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166 JID 2009:200 (15 July) PERSPECTIVE

P E R S P E C T I V E

Immunogenicity Testing in Human PapillomavirusVirus-Like-Particle Vaccine TrialsJohn T. Schiller and Douglas R. LowyLaboratory of Cellular Oncology, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland

Received 16 October 2008; accepted 22 January 2009;electronically published 11 June 2009.

Potential conicts of interest: J.T.S. and D.R.L. are listed asinventors on US governmentowned patents covering thepapillomavirus virus-like-particlebased vaccine technology.These patents have been licensed coexclusively to Merck andGlaxoSmithKline.

Financial support: Center for Cancer Research, NationalCancer Institute.

Reprints or correspondence: Dr. John Schiller, Center forCancer Research, National Cancer Institute, Bldg. 37, Rm. 4106,Bethesda, MD 20892 ([email protected]).

The Journal of Infectious Diseases 2009;200:16671This article is in the public domain, and no copyright is claimed.0022-1899/2009/20002-0003DOI: 10.1086/599988

The recently introduced human papillo-mavirus (HPV) prophylactic vaccines arethe rst widely approved vaccines specif-ically designed to prevent a sexually trans-mitted infection and are only the second(after the hepatitis B virus vaccine) de-signed to prevent human cancer, in thiscase cervical cancer and several other ano-genital and head and neck cancers. Theantigens in the HPV vaccines are nakedicosohedral virus-like particles (VLPs)composed of the major HPV virion pro-tein L1. These subunit vaccines were in-dependently developed and tested by 2companies, GlaxoSmithKline (GSK) and

Merck [1]. The GSK vaccine, Cervarix, isbivalentit contains the VLPs of HPV-16and -18, the types that cause 70% of cervical cancers worldwideand is pro-duced in insect cells. Cervarix also con-tains the proprietary adjuvant AS04,which is composed of monophyosphoryllipid A and an aluminum salt. The Merckvaccine, Gardasil, is tetravalentit con-

tains the HPV-16 and -18 VLPs and theVLPs of HPV-6 and -11, which cause80%90% of genital wartsand is pro-duced in yeast. Gardasil is adjuvanted witha simple aluminum salt. The vaccines aredelivered by intramuscular injection in 3doses over 6 months. Both have been re-markably effective in phase 3 trials con-ducted in young women, providing nearly complete protection against persistentgenital tract infection and premalignantneoplasticdiseaseend pointscausedby theHPV types targeted by the respective vac-cines [2]. Both vaccines have beenlicensedin 1 50 countries, starting with Mercks in2006 and GSKs in 2007, and millions of doses have beensold. However, despite therapid and successful introduction of these2 vaccines, they can reasonably be viewedas introductory products that will likely be followed by second-generation vac-cines that will target more types and/orbe less expensive to produce and deliver.This article will focus on one aspect of HPV VLP vaccines, the assessment of immunogenicity.

WHY IMMUNOGENICITYTESTING IS IMPORTANT

Immunogenicity testing contributes sub-stantially to 5 aspects of HPV vaccine de-velopment and deployment. The rst areais quality control of the vaccine-manufac-turing process and the stability of the vac-cine during storage and distribution. Vac-cine manufacturers tend to preferphysical

characterization for routine quality-con-trol purposes, because reproducible quan-titative results are more easily achievedthan with biological assays. However, di-rect evaluation of the immune response toa vaccine in an animal model or humansubjects remains the most relevant test of vaccine quality. Second, immunogenicity bridging studies are being used to extendvaccine approval forpopulations that werenot evaluated in pivotal phase 3 studies,which were limited to females aged 1526 years [3,4].Thewidespreadregulatoryap-proval of the vaccines for younger ado-lescent girls is based on the noninferi-or immunogencity observed in this agegroup. Regulatory bodies in some coun-tries have also approved the vaccines forolder women and young men, on the basisof the noninferiority of the immune re-sponses to the vaccines. Third, immuno-genicity studies may help to predict theduration of protection. Typical for a newly introduced vaccine, the duration of pro-tection of the HPV vaccines is unknownat present. However, the durability of thepresumably relevant immune response

(discussed below) supports an optimisticprojection. Fourth, immunogenicity stud-ies can lead to the establishment of animmune correlate of protection. Such acorrelation can be of immense help in thedevelopment of second-generation vac-cines by providing an early indication of potential efcacy and, in some cases of closely related vaccines, might evenpermitregulatory approval without costly and


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PERSPECTIVE JID 2009:200 (15 July) 167

