**14. Alternative strategies for HPV prevention**

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

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

**13. Approaches to measure induction of neutralizing antibodies**

that antibodies are sufficient for protection.

for routine use.

A nowadays routine assay is the pseudovirion-based neutralization (PBNA) assay developed byBucket al.thatmeasures transductionefficiencyofPVcapsids encapsidatinga reportergene. In the presence of L1 or L2 neutralizing antibodies or compounds that interfere with virus infection such as carrageenan (see below), transduction of cells is inhibited. This assay is considered the gold-standard for *in vitro* assays and (theoretically) measures any antibody that prevents binding, uptake, uncoating and trafficking of viruses. Although the PBNA has been routinely used for the detection of L2-directed neutralization, recently, Day et al. described a modified *in vitro* neutralization assay with increased sensitivity for L2- (and L1-) specific neutralizing antibodies [144]. In this assay, the virus is treated with exogenous furin conver‐ taseafterinducingaconformationalchange.FurinhasbeenshowntobeessentialforPVinfection and the L2 proteins have a conserved cleavage site at their N-terminus. Cleavage of L2 is a prerequisiteforthebindingofantibodiestothemajorcross-neutralizingepitope17-36.Typically, the L2-specific titers in the L2-PBNA are at 10-100 fold higher compared to the standard PBNA. As described above, early vaccination experiments have been carried out in rabbits and cows, followed by challenge with the corresponding virus, CRPV or BPV. Readout was induction of papillomas. The CPRV model was extended for the use of HPV by 'pseudotyping', i.e. encapsidating CRPV genomes into HPV 16 capsids. By this, rabbits can serve as an *in vivo* model for testing HPV vaccine antigens. Protection against oral papillomas in dogs infected with the canine oral papillomavirus was an essential milestone to demonstrate that VLPs can induce sterilizing immunity against PV infection. Also, by passive transfer it could be shown

However, despite the highly valuable contribution of BPV, CRPV, and COPV models, only a few laboratories around the world had the available means and resources to establish them

The laboratory of John Schiller developed a mouse model for PV infection that can find widespread routine application more easily [13]. In this model, the genital mucosa is infected with pseudovirions encapsidating a luciferase reporter gene. Infection can be quantified by *in vivo* imaging. For efficient infection, microtraumata are induced into the mucosal epithelium,

assays.

Concerns about the limitation of the HPV vaccines (e.g.: type specificity and costs) stimulate constant research on alternative strategies for HPV prevention.

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

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 the HPV infected genital areas [146].

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 promising. This spermicide, largely available in the market during the 90s, has shown to be protective *in vitro* against several STDs as gonorrhea, candidiasis, herpes simplex and HIV [154-157] However, clinical trials showed that *in vivo* N-9 was not protective against HIV and HPV and could even promote higher infection ratio due to inflammatory and toxicity effects [13, 158].

[3] de Villiers EM, Gissmann L, zur Hausen H. Molecular cloning of viral DNA from hu‐

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http://dx.doi.org/10.5772/55852

169

[4] Dürst M, Gissmann L, Ikenberg H, zur Hausen H. A papillomavirus DNA from a cervical carcinoma and its prevalence in cancer biopsy samples from different geo‐ graphic regions. Proceedings of the National Academy of Sciences of the United

[5] Grigg WK, Wilhelm G. Epidemiological study of planter warts among school chil‐

[6] Biberstein. Immunization therapy of warts. Arch Dermatol Syphilol. 1943;50:12-22.

[7] Nicholls PK, Stanley MA. The immunology of animal papillomaviruses. Veterinary

[8] Chambers VC, Evans CA, Weiser RS. Canine oral papillomatosis. II. Immunologic as‐

[9] Roden RB, Greenstone HL, Kirnbauer R, Booy FP, Jessie J, Lowy DR, Schiller JT. In vitro generation and type-specific neutralization of a human papillomavirus type 16

[10] Roden RB, Hubbert NL, Kirnbauer R, Breitburd F, Lowy DR, Schiller JT. Papilloma‐ virus L1 capsids agglutinate mouse erythrocytes through a proteinaceous receptor.

[11] Howett MK, Kreider JW, Cockley KD. Human xenografts. A model system for hu‐

[12] Buck CB, Pastrana DV, Lowy DR, Schiller JT. Efficient intracellular assembly of pap‐

[13] Roberts JN, Buck CB, Thompson CD, Kines R, Bernardo M, Choyke PL, Lowy DR, Schiller JT. Genital transmission of HPV in a mouse model is potentiated by nonoxy‐

[14] Day PM, Kines RC, Thompson CD, Jagu S, Roden RB, Lowy DR, Schiller JT. In vivo mechanisms of vaccine-induced protection against HPV infection. Cell host & mi‐

[15] Inglis S, Shaw A, Koenig S. Chapter 11: HPV vaccines: commercial research & devel‐

[16] Schiller JT, Castellsague X, Villa LL, Hildesheim A. An update of prophylactic hu‐ man papillomavirus L1 virus-like particle vaccine clinical trial results. Vaccine. 2008

[17] Keam SJ, Harper DM. Human papillomavirus types 16 and 18 vaccine (recombinant,

AS04 adjuvanted, adsorbed) [Cervarix]. Drugs. 2008;68(3):359-72.

nol-9 and inhibited by carrageenan. Nature medicine. 2007 Jul;13(7):857-61.

man genital warts. Journal of virology. 1981 Dec;40(3):932-5.

immunology and immunopathology. 2000 Feb 25;73(2):101-27.

virion pseudotype. Journal of virology. 1996 Sep;70(9):5875-83.

man papillomavirus infection. Intervirology. 1990;31(2-4):109-15.

illomaviral vectors. Journal of virology. 2004 Jan;78(2):751-7.

opment. Vaccine. 2006 Aug 31;24 Suppl 3:S3/99-105.

pects of the disease. Cancer research. 1960 Aug;20:1083-93.

States of America. 1983 Jun;80(12):3812-5.

Journal of virology. 1995 Aug;69(8):5147-51.

crobe. 2010 Sep 16;8(3):260-70.

Aug 19;26 Suppl 10:K53-61.

dren. Public health reports. 1953 Oct;68(10):985-8.

Carrageenan is a sulfated polysaccharide compound routinely used as thickening ingredient in food products as well as in sexual lubricants and therefore has an excellent safety record. It is derived from seaweed and studies have shown that it confers protection against HIV and HPV *in vitro* [159, 160]. In a phase III clinical trial, carrageenan did not show any effect against HIV but it was tolerable and safe [161]. However, carrageenan was shown to confer HPV protection in a murine animal challenge model [162] and to minimize the increased susceptibility to HPV infection during or after cytology screening in rhesus monkeys [163].

Recently, a dendrimeric gel microbicide (VivaGel – SPL7013) was developed by Starpharma for prevention of infections by HIV and HSV-2. The efficacy and safety of the gel have been demonstrated *in vitro* and in *in vivo* in animal models [164]. Several clinical trials to evaluate the gel safety, tolerance and efficacy are ongoing. In 2008 Starpharma announced that their product can inhibit HPV infection in *in vitro* assays [165].
