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1 MAJOR ARTICLE Comprison of the Immunogenicity nd Rectogenicity of Cervrix nd Grdsil Humn Ppillomvirus Vccines in HIV-Infected Adults: A Rndomized, Double-Blind Clinicl Tril Lrs Toft, 1 Merete Storgrd, 1 Mrtin Müller, 2 Peter Sehr, 3 Jesper Bonde, 4,5 Mrtin Tolstrup, 1 Lrs Østergrd, 1 nd Ole S. Søgrd 1 1 Deprtment of Infectious Diseses, Arhus University Hospitl, Arhus, Denmrk; 2 Reserch Progrm Infection nd Cncer, Germn Cncer Reserch Center; 3 Chemicl Biology Core Fcility, Europen Moleculr Biology Lbortory, Heidelberg, Germny; nd 4 Deprtment of Pthology nd 5 Clinicl Reserch Centre, Copenhgen University Hospitl Hvidovre, Copenhgen, Denmrk Bckground. We compred the immunogenicity nd rectogenicity of Cervrix or Grdsil humn ppillomvirus (HPV) vccines in dults infected with the humn immunodeficiency virus (HIV). Methods. This ws double-blind, controlled tril rndomizing HIV- dults to receive 3 doses of Cervrix or Grdsil t 0, 1.5, nd 6 months. Immunogenicity ws evluted for up to 12 months. Neutrlizing nti HPV-16/18 ntibodies were mesured by pseudovirion-bsed neutrliztion ssy. Lbortory tests nd diry crds were used for sfety ssessment. The HPV-DNA sttus of the prticipnts ws determined before nd fter immuniztion. Results. Ninety-two prticipnts were included in the study. Anti HPV-18 ntibody titers were higher in the Cervrix group compred with the Grdsil group t 7 nd 12 months. No significnt differences in nti HPV-16 ntibody titers were found mong vccine groups. Among Cervrix vccinees, women hd higher nti HPV-16/ 18 ntibody titers compred to men. No sex-specific differences in ntibody titers were found in the Grdsil group. Mild injection site rections were more common in the Cervrix group thn in the Grdsil group (91.1% vs 69.6%; P =.02). No serious dverse events occurred. Conclusions. Both vccines were immunogenic nd well tolerted. Compred with Grdsil, Cervrix induced superior vccine responses mong HIV-infected women, wheres in HIV-infected men the difference in immunogenicity ws less pronounced. Clinicl Trils Registrtion. NCT Keywords. HPV vccines; HIV; neutrlizing ntibodies; serology; sfety; Grdsil; Cervrix. Persistent infection with oncogenic humn ppillomvirus (HPV) genotypes cuse pproximtely cncers worldwide every yer, including cervicl cncer, nl cncer, vulvr nd vginl cncers, penile cncer, nd certin orophryngel cncers [1 3]. Most Received 8 October 2013; ccepted 11 November 2013; electroniclly published 23 November Correspondence: Lrs Toft, MD, Deprtment of Infectious Diseses, Arhus University Hospitl, Skejby, 8200 Arhus, Denmrk (lrsnise@rm.dk). The Journl of Infectious Diseses 2014;209: The Author Published by Oxford University Press on behlf of the Infectious Diseses Society of Americ. All rights reserved. For Permissions, plese e-mil: journls.permissions@oup.com. DOI: /infdis/jit657 HPV-ssocited cncers re cused by genotypes 16 nd 18 [4, 5] nd the 2 commercilly vilble HPV vccines, Cervrix (GlxoSmithKline) nd Grdsil (Merck), both protect ginst these oncogenic HPV types. Grdsil dditionlly covers HPV-6 nd -11, the mjor custive gents of genitl wrts [6 8]. People infected with humn immunodeficiency virus (HIV) hve highly incresed risk of developing HPVssocited cncers [9]. Despite the incresing use of highly ctive ntiretrovirl therpy (HAART) s routine tretment in HIV-infected individuls nd estblishment of screening progrms for precncerous cervicl lesions, the incidence of cervicl cncer mong HIVinfected women hs not declined in recent yers [10]. Additionlly, the incidence of nl cncer is stedily HPV Vccintion of HIV-Infected Adults JID 2014:209 (15 April) 1165

