**10. Measuring the immune response to vaccine**

should achieve an optimal balance between clinical sensitivity and clinical specificity for detection of CIN2+/CIN3+ in order to minimize redundant or excessive follow-up procedures for hr-HPV-positive women with transient hr-HPV infections and/or without cervical lesions. Thus, as an example, a HPV assay with very high analytical sensitivity can yield a large number of clinically insignificant positive results, which will cause unnecessary clinical follow-up, unnecessary diagnostic procedures and unnecessary treatment of healthy women [126].

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

In forthcoming years, self-sampling may become increasingly important in cervical screening since self-collection for HPV testing (HPV self-sampling) has shown to persuade a subset of non-attendees to participate [127-129]. Targeting non-attendees is important, because they are at higher risk of developing cervical cancer. Additionally, self-sampling may make cervical

An accurate and internationally comparable HPV DNA detection and genotyping methodol‐ ogy is an essential component both in the evaluation of HPV vaccines and in the effective implementation and monitoring of HPV vaccination programs. Genotyping assays used today differ in their analytical performance with regard to type-specific sensitivity and specificity [131]. The evaluation of assay performance needs to be performed in a standardized manner, where different assay performances can be evaluated and results can be compared against a

In 2008, the WHO HPV LabNet conducted a proficiency study based on HPV DNA plasmids containing the genomes from 14 oncogenic HPV types and 2 benign HPV types and open for participation to laboratories worldwide [132]. This study demonstrated that it is possible to perform global proficiency studies with unitage traceable to ISs based on plasmid DNA and that such studies can provide an overview of the status of the HPV detection and typing methodology worldwide. More recently, based on a proficiency panel composed of the same HPV DNA plasmid material used in 2008, with the amount of DNA titrated in amounts traceable to the IS. The use of the same panel material allowed a reproducible, standardized evaluation of assay sensitivity over time. Specificity was defined as absence of incorrect typing. The sample preprocessing was evaluated with extraction controls of cervical cancer cell lines. The panel was distributed to 105 laboratories worldwide and analyzed using a range of HPV DNA typing assays in a blinded manner [133]. Among laboratories that used the same assay in both years, 27% were proficient in 2008, whereas 30% were proficient in 2010. They also saw a strong trend toward increased sensitivity of assays. For example, among the laboratories using the same assay in 2008 and 2010, 50 IU of HPV-16 could be detected by all (100%) laboratories in 2010, whereas 86% of laboratories could detect 50 IU of HPV-16 in 2008. However, for several laboratories, the increased sensitivity was accompanied by increased amounts of false-positive results, resulting in nonproficiency, suggesting that recommenda‐ tions for HPV laboratory testing include an increased emphasis on the use of negative controls

The demands on sensitivity of HPV typing assays vary depending on the purpose of the testing. The WHO HPV LabNet proficiency panels are designed to evaluate the performance of HPV typing tests used in HPV vaccinology and HPV surveillance. In vaccinology, high analytical sensitivity is needed, as failure to detect prevalent infections at trial entry may result

screening accessible to women in developing regions [130].

known and accepted standard over time.

in the assays.

HPV serology is an essential technology for both HPV vaccinology and HPV epidemiology. Definitions of HPV-naïve subjects eligible for HPV vaccination trials include seronegativity for HPV. Immunogenicity of HPV vaccines has been used to bridge results from efficacy trials in adolescents to children and to evaluate different batches of HPV vaccines. Antibody measurements are also important in vaccinology research, e.g. for characterizing the immune response with respect to studies of seroconversion and antibody increases, cross-reactions, immune memory and immune persistence as well as kinetics of antibody responses and establishment of correlates of protection. Finally, HPV seroepidemiology is also useful for understanding the epidemiology of HPV infections in populations to be targeted by HPV vaccination programs. The lack of a standardized assay to measure HPV antibody levels has hindered both epidemiological studies of HPV infection and comparison of results from different HPV vaccine trials [134]. WHO Guidelines for HPV vaccines suggest that "initial assessment of immune responses to HPV VLP vaccines should be based on measurement of neutralizing antibodies in serum". The available data [134-136] suggest that neutralizing and ELISA antibody titres are usually highly correlated when the ELISA antigen target is confor‐ mationally intact VLPs. Due to the complexity and labour-intensiveness of neutralization assays, VLP-based ELISAs have been preferred in large epidemiological studies. E.g., a study of HPV seroprevalences was conducted by measuring HPV 16 antibodies with an HPV16 L1 VLP-based ELISA to estimate the public-health impact of HPV vaccination strategies [137].