Figure 1. Relationships among virus-like-particle (VLP) antibodies detected by enzyme-linked immunosorbent assay (ELISA), competitive Lumimmunoassay (cLIA), and secreted alkaline phosphatase neutralization assay (SEAP-NA). The large background circle depicts the entire spectrum antibodies induced by VLP vaccination, and the interior circles depict the subsets of antibodies detected by ELISA, SEAP-NA, and cLIA. Note thatsize of the circles and the extent of their overlap will vary depending on the specic performance characteristics of the assays for a given humanpapillomavirus type. Abs, antibodies.

time-consuming efcacy trials. Fifth, im-munogenicity testing can be used to assessthe relative merits of competing vaccinesin the absence of a head-to-head efcacy trial. As discussed below, caution must betaken in comparing vaccines that havebeen evaluated by different manufactur-ers using different assays. However, in aninteresting development GSK has an-nounced that it is undertaking a ran-domized clinical trial in young womenthat will directly compare the immuno-genicity of Cervarix and Gardasil [5].

WHY THE FOCUS IS ON

ANTIBODY RESPONSES

HPV VLPs induce potent T and B cellresponses in animal models and humansubjects [6]. However, immunogenicity testing in clinical trials has focused pri-

marily on antibody responses for severalreasons. One is that the protection in-duced by established prophylactic viralvaccines is largely, if not entirely, mediatedby antibodies that prevent virus infection[7]. Another is that the protection fromexperimental challenge in animal papil-lomavirus models can be passively trans-ferred in serum or puried immunoglob-ulin (Ig) G from VLP-vaccinated animals,

which indicates that VLP-induced anti-bodies alone are sufcient to confer pro-tection [6]. In addition, the characteristicsof the antibody response induced by VLPvaccination are consistent with the pro-tection in humans being antibody medi-ated in that there is a correlation betweenthe type-restricted protection that hasbeen seen in clinical trials and a similarspectrum of type restriction in in vitro an-tibody-neutralization assays [6]. The highrate of protection seen in clinical trials alsocorrelates with the 1 99.5% rate of sero-conversion detected in VLP vaccinees.

Antibodies induced after intramuscularVLP injection may protect from cervi-covaginal HPV infection by a combina-tion of 2 mechanisms [6]. The rst istransudation of serum antibodies intogenital tract mucus, which is quite pro-

nounced at the cervix [8]. The secondis that infection is thought to requiretrauma that exposes the epithelial base-ment membrane to the virus, and thiswounding is expected to result in directexudation of systemic antibodies at thesite of virus infection [9].

The expression pattern of L1, the viralprotein that comprises the VLPs, makes itunlikely that T cell responses to the VLPs

contribute to protection. Rather than be-ing expressed in the proliferating basalcellkeratinocytes, where infection is main-tained, L1 is detectably expressed only interminally differentiating keratinocytes of an infected stratied squamousepithelium[10]. It therefore seems unlikely that T celleffector responses would substantially in-uence infection or lesion development.Consistent with this conjecture, VLP vac-cination wasnoteffective against prevalentinfection [11]. It is likely the case that Thelper responses contribute to the uni-formly robust B cell responses seen in VLPvaccinees. However, even in this regardthere is evidence that VLPs, like other well-ordered multivalent antigens, can induceT cellindependent antibody responses[12]. In summary, the focus on antibody responses in immunogenicity studies of

VLP vaccines seems well justied.

ASSAYS TO MEASURE VLP

ANTIBODY RESPONSES

Three assays to measure serum antibodiespredominate in the evaluation of VLP an-tibody responses in clinical trials. Eachmeasures an overlapping but distinct sub-set of the antibody responses to the VLPs


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(gure 1), and each has a unique set of strengths and weaknesses.

Enzyme-linked immunosorbent assay (ELISA). GSK has used a VLP-basedELISA as the principal assay of immu-nogenicity in its trials [13]. This assay

measures antibodies that bind to a VLPantigen xed to a solid surface. Thebound antibodies are detected by the ad-dition of a secondary antibody that rec-ognizes the constant region of a humanantibody of a specic class (e.g., IgG) orsubclass (e.g., IgG1). The secondary an-tibody has an enzyme (e.g., alkalinephos-phatase) conjugated to it, and the en-zymes activity is monitored by a changein the substrate (e.g., a color change thatcan be measured in a spectraphotome-

ter). The major advantages of the ELISAare that it is sensitive, rapid, and repro-ducible and can be automated. Correla-tions across laboratories can be increasedby reporting results relative to the re-sponses to type-specic standard antisera[14].