2 incresing in HIV-infected men nd women [11 13] nd some countries, including the United Sttes nd Austrli, now recommend routine HPV vccintion of HIV-infected individuls. However, little is known bout immunity induced by HPV vccintion in HIV-infected dults. Both HPV vccines re bsed on virus-like prticles using type-specific HPV L1 cpsid protein s vccine ntigen. Cervrix is formulted with n luminum slt nd Toll-like receptor (TLR) 4 gonist bsed djuvnt system clled AS04, nd Grdsil is formulted with morphous luminum hydroxyphosphte sulfte s djuvnt [7, 14]. The vccines re best dministered before sexul debut, nd vccintion does not cler existing HPV infections [15]. However, recent trils show tht dults lso benefit from vccintion if they hve no preexisting infection with vccine-type HPVs [16, 17] nd tht vccintion reduces the risk of subsequent HPV-ssocited disese mong people treted surgiclly for precncerous cervicl nd nl lesions [18, 19]. The exct mechnisms of vccine-induced protection hve not been estblished, but neutrlizing ntibodies re believed to be of crucil importnce [20 22], nd higher ntibody titers following immuniztion my be surrogte mrker for prolonged protection [23, 24]. In helthy women, Cervrix ws shown to induce higher ntibody titers ginst HPV-16 nd -18 compred to Grdsil [23, 24]. Both vccines re reportedly sfe nd immunogenic in HIVinfected individuls with high seroconversion rtes, lbeit lower ntibody titers thn in ge-mtched HIV- persons [25 29]. To our knowledge, no studies hve reported comprtive immunogenicity nd sfety dt on Cervrix nd Grdsil in HIV-infected persons, nd no efficcy studies hve been published using either vccine. We nd others hve shown tht TLR9-djuvnted vccines induce superior ntibody responses in HIV-infected individuls [30, 31]. Thus, the TLR4-directed djuvnt in Cervrix my be prticulrly useful in this popultion, but sex-specific differences in TLR signling nd ltered TLR4 responsiveness in HIV-infected individuls my ffect vccine responses [32, 33]. This study ws undertken to compre the immunogenicity nd rectogenicity of Cervrix nd Grdsil in HIV-infected dult men nd women. METHODS Study Design This ws n investigtor-initited, rndomized, double-blind, hed-to-hed tril rndomizing persons with HIV to vccintion with either Cervrix or Grdsil. The study protocol ws pproved by the Dnish Medicines Agency, the Regionl Ethicl Committee, nd the Dnish Dt Protection Agency. The study ws monitored for regultory complince nd dt qulity by the Division of Good Clinicl Prctice t Arhus University Hospitl. Setting nd Prticipnts The study ws conducted t the Deprtment of Infectious Diseses, Arhus University Hospitl, Denmrk. HIV- ptients seen t the outptient clinic during 2011 were invited by letter to prticipte. Consenting HIV-sero volunteers 18 yers or older were eligible for enrollment. We excluded individuls who (1) hd previously been vccinted ginst HPV, (2) hd history of mlignncy or utoimmune disese, (3) hd received systemic immunosuppressive tretment, (4) hd previously hd n llergic rection to vccintion, (5) were pregnnt, brestfeeding, or unwilling to use relible contrception methods for the durtion of the tril, nd/or (6) hd n HIV RNA level of >200 copies/ml if receiving ntiretrovirl tretment. Rndomiztion nd Intervention Prticipnts were strtified ccording to use of HAART nd sex nd therefter rndomized 1:1 in blocks of 4 to receive Cervrix or Grdsil. Rndom lloction sequences were computergenerted by the hospitl phrmcy. Prticipnts were ssigned their study identifiction number ccording to the chronologicl order in which they were enrolled. Prticipnts