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  Vaccine 23 (2005) 3634–3641 Immune responses induced by lower airway mucosal immunisationwith a human papillomavirus type 16 virus-like particle vaccine Denise Nardelli-Haefliger a , ∗ , Floriana Lurati b , 2 , Daniel Wirthner a , 3 , Franc¸ois Spertini b , 2 ,John T. Schiller c , 4 , Douglas R. Lowy c , 4 , Franc¸oise Ponci a , 1 , Pierre De Grandi a , 3 a  Department of Gynecology, Centre Hospitalier Universitaire Vaudois, CH-1011 Lausanne, Switzerland  b  Division of Immunology and Allergy, Centre Hospitalier Universitaire Vaudois, CH-1011 Lausanne, Switzerland  c  Laboratory of Cellular Oncology, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA Received 19 July 2004; received in revised form 20 December 2004; accepted 24 February 2005Available online 14 March 2005 Abstract Cervical cancer results from cervical infection by human papillomaviruses (HPV), especially HPV16. Previous studies have shown thatintramuscular vaccination of women with an HPV16 virus-like particle (VLP) vaccine induced a strong IgG response and protected againstgenital HPV16 infection. However, an alternative route of administration that avoids parenteral injection while inducing mucosal immunitymight facilitate vaccine implementation in some settings, and partially overcome the substantial variation in HPV16 antibodies at the cervixseen in ovulating women. In this study, women were vaccinated with escalating doses of HPV16L1 VLPs via nasal nebulisation, bronchialaerosolisation, or a combination of intramuscular and aerosol vaccination. The alternative routes of vaccination were well tolerated and manyof the volunteers who received aerosol vaccinations exhibited serum antibody titers that were comparable to those induced by intramuscularvaccination. A mucosal immune response was induced by aerosol vaccination as demonstrated by the induction of anti-HPV16 VLP IgAsecretingcellsinPBMCandSIgAinsecretions.OurdatasuggestthataerosoladministrationofHPVVLPsmayrepresentapotentialalternativeto parenteral injection.© 2005 Elsevier Ltd. All rights reserved. Keywords:  Aerosol vaccination; Virus like particles; Human papillomavirus; Cervical cancer 1. Introduction Cervical cancer is the second leading cause of cancerdeaths in women world-wide, and virtually all of these tu-mours are attributable to infection with a sub-set of humanpapillomavirues (HPV), of which HPV16 is found most fre-quently[1,2].AneffectivevaccineagainsttheseHPVswould therefore be expected to have a dramatic impact on the inci-denceofthiscanceranditsprecursorlesions,aswellasonthe ∗ Corresponding author. Tel.: +41 21 314 40 81; fax: +41 21 314 40 95.  E-mail address:  dnardell@hospvd.ch (D. Nardelli-Haefliger). 1 Fax: +41 21 314 40 95. 2 Fax: +41 21 314 07 91. 3 Fax: +41 21 314 34 34. 4 Fax: +1 301 480 53 22. less common tumours attributable to these viruses (reviewedin[3]).Theleadingcandidateisasub-unitprophylacticHPV virus-like particle (VLP) vaccine (reviewed by [4,5]).Early phase clinical trials of HPV VLP vaccines havefound that a series of three intramuscular (i.m.) immunisa-tions are well tolerated and can be highly immunogenic evenwithoutadjuvant[6–8].AproofofprincipleefficacytrialhasshownthatwomenfullyvaccinatedwithanHPV16VLPvac-cine were highly protected against genital mucosal infectionby this viral type [9]. However, the requirement for multipleinjections for a vaccine whose anticipated target populationwill be older than those of most vaccines may represent asubstantial hurdle for widespread implementation, particu-larly in the developing world, which accounts for more thanthree-quarters of the world-wide cases of cervical cancer [2].In addition, it has recently been found that although i.m. 0264-410X/$ – see front matter © 2005 Elsevier Ltd. All rights reserved.doi:10.1016/j.vaccine.2005.02.019   