WHO has been coordinating work to develop standard assays that will help in assessing vaccine quality and monitoring impact after vaccination [138]. In 2006, WHO established a global HPV laboratory network (LabNet) with a main focus being the harmonization and standardization of laboratory testing procedures to support consistent laboratory evaluation of regional disease burden and monitoring of the performance of HPV vaccines. At a WHO consultation in January 2008, a group of experts recommended that the HPV LabNet should develop or identify standardized assays for general use and that efforts towards standardiza‐ tion on VLP-ELISA should be a high priority of the WHO HPV LabNet [139]. Following the recommendation, the WHO HPV LabNet launched a serology standardization program encompassing: (i) an international collaborative study to evaluate and refine a direct HPV 16 VLP-ELISA suggested Standard Operating Procedure (SOP), (ii) an international request for donations of VLPs to be used as international reference reagents for serology, followed by characterization and selection of optimal reagents, and (iii) an international collaborative proficiency study on HPV 16 serology, where participating laboratories used the same standardized SOP and the same VLP reference reagent [140].

A measure to address the concern of vaccination efficacy for older women is to consider vaccinating women at middle ages. Undoubtedly, vaccinating mid-adult women misses the most at-risk period of a woman's life for contracting HPV infection at sexual debut. It is, however, shown that one must not dismiss lightly a mid-adult woman who wants to take the vaccination to prevent new or repeat HPV infection [161]. The vaccines have been shown to be immunogenic in adult women and significant prevention of persistent HPV-16 and HPV-18 infection has been demonstrated in a randomized control trial [162]. The effectiveness of the vaccines among these women in reducing the incidence of HGAs is being awaited with great interest. The shifting burden of cervical cancer to the young and the old in the screened

HPV Diagnosis in Vaccination Era http://dx.doi.org/10.5772/55818 75

Immunization of boys with VLPs elicits a serum immune response similar to that in girls. Because genital HPV infection is sexually transmitted, immunization of men may help to prevent infection of women. Modeling studies on herd immunity, i.e. indirect protection of those who remain susceptible, owing to a reduced prevalence of infections in the risk group for disease, have been published [164]. The utility of immunization of males depends upon the assumed population coverage of vaccination, with successively smaller additional benefits seen in scenarios with high population coverage [165]. Modeling of programs with high population coverage (90%) have found that addition of male vaccination gives a more rapid infection control and have suggested that both sex vaccination programs may be required to

Limitations of the current HPV vaccines include the need for multiple parenteral doses, the lack of protection against some HPV types that cause cervical cancer, and a relatively high cost. The opportunity to overcome 1 or more of these limitations provides a rationale for developing candidate second-generation vaccines. Low-cost second-generation vaccines that could induce long-term protective immunity with fewer doses would be especially attractive for the developing world. Vaccines with activity against a broader range of the HPV types that cause cervical cancer could increase their effectiveness throughout the world, and Merck has indicated that a nonavalent (9 HPV targets) VLP vaccine is currently in clinical trials [166]. If successful, such a vaccine might reduce the frequency of potentially oncogenic infections to a degree that would permit a drastic reduction in cervical cancer screening, which accounts for

HPV differs from most other vaccine-preventable diseases in that the major diseases to be prevented occur many decades after infection. Whereas clinical trials have documented prevention of infection and intermediate disease end-points (condylomas and precancers), surveillance following vaccine implementation will be required to document the expected gains in cancer prevention if there is appropriate population coverage. Surveillance will also provide data to indicate if type replacement or escape mutants occur. Other important tasks for the HPV surveillance include monitoring of the duration of protection, long-term safety

most of the cost of HPV-associated disease in developed countries [167].

population should disappear in the HPV vaccination era [163].

achieve an ultimate eradication of the infection [137].

**11. Future prospects and conclusions**

Serologic assays for the evaluation of HPV vaccine responses are currently limited to an enzyme-linked immunosorbent assay (ELISA) [141], three multiplex assay systems [142-144], and a pseudovirus neutralization assay [145], and emerging data suggest that each system has some utility for characterizing HPV vaccine antibody specificity [136, 146]. Protection against vaccine types is thought to be mediated by neutralizing antibodies [147], and while the mechanism of vaccine-induced cross-protection is uncertain, the measurement of antibodies against nonvaccine types may be useful as a potential correlate or surrogate of cross-protection [148, 149]. Recently, Bissett *et al* [150] obtain a sera panel. These plasma pools could be useful as reference reagents. They are currently available as 250-µl aliquots of liquid plasma archived at −80°C and can be obtained from the National Institute for Biological Standards and Control.