nd investigtors were msked to the ssigned vccine throughout the study. Prefilled vccines were provided by the hospitl phrmcy. Prticipnts received vccintion with either Cervrix or Grdsil t months 0, 1.5, nd 6 nd they were seen t month 7 nd 12 for immunogenicity nd sfety follow-up. Trined study nurses dministered ll vccines by intrmusculr injection into the left or right rm. Before ny tril procedures, blood smples were collected for immunogenicity mesurements nd sfety lbortory tests, including HIV RNA, CD4 + cell count, hemoglobin, erythrocyte volume frction, white blood cell count, lkline phosphtse, lnine minotrnsferse, nd cretinine prmeters. Assessment of Neutrlizing Antibodies Serum smples were het-inctivted for 60 minutes t 56 C nd stored t 80 C before shipment for blinded nlysis t the joint Chemicl Biology Core Fcility of the Germn Cncer Reserch Center nd the Europen Moleculr Biology Lbortory, Heidelberg, Germny, using pseudovirion-bsed neutrliztion ssy (PBNA). Pseudovirions (PSVs) were prepred nd closely qulity-monitored s previously described [34 36]. All liquid hndling nd plte reding ws done using utomted devices nd plte reders (PerkinElmer) [37]. Serum smples were diluted 10 times in 3-fold increments in complete Dulbecco s modified Egle medium cell culture medium in 384-well polypropylene pltes to chieve finl serum dilution in the PBNA of 1:40 to 1: Two microliters of diluted serum nd 18 µl PSVs diluted in cell culture medium ws mixed in 384-well ssy pltes. After 1 hour of incubtion t room temperture, 20 µl HeL T cells ws dded t finl density of 1500 cells/well nd 1166 JID 2014:209 (15 April) Toft et l

3 incubted for 2 dys t 37 C/5% CO 2. Finlly, 20 µl/well glow substrte solution (PJK) ws dded nd the luminescence red in n Envision plte reder. Triplicte pltes originting from the sme serum dilution were used for ech HPV type. Inhibition (%) ws clculted by normliztion of the luminescence to the men of the control wells on ech plte. The medin of the triplicte vlues ws used for curve fitting, nd the clcultion of the effective dilution giving 50% inhibition vlue for ech serum ws performed in ActivityBse (IDBS). Assessment of HPV DNA Sttus Anl nd cervicl swbs for HPV DNA testing were collected t bseline nd t month 7 by inserting Regulr FLOQSwb (Copn Dignostics) into the nl cnl or the cervix uteri. Swbs were plced into tubes contining UTM-RT trnsport medium (Copn Dignostics). One milliliter of ech smple ws spun down (5 minutes t rpm), superntnts were removed, nd cell pellets were resuspended in 180 µl phosphtebuffered sline nd 20 µl Proteinse K (Roche Dignostics). Smples were mixed nd incubted for 1 hour t 56 C nd 1 hour t 90 C. HPV DNA ws purified using MgNA Pure LC 96 nd MgNA Pure LC Totl Nucleic Acid Isoltion Kit (Roche Dignostics, Switzerlnd). Five microliters of purified DNA ws used for the PCR mplifiction in concordnce with the mnufcturer s specifictions with subsequent microrry detection for ny of 35 defined HPV genotypes included in the Genomic CLART HPV2 Genotyping micro rry (Genomic). Assessment of Sfety Prticipnts were observed for 30 minutes fter ech immuniztion to evlute immedite dverse events. Solicited dverse events occurring during the first 4 dys fter ech immuniztion (injection site pin, swelling, erythem, fever, hedche, nuse, mylgi, rthrlgi nd rsh) nd other signs of illness nd/or chnges in mediction occurring within 15 dys fter immuniztion were recorded on diry crds. Solicited nd unsolicited dverse events, s well s lbortory tests, were grded ccording to the common toxicity criteri version 2.0. All solicited locl (injection site) nd influenz-like (fever, rthrlgi, chills, nd ftigue) rections were considered cuslly relted to vccintion. Other dverse events were evluted by the primry investigtor nd grded s unlikely or probbly relted to the study vccines. Sttisticl Anlysis The primry objective ws to compre the serologicl nti HPV-16 nd -18 neutrlizing ntibody titers mong vccine groups t month 7. Secondry objectives were to compre ntibody titers t month 12 nd to strtify vccine responses by bseline HPV serology mong HPV DNA prticipnts. We powered our tril to detect 35% difference in men geometric men titers (GMTs) between the 2 vccine groups t month 7. Setting type I error probbility (α) to.05 (2-sided), type II error probbility (β) to.10(power=1 β =.90), nd ssuming stndrd devition of 50% of the men GMT, we found tht 43 ptients per group were required to detect this difference. Continuous vribles were summrized using medins with interqurtile rnges. Dichotomous nd ctegoricl vribles were summrized using numbers nd percentges. We conducted per-protocol nlyses of ll rndomized subjects for whom immunogenicity dt were vilble t bseline nd subsequent timepoint. We clculted crude GMTs on the primry endpoints. An djusted estimte ws obtined using multivrite liner regression tht included the following vribles: current smoker, bseline CD4 + cell count, current use of HAART (yes or no), body mss index, sex, ge, type-specific bseline HPV ntibody titer, nd HPV DNA sttus. Finlly, s the AS04 djuvnt my hve differentil effects in men nd women, we conducted the bove-mentioned nlysis strtified on sex. Incident nd persistent HPV-16 nd -18 infection ws summrized ccording to vccine group. We used the χ 2 test to compre GMTs nd the proportion of dverse events between the vccine groups. We used 2-wy t test to nlyze for bseline group differences nd temporl chnges in CD4 + cell count nd log-trnsformed HIV RNA from bseline, nd performed subnlyses of the 2 vribles strtified ccording to HAART use. We used Stt softwre, version 12.2 (SttCorp), for sttisticl nlyses. RESULTS Study Popultion Of the 512 individuls invited to prticipte, 121 signed up nd were screened for eligibility, nd 92 of these were subsequently enrolled in the tril between 10 August nd 2 November 2011 (Figure 1). The tril ended on 3 December 2012 when the lst prticipnt ws seen for follow-up. Two prticipnts did not complete the 3-dose vccintion series: 1 withdrew her consent nd 1 ws inppropritely enrolled (medicl condition incomptible with intrmusculr injections). One ws lost to followup nd missed the 12-month visit. The Grdsil group hd higher proportion of smokers thn the Cervrix group (16 vs 6; P =.026). The 2 groups were similr in immune sttus nd ll other bseline chrcteristics t the time of inclusion in the study (Tble 1). Bseline chrcteristics did not differ significntly between men nd women. Immunogenicity Primry endpoints re shown in Figure 2. Both vccines incresed HPV-16 GMTs from bseline to 7 nd 12 months, nd no significnt differences in nti-hpv-16 ntibody titers were found between vccine groups. Anti-HPV-18 GMTs were HPV Vccintion of HIV-Infected Adults JID 2014:209 (15 April) 1167