D. Nardelli-Haefliger et al. / Vaccine 23 (2005) 3634–3641  3635 VLPvaccinationinducesreadilydetectableIgGatthecervix,the level of antibody decreases several fold during ovula-tion, which might impair the protective effects of the vaccine[10].Mucosalvaccinationmightrepresentapotentialapproachto overcome these difficulties. While both systemic and mu-cosal immunisation can induce serum-derived Igs in the fe-male genital tract, the mucosal route has the theoretical ad-vantage of being given without injection and of inducinglocally produced specific secretory IgA (SIgA) in mucosalsecretions [10–15]. Evidence in animals and in people sug- gests that menstrual cycle-dependent changes of SIgA in thefemale genital tract differ from those of serum-derived Igs,which implies that the presence of specific SIgA might atleast partially compensate for the decrease in serum-derivedIgsatthecervixintheovulatoryphaseofthemenstrualcycle([14,16] and references therein, [17]). Among the different mucosal routes of immunisation,nasal vaccination has been reported frequently to be an ef-fective method for inducing specific immunity in the genitaltract[13,18–25].However,wehavefoundinfemalemicethatHPV16VLPmucosalimmunisationviathelowerrespiratorytract can induce high antibody levels in the genital tract andthat this route is more efficient than the nasal route [13,26],butitisnotknownwhetheranyimmunogendeliveredviathelowerrespiratorytractcaninduceantibodiesinthehumanfe-malegenitaltract.Wehavethereforeundertakenapilotstudyto compare the safety and immunogenicity of HPV16 VLPsadministered by nasal spray or aerosol to female volunteerswith the response obtained by systemic immunisation. 2. Material and methods 2.1. Volunteers The clinical protocol was approved by the local ethicalcommittee.Healthyadultfemalevolunteers,18–45yearsold,with no history of positive Papanicolaou (Pap) smear wererecruited. Written informed consent was obtained for eachparticipant after the study had been explained in detail. Ex-clusion criteria included: atopic diseases (food, perennial orseasonal allergies, rhinitis, asthma, or atopic dermatitis), ab-normalPapsmear,HIVorHPV16seropositivity,anychronicmedication,orpregnancy.Thevolunteerswererecruitedovera17-monthperiodandsuccessivelyassigned(fivepergroup)toeithernasalsprayoraerosolvaccinationswhilethevaccinedose was escalated (2, 50 and 250  g HPV16 VLPs). A finalgroup was assigned to receive an i.m. priming dose (50  gVLP) followed by a mucosal boosting dose (50  g VLP byaerosol, see below). Following the screening visit, 33 vol-unteers were included in this study, while 11 were excludedbecause of HPV16 VLP IgG titers >50 ( n =4) or abnormalPap smear ( n =7). The responses were compared with a par-allel group of nine volunteers who were immunised i.m. atweek 0 and week 4 with two doses of 50  g [10]. The eli- gibility criteria were similar, and the same bulk vaccine lotwas used for the i.m. immunisations and the 50 and 250  gnasal and aerosol vaccinations. The shorter interval beforeboosting after mucosal delivery (2 weeks) was based on ourprevious findings in the murine model [13]. 2.2. Vaccination Clinical lots of recombinant HPV16 L1 VLPs vaccinewere purified from insect cells and suspended in saline with-out adjuvant, as described previously [6,10]. Nasal adminis- tration was via a Devilbiss ® nebulizer sprayed alternativelyinto each nostril. Aerosol administration to the lower airwaywas performed with a Systam ® nebulizer (aerosolisation bysonication) and delivered via a mouth piece. All volunteersreceived two immunisations. The first group of volunteers( n =9) was randomly assigned to receive 2  g VLP dosesof vaccine with the nasal spray ( n =4) or the aerosol ( n =5)at week 0 and week 2. Serum and saliva were collected atweeks 0, 2, 6 and 8, while cervical