to produce quantitative

Concerns about the limitation of the HPV vaccines (e.g.: type specificity and costs) stimulate

Condoms, spermicides, microbicides, circumcision and contraceptives are included in the extensive list of preventive measures that have been shown to curb HPV infection and

Condoms are known to be protective against many sexual transmitted diseases such as HIV, gonorrhea, chlamydia and tricomoniasis. However, a cross- sectional analysis conducted in men (18-70 years old) from Brazil, Mexico and United States, showed that HPV infection can be reduced but not completely prevented by the use of condoms. Several factors can be attributed to the low efficacy of condoms in preventing HPV infection, including inappropriate usage leading to condom breakage and slippage and the fact that condoms cannot cover all

Circumcision has been reported to play a role in preventing sexual transmission of HIV, herpes simplex and HPV [147-149]. A recent trial reported that circumcised males have a reduced prevalence of oncogenic HPV types by 32% to 35% and that this effect might be transferred to the partners of circumcised men [150]. Even though the positive effect of the circumcision against HPV persistence has been confirmed by several studies [151-153], ethical issues and complications make circumcision a procedure that most likely will not be routinely adopted. Different microbicides have been studied for their properties to protect against sexual transmitted infections (STIs). Among those, the spermicide the nonoxynol-9 (N-9) was the most

estimates of protection as they allow only very limited titration of sera.

geenan in the lubricant which is used in the pelvic exam.

model correlate with protection *in vivo* in humans.

**14. Alternative strategies for HPV prevention**

persistence.

the HPV infected genital areas [146].

constant research on alternative strategies for HPV prevention.