4 Figure 1. Disposition of study prticipnts. Abbrevitions: HAART, highly ctive ntiretrovirl therpy; HIV, humn immunodeficiency virus. higher in the Cervrix group compred with the Grdsil group t 7 nd 12 months. The GMT rtio ws 4.31 t 7 months (95% confidence intervl [CI], ) nd 4.15 t 12 months (95% CI, ). Adjustment for potentil confounders (current smoker, bseline CD4 + cell count, current use of HAART, body mss index, sex, ge, type-specific bseline HPV ntibody titer, nd HPV DNA sttus) did not ffect the results. An nlysis of ntibody titers ccording to bseline serosttus nd nogenitl DNA sttus is shown in Tble 2. Agin, we found no significnt differences in nti HPV-16 ntibody titers between vccine groups. Among those sero nd HPV-18 DNA t bseline, nti HPV-18 ntibody titers were higher in the Cervrix group thn in the Grdsil group. The GMT rtio ws 7.60 t 7 months (95% CI, ) nd 8.90 t 12 months (95% CI, ). Results of n nlysis strtifying ntibody responses by sex re shown in Figure 3. Among prticipnts vccinted with Cervrix, women hd 3.16-fold (95% CI, to 6.40-fold) higher nti-hpv-16 ntibody titers t 7 months, nd 4.43-fold (95% CI, to 10.6-fold) higher nti HPV-16 ntibody titers t 12 months when compred to men. Anti HPV-18 ntibody titers in the Cervrix group were 2.07-fold (95% CI,.88- to 4.85-fold) nd 3.57-fold (95% CI, to 9.34-fold) higher mong femle compred to mle prticipnts. We found no differences in ntibody titers between men nd women in the Grdsil group JID 2014:209 (15 April) Toft et l

5 Tble 1. Bseline Chrcteristics of the Study Popultion Chrcteristic Cervrix (n = 45) Grdsil (n = 46) Sex Mle 30 (66.7) 31 (67.4) Femle 15 (33.3) 15 (32.6) Rce White 38 (84.4) 37 (80.4) Other 7 (15.6) 9 (19.6) Age, y, medin (IQR) 47.0 ( ) 44.5 ( ) Body mss index, kg/m2, 23.8 ( ) 24.1 ( ) medin (IQR) Current smoker 6 (13.3) 16 (34.8) CD4 + cell count, cells/µl, 600 ( ) 585 ( ) medin (IQR) Undergoing HAART Yes 40 (88.9) 40 (87.0) No 5 (11.1) 6 (13.0) HIV RNA level, log 10 copies/ml, medin (IQR) In ptients receiving HAART In ptients not receiving HAART 4.97 ( ) 4.58 ( ) HPV serosttus nd HPV DNA sttus HPV-16 Sero, HPV DNA 22 (51.2) 19 (44.2) Sero, HPV DNA 0 2 (4.6) Sero, HPV DNA 17 (39.5) 15 (34.9) Sero, HPV DNA 4 (9.3) 7 (16.3) HPV-18 Sero, HPV DNA 22 (51.2) 18 (41.9) Sero, HPV DNA 0 1 (2.3) Sero, HPV DNA 19 (44.2) 20 (46.5) Sero, HPV DNA 2 (4.6) 4 (9.3) HPV-16 nd -18 Both sero, 16 (37.2) 15 (34.9) HPV DNA Both sero, HPV 0 0 DNA Both sero, HPV DNA 13 (30.2) 12 (27.9) Both sero, HPV DNA 1 (2.3) 2 (4.6) Dt re presented s No. (%) of ptients unless otherwise indicted. Abbrevitions: HAART, highly ctive ntiretrovirl therpy; HIV, humn immunodeficiency virus; HPV, humn ppillomvirus; IQR, interqurtile rnge. Two prticipnts in the Cervrix group nd 3 prticipnts in the Grdsil group with inconclusive bseline HPV DNA sttus re not included. Figure 2. Humn ppillomvirus (HPV) serotype-specific neutrlizing ntibody geometric men titers (GMTs). HPV serotype-specific neutrlizing ntibody GMTs were computed using the log 10 trnsformtion of ED 50 vlues for nti HPV-16 nd HPV-18 serum neutrlizing ntibodies s mesured by pseudovirion neutrliztion ssy t months 0, 1.5, 6, 7, nd 12. **P <.001, ***P < One study prticipnt from the Cervrix group withdrew fter the second visit, nd 1 study prticipnt from the Grdsil group ws lost to follow-up fter the fourth visit. Abbrevition: ED 50, effective dilution giving 50% inhibition. Grdsil group t 7 nd 12 months, nd nti HPV-18 GMTs were 3.93-fold (95% CI, to 15.2-fold) nd 6.02-fold (1.05- to 14.6-fold) higher in the Cervrix compred to the Grdsil group t 7 nd 12 months, respectively. Among mle prticipnts, We found no significnt differences in nti-hpv-16 GMTs between the vccine groups. Anti- HPV-18 titers were 4.54-fold (95% CI, to 9.59-fold) nd 3.55-fold (95% CI, to 8.53-fold) higher in the Cervrix group thn in the Grdsil group t 7 nd 12 months, respectively. Adjustment for potentil confounders (current smoker, bseline CD4 + cell count, current use of HAART, body mss index, sex, ge, type-specific bseline HPV ntibody titer, nd HPV-DNA sttus) did not ffect sex-specific results. Among femle prticipnts, nti-hpv-16 GMTs were fold (95% CI,.76-fold to 11.6-fold) nd 3.90-fold (95% CI, to 14.6-fold) higher in the Cervrix compred to the Anogenitl HPV DNA Detection t 7 Months Eighty prticipnts hd vilble nogenitl HPV DNA detection t both bseline nd 7 months. Tble 3 summrizes HPV Vccintion of HIV-Infected Adults JID 2014:209 (15 April) 1169