secretions were collectedatweeks0and8.PBMCweretakenatweek0and10daysaf-ter the second immunisation. As there were only mild symp-toms from vaccination, a second group of volunteers ( n =7)was randomly assigned to receive 50  g VLP doses with thenasal spray ( n =4) or the aerosol ( n =3), and sampling wasperformed as described. Because an adverse reaction wasobserved in one of the volunteers after the second aerosolimmunisation (see results), the subsequent group ( n =7) in-cluded three volunteers who received the 50  g VLP dosewith the aerosol, and four who received 250  g dose withthe nasal spray. In the absence of additional adverse reac-tions to the aerosol administration, a new group ( n =5) wasimmunised with 250  g VLP doses with the aerosol. Finally,an additional group ( n =4) received one i.m. priming dosewith 50  g VLP at week 0 and was boosted at week 4 withan aerosol dose of 50  g VLP. Serum and saliva in this lastgroup were collected at weeks 0, 4 and 8, cervical secretionswere taken at weeks 0 and 8, and PBMC were collected atweek 0 and 10 days after the aerosol vaccination. 2.3. Sampling Cervical samples were collected with Weck-cell spongesduringagynaecologicalexamination,aspreviouslydescribed[10]. Saliva was collected with Weck-cell sponges in a spearformat. Two sponges were placed under the tongue and leftin place for 2–3min. Saliva was extracted and stored withprotease inhibitors at  − 70 ◦ C. 2.4. Adverse reactions Each volunteer had a physical examination prior to vac-cination and also at weeks 0 and 2, and 10 days after thesecond vaccination. For nasal spray vaccination, the exami-nation included an anterior rhinoscopy to assess the integrityof the nasal mucosa and the quality of the secretions. For  3636  D. Nardelli-Haefliger et al. / Vaccine 23 (2005) 3634–3641 Fig. 1. Comparison of the HPV16 VLP specific titers induced by the different vaccination protocols. Specific IgG and IgA titers in serum are shown in A andB, respectively. Specific titers in cervical secretions normalised to their content in total Igs (U/   g) are shown in C (IgG), D (IgA) and in E (SIgA). The differentvaccination routes and/or VLP doses are indicated below the  x -axis. Fisher’s test exact  p -values are indicated.   D. Nardelli-Haefliger et al. / Vaccine 23 (2005) 3634–3641  3637 aerosol vaccination, the examinations included a pharyngealexamination,apulmonaryauscultationandameasureofpeak airflow. The volunteers were also given symptom diaries tocomplete. Those receiving nasal spray vaccination were in-structed to record nasal itch, runny nose, stuffy nose, sneez-ing, and dry nose, while those receiving aerosol vaccinationrecorded throat itch, sore throat, dyspnea, and cough. Eachsymptomwascategorisedasweak,mild,orstrong,andasoc-curring immediately, 1h, or 4–5h after vaccination, as wellas during each of the three following days. In addition, allvolunteerswereinstructedtorecordanyothersymptomsandtheir daily temperature. 2.5. ELISA The levels of total and HPV16L1 VLP specific IgA andIgGweredeterminedbyELISA,aspreviouslydescribed[10].The total or HPV16 VLP specific SIgA was developed byadding anti-human secretory component (DAKO #A0187)as secondary antibody. Each sample was tested in duplicate,with purified human serum IgA (Cappel #55906), IgG (Cap-pel #55908) or SIgA (Cappel #55905) included in the as-says as a reference standard. For HPV16 VLP specific Igs,two-fold serial dilutions were tested, starting at a dilution of 1:50 for sera, 1:5 for cervical secretions, and 1:20 for saliva.There was less than 15% variation between duplicate sam-ples. The titers were arbitrarily determined as the reciprocalend-point dilution that yielded an OD>0.100 (two–three-foldthemeanpreimmuneOD+3S.D.).Volunteerswerecon-sideredtobeHPV16seropositiveatrecruitmentiftheirserumyielded an OD>0.100 at a 1:50 dilution. OD correspondingto the mean preimmune OD+3 S.D. was used to determinethe titers for SIgA in cervical secretion (OD>0.150) and forIgA (OD>0.200) and SIgA (OD>0.300) in saliva. In addi-tion,forcomparativepurposes,thespecifictitersinsecretionswere normalised to their content in total Ig and expressed asU/   goftotalIgs,tocompensateforthevariationinIgcontentbetween samples (see Fig. 1). 