Although the ELISA can measure neu-tralizing antibodies,which for HPV L1 ex-clusively recognize conformation-depen-dent virion surface epitopes, it has the dis-advantage of also potentially measuringnonneutralizing antibodies that are elic-ited by VLPs and that may be recognizedby VLP antigens in the ELISA. The frac-tion of reactivity that is attributable tononneutralizing antibodies (which wouldnot be protective) would depend on thedegree to which the VLPs used in the vac-cine and/or as ELISA antigen are properly folded and assembled. Some ELISAs canproduce misleading results. For instance,VLP ELISAs can exhibit extensive cross-

reactivity among genital HPV types,whereas protection in the trials is moretype restricted [15]. Such cross-reactivity between types may be a signicant issuein evaluating the immunogenicity of mul-tivalent vaccines, because it precludes cat-egorical assignment of reactivity to a par-ticular VLP type in the vaccine.

A unique feature of the ELISA is thatit detects reactivity to only 1 class or sub-

class of antibodies. Most studies focus ondetecting only IgG, the predominant im-munoglobulin class induced in the serumof most vaccinees. Contributions of otherclasses, such as IgA, to protection are notroutinely evaluated, potentially diminish-

ing the association between antibody re-sponse and protection.Competitive Luminex immunoassay

(cLIA). Merck has mainly used a cLIAto evaluate VLP antibody responses in itsclinical trials [16]. The assay involves x-ing each of the 4 VLP types in the vaccineto Luminex microspheres (Invirtogen)with distinct uorescent properties.Serumsamples from the vaccinees are evaluatedfor their ability to prevent VLP bindingby a type-specic neutralizing monoclonal

antibody that has a phycoerythrin uo-rescent tag. Thus, the strength of the an-tibody response is inversely proportionalto the detection of the monoclonal anti-body binding signal. This assay has 2 major advantages. First, it is highly type spe-cic, because it is based on monoclonalantibodies that were specically chosen tonot cross-react with the VLPs of even very closely related HPV types. Second, indi-vidual reactivities to multiple VLPs can besimultaneously evaluated in a single re-action, and the assay is amenable to high-throughput processing. Unlike the VLPELISA, the cLIA simultaneously measuresVLP-binding antibodies of all immuno-globulin classes.

The major disadvantage of the assay isthat it measures only the subset of neu-tralizing antibodies that compete with thespecic monoclonalantibody forVLPsur-face binding (gure 1). The responses of some vaccinees could be dominated to

varying degrees by VLP-binding (and po-tentially neutralizing) antibodies that donot compete with the monoclonal anti-body. Thus, the assay can underrepre-sent the potentially protective antibody response induced by the vaccine. It is alsounclear to what degree the epitopes rec-ognized by the different type-specicmonoclonal antibodies overlap the im-munodominant epitopes in humans.

Therefore, titers of antibody to the dif-ferent VLP types in a given vaccine can-not be directly compared in a cLIA.

Neutralization assay. The third assay is an in vitro neutralization assay. The rstreproducible quantitative neutralization

assay that is not limited by availability of infectious capsids was recently developedand is becoming widely adopted by bothcompanies and academic laboratories[15]. It involves the cell culture produc-tion of high-titer infectious L1/L2 pseu-dovirions that have encapsidated a genewhose activity can easily be measured asa marker of infection. In the most widely employed variation, the pseudovirus car-ries the gene for secreted alkaline phos-phatase (SEAP), which enables infection

to be quantitated by measuring the ability of culture supernatants to cleave a color-igenic substrate. The sensitivity of theSEAP neutralization assay(SEAP-NA), interms of titers measured in response tonatural infection or VLP vaccination, issimilar to that of standard VLP-basedIgG ELISAs [15, 17, 18]. The assay hasbeen adapted to a high-throughput 96-well-plate format. Nevertheless, a majordisadvantage of this assay is that it is con-siderably more laborious than eitherELISA or cLIA. However, the results of this assay are the most likely to correlatewith protection, because it presumably measures all neutralizing antibodies (re-gardless of immunoglobulin class) andonly potentially protective antibodies.