6 Tble 2. Geometric Men Antibody Titers (GMTs) nd GMT Rtios According to Bseline Seropositivity nd Anogenitl Humn Ppillomvirus DNA Detection HPV Type HPV-16 HPV-18 incident nd persistent HPV-16 nd -18 infection. All incident HPV-16/18 infections occurred mong mle prticipnts. Sfety No serious dverse events were detected in this study. Both vccines were generlly well tolerted nd very few mild systemic rections were observed (Tble 4). Injection site rections were more common in the Cervrix group thn in the Grdsil group (91.1% vs 69.6%; P =.02). No sex-relted differences in injection site rections were detected (dt not shown). DISCUSSION Bseline HPV Serosttus nd DNA Sttus Sero, HPV DNA Sero, HPV DNA Sero, HPV DNA Sero, HPV-DNA Sero, HPV DNA Sero, HPV DNA Sero, HPV DNA Sero, HPV-DNA Month Cervrix In the present study, we found tht Cervrix ws significntly more immunogenic thn Grdsil in HIV-infected women. However, this difference in immunogenicity ws less pronounced in HIV-infected men where the 2 vccines induced Grdsil No. GMT (95% CI) No. GMT (95% CI) similr ntibody responses ginst HPV-16 but higher nti HPV-18 ntibodies for Cervrix thn Grdsil. Mild injection site rections were more common in the Cervrix group, but the overll rectogenicity of both vccines ws cceptble for both ptients nd physicins. Collectively, our findings suggest tht Cervrix is superior to Grdsil in terms of inducing protective immunity ginst oncogenic HPV-16 nd HPV-18 infection in HIV-infected women. However, whether this difference trnsltes into enhnced or prolonged protection ginst cervicl cncer is still unknown. To our knowledge, this is the first clinicl tril compring immunogenicity dt of Cervrix nd Grdsil in HIV-infected individuls. One strength of the study ws tht ll prticipnts nd investigtors were msked to the ssigned test drugs throughout the tril, leding to unbised sfety nd immunogenicity mesurements. The study ws dequtely powered to detect 35% difference in GMTs between the vccine groups, GMT Rtio (95% CI) ( ) ( ) 1.26 ( ) ( ) ( ) 1.50 ( ) ( ) ( ) 1.10 ( ) ( ) ( ) 1.59 ( ) ( ) ( ) 2.15 ( ) ( ) b ( ) ( ( ) ( ) 7.60 ( ) ( ) ( ) 8.90 ( ) (95 337) ( ) 0.68 ( ) ( ) ( ) 2.34 ( ) ( ) ( ) 2.02 ( ) GMTs were computed using the log 10 trnsformtion of titers. GMT rtio is Cervrix GMT divided by Grdsil GMT. Two prticipnts in the Cervrix group nd 3 prticipnts in the Grdsil group with inconclusive bseline HPV DNA sttus were not included in the nlysis. Abbrevitions: CI, confidence intervl; GMT, geometric men titer; HPV, humn ppillomvirus. CIs not reported for subgroups with <5 prticipnts. b GMT rtios not clculted if 1 or more subgroups hd <5 prticipnts JID 2014:209 (15 April) Toft et l

7 Figure 3. Humn ppillomvirus (HPV) serotype-specific neutrlizing ntibody geometric men titers (GMTs) strtified ccording to sex. GMTs were computed using the log 10 trnsformtion of ED 50 vlues for nti HPV-16 nd nti HPV-18 serum neutrlizing ntibodies mesured by pseudovirion neutrliztion ssy t months 0. 