2.6. Enzyme-linked immunospot assay (ELISPOT) Trafficking antibody-secreting cells (ASC) that secretedanti-HPV16 VLP IgA were measured by the ELISPOTmethod [27]. PBMC separated by a Ficoll gradient (Ficoll- paque, Pharmacia, Uppsala, Sweden) were added to VLP-coated Maxisorb plates (NUNC, 4 × 10 5 cells/well), andspecific IgA secreted by individual lymphocytes were de-tected with a biotinylated goat anti-human IgA (Chemi-con AP #110B), and visualised by alkaline phosphatase-conjugated streptavidin (DAKO) with 5-bromo-4-chloro-3-indolyphosphate as substrate. Detection of 20 or morespots/4 × 10 6 cells after vaccination was defined as a posi-tiveresponse,asitcorrespondedtothemeannumberofspotscounted in the PBMC of the vaccinee before vaccination+3S.D. 2.7. Statistics Toallowstatisticalcomparisonwithlargergroupsbetweenvaccination protocols, all doses in the nasal group were con-sidered together, while for the aerosol protocol the 50 and250  gdoseswereconsideredtogether.Comparisonwiththei.m. group were made with a Fisher’s exact test for serocon-version (HPV16 VLP-specific IgG and IgA, see Fig. 1A and B)andfordetectableresponsesincervicalsecretions(HPV16VLP specific cervical IgG and IgA, see Fig. 1C and D) using GraphPad Prism. 3. Results 3.1. Safety profile of the vaccine This was a dose escalation study of nasal (nasal groups)and aerosol (aerosol groups) immunisation of HPV16 VLPs(two doses of 2, 50, or 250  g) as well as the combination of a 50  g priming i.m. injection followed by a 50  g aerosolbooster (systemic/aerosol group). The responses were ableto be compared against two doses of 50  g i.m. vaccination(systemic group) from a parallel study [10]. The nasal andaerosol vaccinations were generally well tolerated, with noserious adverse events. Among the 12 volunteers who re-ceived VLPs as a nasal spray, the only local symptom notedwas some mild minor local discomfort, and one volunteerreported a mild possibly related systemic adverse event (fa-tigue). Among the 15 volunteers in the aerosol groups andthe 4 volunteers in the systemic/aerosol group, the only localsymptom was mild pharyngeal discomfort. One volunteer, inthe 50  g aerosol group, reported a systemic side effect of moderate intensity that was probably related to the vaccine:5h after her second dose she experienced dyspnea, chills,fever (39.8 ◦ C), myalgias, and arthralgias that were relievedby self-administration of aspirin. The patient first informedus about these symptoms 36h after they began; by that timeshe had no symptoms, the physical examination was normal,and blood tests suggested mild systemic inflammation (ery-throcytesedimentationrate28mm/h { normal:<20mm/h } ,Creactive protein 46mg/l  { normal: <10mg/l } ). Interestingly,this volunteer exhibited a very high anti-VLP response in herserum and cervical secretions (see Tables 1 and 2). On theday following her first vaccination, another volunteer, in the2  g aerosol group, reported mild fatigue, which persistedfor 48h and was considered possibly vaccine-related. 3.2. Immunogenicity of the vaccine We measured ELISA titers of HPV16 VLP-specific an-tibodies in serum and at the cervix; for both of these fluidsa good correlation has been shown between this assay andHPV16 neutralisation [6,28,29]. In contrast to reports with other antigens nasal vaccination was poorly immunogenicfor most volunteers, including the 250  g nasal group (see
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