Type-specificL1VLP-antibodies reachmaximumtitresatmonth7,i.e.1monthafteradministra‐ tionofthe thirddose.Titresdeclineuntilmonth24 andremainrather stable thereafter[151, 152]. At 3 years, antibody titres remain two- to 20-fold higher than in placebo controls [152]. Com‐ plete protection against HPV16 associated CIN lesions was observed over the whole followup duration of two Phase IIb trials: 6 years for the monovalent HPV16 vaccine, 5 5 years for the bivalent HPV16/18 vaccine [153, 154] and 4 years for the quadrivalent vaccine. Follow-up is continuing,andcontinuedprotectionagainstHPV16/18-associateddiseaseend-pointshasbeen shown for the entire available observation time, even when specific antibody titres fall.

HPV infection is the most prevalent soon after sexual debut of a girl [155]. The current HPV vaccines, being prophylactic in nature, should be used before exposure of girls to HPV infection. National mass vaccination programs targeting adolescent girls between 12 and 17 years of age are available in most of the developed countries. The vaccine coverage varies greatly between countries, but school-based schemes have thus far demonstrated the highest coverage rate of 80% or higher [1, 156-158]. Australia is the first country to implement a schoolbased mass vaccination program with Gardasil in April 2007 and, within 3 years, has already witnessed a reduced incidence of high-grade abnormalities (HGAs) among women below 18 years of age [1].

High coverage rates of adolescent vaccination with rubella and hepatitis B suggest that, with appropriate parental and public education, adequate HPV vaccine uptake rate of 50% or more is achievable. This coverage rate is critical for mass vaccination to attain cost-effectiveness as a public measure in controlling cervical cancer burden [159]. It will also remove the imbalance in accessing screening and effectively diminish the unequal distribution of cervical cancer burden among women in the metropolitan and rural areas, among women of different ethnic groups, and among women of various socio-economic status in developed countries. Adoles‐ cent HPV infection protects women from the first exposure to HPV infection at sexual debut and abolishes the opportunity for latent infection to occur with a resultant reduction of cervical cancer in old age. The current HPV vaccines induce very high levels of anti-HPV-16 and anti-HPV-18 antibody levels to sustain protection from infection by these aggressive oncogenic HPV subtypes for at least 20 years to cover the average age period of sexual debut [160].

A measure to address the concern of vaccination efficacy for older women is to consider vaccinating women at middle ages. Undoubtedly, vaccinating mid-adult women misses the most at-risk period of a woman's life for contracting HPV infection at sexual debut. It is, however, shown that one must not dismiss lightly a mid-adult woman who wants to take the vaccination to prevent new or repeat HPV infection [161]. The vaccines have been shown to be immunogenic in adult women and significant prevention of persistent HPV-16 and HPV-18 infection has been demonstrated in a randomized control trial [162]. The effectiveness of the vaccines among these women in reducing the incidence of HGAs is being awaited with great interest. The shifting burden of cervical cancer to the young and the old in the screened population should disappear in the HPV vaccination era [163].

Immunization of boys with VLPs elicits a serum immune response similar to that in girls. Because genital HPV infection is sexually transmitted, immunization of men may help to prevent infection of women. Modeling studies on herd immunity, i.e. indirect protection of those who remain susceptible, owing to a reduced prevalence of infections in the risk group for disease, have been published [164]. The utility of immunization of males depends upon the assumed population coverage of vaccination, with successively smaller additional benefits seen in scenarios with high population coverage [165]. Modeling of programs with high population coverage (90%) have found that addition of male vaccination gives a more rapid infection control and have suggested that both sex vaccination programs may be required to achieve an ultimate eradication of the infection [137].

Limitations of the current HPV vaccines include the need for multiple parenteral doses, the lack of protection against some HPV types that cause cervical cancer, and a relatively high cost. The opportunity to overcome 1 or more of these limitations provides a rationale for developing candidate second-generation vaccines. Low-cost second-generation vaccines that could induce long-term protective immunity with fewer doses would be especially attractive for the developing world. Vaccines with activity against a broader range of the HPV types that cause cervical cancer could increase their effectiveness throughout the world, and Merck has indicated that a nonavalent (9 HPV targets) VLP vaccine is currently in clinical trials [166]. If successful, such a vaccine might reduce the frequency of potentially oncogenic infections to a degree that would permit a drastic reduction in cervical cancer screening, which accounts for most of the cost of HPV-associated disease in developed countries [167].