7, nd 12. *P <.05; **P <.001. One womn from the Cervrix group withdrew before month 7, nd 1 mn from the Grdsil group ws lost to follow-up before month 12. Abbrevition: ED 50, effective dilution giving 50% inhibition. nd it is unlikely tht the observed differences in ntibody titers were due to chnce. The rndomiztion ws considered successful becuse the 2 vccine groups were comprble in size Tble 3. Week 28 Humn Ppillomvirus DNA t 7 Months, According to Bseline Results Result t 7 mo Cervrix, Proportion (%) of Prticipnts Grdsil, Proportion (%) of Prticipnts HPV-16 DNA detected t 7 mo All 4/39 (10) 8/41 (20) Persistent infection 2/4 (50) 7/9 (78) Incident infection 2/35 (6) 1/32 (3) HPV-18 DNA detected t 7 mo All 3/39 (8) 4/41 (10) Persistent infection 1/2 (50) 3/5 (60) Incident infection 2/38 (5) 1/36 (3) Results presented for those with known bseline HPV DNA sttus. Six HPV DNA tests were either missing or inconclusive t 7 months. Persistent infection is the number with DNA detected t 7 months mong those with DNA of the sme type detected t study entry. Incident infection is the number with DNA detected t 7 months mong those with DNA of tht type not detected t entry. Abbrevition: HPV, humn ppillomvirus. nd most bseline chrcteristics. The study ws conducted t single tril site, minimizing vritions in tril procedures nd blood smple nlyses. The study lso hd severl limittions. First, the study ws not powered to compre the 2 vccines on clinicl endpoints nd, with only 15 femle subjects per vccine group, it ws not powered to detect sex-relted differences. Serologic correltes hve not been estblished for protection ginst HPV infection, nd the significnce of the mgnitude of neutrlizing ntibody titers remins elusive. We did not hve long-term follow-up nd cnnot predict decreses in ntibody responses over time. Finlly, we hd no HIV- controls nd thus we cnnot directly compre immunogenicity of the HPV vccines in HIVinfected individuls to tht in the generl popultion. A plusible explntion for the difference in immunogenicity between Grdsil nd Cervrix could be the vccine djuvnts, Cervrix being formulted with the highly immunogenic TLR4-stimulting AS04 djuvnt system. The overll difference in ntibody titers ws significnt for HPV-18 but not for HPV-16, perhps owing to the fct tht Grdsil contins twice s much HPV-16 ntigen s Cervrix (40 µg vs 20 µg, respectively) wheres both vccines contin 20 µg of HPV-18 ntigen. The PBNA used in this study hs incresed sensitivity compred to the trditionl neutrliztion ssys [37]. Accordingly, we report higher titers thn previously found mong helthy HPV Vccintion of HIV-Infected Adults JID 2014:209 (15 April) 1171

8 Tble 4. Injection-Relted Adverse Events Adverse Event First Vccintion Second Vccintion Third Vccintion No. (%) of Ptients No. (%) of Ptients No. (%) of Ptients Cervrix (n = 45) Grdsil (n = 46) P Vlue Cervrix (n = 45) Grdsil (n = 46) P Vlue Cervrix (n = 44) Grdsil (n = 46) P Vlue At lest 1 dverse event 38 (84.4) 25 (54.3) (52.3) 13 (28.2) (61.4) 18 (39.1).04 Injection site pin 38 (84.4) 15 (32.6) < (50.0) 9 (19.6) (56.8) 17 (37.0) ns Injection site swelling 10 (22.2) 3 (6.5).04 9 (20.5) 2 (4.3).03 9 (20.5) 4 (8.7) ns Injection site erythem 4 (8.9) 5 (10.9) ns 7 (15.9) 2 (4.3) ns 8 (18.2) 2 (4.3) <.05 Influenz-like symptoms 6 (13.3) 4 (8.7) ns 0 2 (4.3) ns 0 0 Hedche 1 (2.2) 3 (6.5) ns 0 2 (4.3) ns 0 0 Nuse 1 (2.2) 1 (2.2) ns ns Solicited dverse events occurring during the first 4 dys fter ech immuniztion. P Vlues for At lest 1 dverse event nd Injection site pin were clculted using χ 2 test. Fischer s exct test ws used for clculting the P Vlues in Injection site swelling, Injection site erythem, influenze-like symptoms, hedche nd nuse due to smll numbers of events. Abbrevition: ns, not significnt (P vlue.05). Influenz-like symptoms included pyrexi, rthrlgi, chills, nd ftigue. women [23]. The Cervrix/Grdsil ntibody rtios mong HIV-infected women were comprble to