The in vitro neutralization assay wasfound to be considerably more type spe-cic than VLP ELISAs using research-grade VLPs [15]. However, a strong cor-relation between individual ELISA and

neutralizing-antibody titers was observedin trials of the GSK vaccine [17, 18]. Insome situations, the neutralization assay may be less type specic than the cLIA.For instance, serum samples from HPV-18 VLP vaccinees had low but readily de-tected neutralizing-antibody titers in anHPV-45 pseudovirus neutralization assay,whereas there was no cross-reactivity inthe cLIA using the type-specic HPV-


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18 monoclonal antibody [19]. However,HPV-18 and -45 are very closely related,and signicant protection from HPV-45infection was demonstrated in the GSKclinical trials [20]. Thus, cross-reactivity detected in the neutralization assay ap-pears to correlate with biologically signif-icant cross-protective responses. Titersmeasured in neutralization assays of dif-ferent HPV types cannot alwaysbe directly compared. This is because thepseudoviruspreparations of the different types cancontain different percentages of noninfec-tious capsids, which can compete for neu-tralizing-antibody binding. An assaybasedon a pseudovirus preparation with a highparticle-to-infectivity ratio will result in alower apparent neutralizing-antibody ti-

ter than will an assay based on a prepara-tion with a low particle-to-infectivity ra-tio [15].

QUESTIONS AND

CONTROVERSIES

Although there are a numberof interestingissues involving immunogenicity assess-ment of the HPV VLP vaccine clinical tri-als, 3 predominate at present. First, is thereis one assay that should be considered the

reference standard? National regulatory agencies have endorsed in a de facto fash-ion both the cLIA and the ELISA in thatimmunogenicity bridging data generatedwith them have served as the basis forextending authorization of the Merck andGSK vaccines to groups not evaluated inthe efcacy trials [2]. Both assays haveper-formance characteristics that make themappropriate for high-throughput analy-sis of the large numbers of samples thatare typically assessed in immunogenicity

bridging studies. However, it is importantto note that neither assay has been vali-dated as an immune correlate of protec-tion. In large measure, this lack of vali-dation may result from the exceptionalefcacy of the vaccines. There have beenvery few cases of breakthrough infectionor disease in the VLP vaccinees, perhapstoo few to reasonably assess the assays asimmune correlates. World Health Orga-

nization guidelines for HPV vaccinessuggest that neutralizing assays should beconsidered the reference standard for as-sessing potentially protective antibodiesinduced by the vaccines [21]. Therefore,as breakthrough cases accumulate it may

be preferable to consider using SEAP-NAas the primary assay for evaluating im-mune correlates of protection against in-fection and disease. Although the GSKELISA results correlated very well withSEAP-NA results for both serum and cer-vical mucus specimens collected from arandom sample of young women vacci-nated with Cervarix [18, 22], the ELISAmay somewhat overestimate the protec-tive antibody response (i.e., generatefalse-positive results) if some individuals

predominantly generate a response tononneutralizing epitopes. Conversely, thecLIA may underestimate the protectiveantibody response in some individuals(i.e., generate false-negative results) if theimmunodominant epitopes in certain in-dividuals do not substantially overlap theepitopes recognized by the competingmonoclonal antibody used for that HPVtype in the assay. Determination of animmune correlate of protection might beaccomplished in a nested case-controlstudy evaluating the infrequent vaccineeswith breakthrough infection or diseaseand a randomized subset of the vaccineeswho remain free of infection and disease.Such a study would not overly tax theperformance capacity of the neutraliza-tion assay. Ideally, it would be preferableto evaluate both serum and local cervicalantibody levels, because it is possible thatsome of the rare breakthrough infectionsmay result from unusually poor transu-

dation of serum IgG into the cervicalmucus.

Merck has initiated clinical trials of anonavalent vaccine containing VLPs of anadditional 5 high-risk types (31, 33, 45,52, and 58). The cLIA will be useful inevaluating nonoverlapping type-specicantibody responses. This analysis will ad-dress the question of whether an increasein valency diminishes the antibody re-

sponses to the 4 original VLP types. How-ever, it would also seem important to eval-uate the potential added value of eachadditional VLP type (or the combinationof VLP types) by assessing the ability of the various VLPtypes (or thecombination

of VLP types) to induce cross-neutralizingantibodies against related types by meansof the SEAP-NA. For instance, would avaccine that includes types 16, 31, and 52induce a sufciently high titer of HPV-58cross-neutralizing antibodies that this typecould be omitted from the second-gen-eration vaccine? Similarly, could a second-generation GSK vaccine including HPV-6VLPs potentially protect against bothHPV-6induced and HPV-11inducedgenital warts, analogous to the protection

against HPV-45 observed with the GSKHPV-16/18 vaccine? Insight into thesepossibilities cannot be obtained from acLIA or ELISA.