those reported by Einstein [23]. Until universl stndrds for HPV serology re implemented, bsolute comprison of neutrlizing titers obtined in different studies remins difficult. We cnnot compre the ntibody titers to the vilble immunogenicity dt of Grdsil in HIV-infected individuls. We specificlly tested for ntibodies with neutrlizing ctivity giving functionl titer, wheres the previously published studies hve used either competitive Luminex-bsed immunossy, presenting dt in milli-merck units per milliliter [25 28], or n enzymelinked immunosorbent ssy [29]. Sex differences in humorl responses to virl vccintions in prticulr hve been reported in numerous studies nd severl potentil mechnistic explntions hve been hypothesized, tht is, gondl hormone levels nd yet-undefined ntigenspecific interctions with the immune system [38, 39]. It ws surprising, however, tht the observed sex-relted differences in ntibody titers were only found mong those vccinted with Cervrix. Some dt suggest tht AS04-djuvnted vccines could hve incresed effect on women; for exmple, AS04- djuvnted genitl herpes vccintion reduced the rte of cquisition of genitl herpes in women but hd no effect in men [40]. AS04 contins TLR4-stimulting djuvnt (monophosphoryl lipid A), nd TLR4 signling pthwys re ltered by HIV infection [33]. Sex-specific differences in TLR-induced cytokine production in HIV-infected individuls hve previously been described, women showing higher responses thn men [41]. Furthermore, TLR4 expression nd responsiveness is incresed following dministrtion of exogenous estrdiol in rodents [32], thus suggesting potentil mechnisms behind the observed differences in our study. Also, we cnnot rule out tht the observed sex differences my be prtilly relted to HIV infection. Severl studies hve indicted tht men nd women respond differently to both HIV infection nd ntiretrovirl therpy [42, 43]. Our study emphsizes the need to crefully design nd interpret clinicl studies with enhnced focus on differences between mle nd femle prticipnts. Ultimtely, this study supports the estblishment of efficcy trils to determine the impct of routine HPV vccintion on the incidence of HPV-relted cncers in HIV-infected dults, s supplement to cervicl nd nl cncer screening progrms. Notes Acknowledgments. We thnk the prticipnts for their involvement in the tril. We lso thnk the study nurses, Iben Loftheim nd Inge Arbs, for their importnt job vccinting the prticipnts, nd lb technicin Lene Svinth-Jøhnke for the excellent job hndling ll the smples. Finncil support. This work ws supported by Arhus University, Henrik Henriksen s Foundtion, The Hede Nielsen Fmily Foundtion, Ase nd Ejnr Dnielsen s Foundtion, Jørgen Holm nd Wife s Foundtion, Lykfeldt nd Wife s Foundtion, nd the Dnish Medicl Assocition. Potentil conflicts of interest. All uthors: No reported conflicts. All uthors hve submitted the ICMJE Form for Disclosure of Potentil Conflicts of Interest. Conflicts tht the editors consider relevnt to the content of the mnuscript hve been disclosed. References 1. Munoz N, Cstellsgue X, de Gonzlez AB, et l. Chpter 1: HPV in the etiology of humn cncer. Vccine 2006; 24(suppl 3):S3/ Wlboomers JM, Jcobs MV, Mnos MM, et l. Humn ppillomvirus is necessry cuse of invsive cervicl cncer worldwide. J Pthol 1999; 189: Formn D, de Mrtel C, Lcey CJ, et l. Globl burden of humn ppillomvirus nd relted diseses. Vccine 2012; 30(suppl 5):F Hoots BE, Plefsky JM, Piment JM, Smith JS. Humn ppillomvirus type distribution in nl cncer nd nl intrepithelil lesions. Int J Cncer 2009; 124: JID 2014:209 (15 April) Toft et l

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