The second major issue is whether lossof a detectable antibody response to a spe-cic VLP type indicates loss of protectionagainst that type. This question wasbrought to the forefrontby the report that,by 5 years after vaccination, 35% of Gar-dasil-vaccinated women had lost detecta-ble antibodies to HPV-18 VLPs, as mea-sured in Mercks cLIA [23]. In contrast,only 1% of vaccinees had undetectableHPV-16specic cLIA responses at thistime point. Also, 5-fold higher peak titerswere measured for HPV-16 than for HPV-18.There are2 reasonable explanationsforthese ndings. One is that Mercks HPV-18 VLPs are intrinsically less immuno-genic than its HPV-16 VLPs. The HPV-18VLPs do not disassemble under the lowsalt-reducing conditions used to disassem-

ble HPV-6, -11, and -16 VLPs during themanufacturing process [24]. This ndingsuggests that HPV-18 VLPs may havesome structural properties that differfromthe other Merck VLPs and from the GSKHPV-18 VLPs produced in insect cells,which disassemble under similar condi-tions. The second possibility is that theHPV-18 cLIA is simply less sensitive and/or does not detect a substantial fraction


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of the protective antibody responses in asubset of women. Consistent with the lat-ter interpretation is the observation thatprotection against HPV-18 infection doesnot appear to be waning in years 4 and 5after Gardasil vaccination [25]. It is pos-

sible that, in selecting an HPV-18 mono-clonal antibody that does not cross-reactwith the closely related type HPV-45, anantibody was chosen that is less represen-tative of the polyclonal antibody responseto HPV-18 VLP vaccination, comparedwith the degree to which the HPV-16monoclonal antibody is representative of the polyclonal antibody response to HPV-16 VLPs. It may be possible to distinguishbetween these alternatives by conductingHPV-16specic and HPV-18specic

SEAP-NAs, which measure neutralizingantibodies regardless of where they bindon the capsid. However, the caveat thatSEAP-NA titers are inuenced by thepresence of noninfectious interferingparticles would have to be taken into ac-count. Interestingly, Cervarix inducedmean serum titers of antibody to HPV-18 and -16 that differed by only 2-fold,as accessed by GSKs ELISA and theSEAP-NA, and titers remained in the de-tectable range for both assays over the 4 years women were examined after vac-cination [22, 26]. A competitive ELISAbased on J4, the same monoclonal anti-body used in Mercks cLIA, was less sen-sitive than GSKs direct ELISA or theSEAP-NA [18].

The third major question is whether avaccine that is more immunogenic is nec-essarily a better vaccine. This issue willlikely receive increased attention once theresults of a GSK clinical trial directly com-

paring the immunogenicity of Cervarixand Gardasil are announced [27]. If othercharacteristics of 2 vaccines are compa-rable, in general one would favor the vac-cine that is more immunogenic. However,because no immune correlate of protec-tion has been established for these vac-cines, we do not know the minimal levelsof antibodies needed for solid protectionand therefore do not knowwhether higher

levels would induce longerprotection.Forboth vaccines, the mean VLP antibody ti-ters reached a plateau by 2 years after vac-cination, and there has been no sign of waning protection 45 years after vacci-nation [20, 28, 29]. Therefore, both vac-cines might be sufciently immunogen-ic to provide long-term or even lifetimetype-specic protection. Also, virologistsgenerally consider antibody titerson a log-arithmic scale, and differences in titers of a few fold might be considered of ques-tionable biological signicance.

Because cross-neutralizing titers arelower than vaccine typespecic titers,there is a greater likelihood that they may wane to nonprotective levels over time.One could imagine that subtle differences

in the folding of the conformation-de-pendent neutralizing L1 epitopes underthe inuence of the chaperones in yeastand insect cells might inuence the dis-play of the immunosubdominant cross-neutralization epitopes by the 2 vaccines.These differences could inuence the ti-ters of antibodies to nonvaccine types in-duced. Therefore, it will be important toalso evaluate the titers of neutralizing an-tibodies to nonvaccine types. There are anumber of HPV-16related types and a

number of HPV-18related types that areeach associated with 1%5% of cervicalcancers, and some of these types mightbe preferentially neutralized by the an-tibodies induced by 1 of the 2 vaccines.However, HPV-16 and -18 are only dis-tantly related to HPV-6 and -11, whichmakes it unlikely that Cervarix, as cur-rently constituted, would induce cross-neutralizing antibodies to these genitalwartassociated types.

CONCLUSION

There is a strong rationale for focusing onthe antibody responses to the HPV VLPvaccines. Each of the 3 types of antibody assays discussed here will be useful in theevaluation of current and future prophy-lactic HPV vaccines. However, it will beimportant to remain cognizant of theunique strengths and limitations of each

assay when interpreting the immunoge-nicity results of clinical trials.

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Schiller JT - 09