Preferential Amplification of CD8 Effector-T Cells after Transcutaneous Application of an Inactivated Influenza Vaccine: A Randomized Phase I Trial

Background Current conventional vaccination approaches do not induce potent CD8 T-cell responses for fighting mostly variable viral diseases such as influenza, avian influenza viruses or HIV. Following our recent study on vaccine penetration by targeting of vaccine to human hair follicular ducts surrounded by Langerhans cells, we tested in the first randomized Phase-Ia trial based on hair follicle penetration (namely transcutaneous route) the induction of virus-specific CD8 T cell responses. Methods and Findings We chose the inactivated influenza vaccine – a conventional licensed tetanus/influenza (TETAGRIP®) vaccine – to compare the safety and immunogenicity of transcutaneous (TC) versus IM immunization in two randomized controlled, multi-center Phase I trials including 24 healthy-volunteers and 12 HIV-infected patients. Vaccination was performed by application of inactivated influenza vaccine according to a standard protocol allowing the opening of the hair duct for the TC route or needle-injection for the IM route. We demonstrated that the safety of the two routes was similar. We showed the superiority of TC application, but not the IM route, to induce a significant increase in influenza-specific CD8 cytokine-producing cells in healthy-volunteers and in HIV-infected patients. However, these routes did not differ significantly for the induction of influenza-specific CD4 responses, and neutralizing antibodies were induced only by the IM route. The CD8 cell response is thus the major immune response observed after TC vaccination. Conclusions This Phase Ia clinical trial (Manon05) testing an anti-influenza vaccine demonstrated that vaccines designed for antibody induction by the IM route, generate vaccine-specific CD8 T cells when administered transcutaneously. These results underline the necessity of adapting vaccination strategies to control complex infectious diseases when CD8 cellular responses are crucial. Our work opens up a key area for the development of preventive and therapeutic vaccines for diseases in which CD8 cells play a crucial role. Trial Registration Clinicaltrials.gov NCT00261001

I agree: to conduct the study as outlined in the protocol and in compliance with GCPs and with applicable regulatory requirements; to provide the protocol and all investigational drug and administration mode information relating to pre-clinical and prior clinical experience, if any furnished to me by the sponsor, to all physicians responsible to me who participate in this study. I will discuss all information with them to assure that they are fully informed regarding the administration mode, drug and the conduct of the study; to appropriately direct and assist the staff under my control, who will be involved in the study; to use the trial material including drug supplies only according to the instructions of the protocol; to permit monitoring, auditing and inspection; to retain the trial-related essential documents until the Sponsor informs me that these documents are no longer needed; I have been informed that certain regulatory authorities require the Sponsor to obtain and supply details about the investigator's ownership interest in the Sponsor or the experimental mode of administration or the study drug, and more generally about his/her financial ties with the Sponsor. ORVACS will use and disclose the information solely for the purpose of complying with regulatory requirements.
Hence I: Agree to supply ORVACS with any information regarding ownership interest and financial ties connected with the study or with ORVACS.
Agree to promptly update this information if any relevant changes occur during the study; and Agree that ORVACS may disclose this information about such ownership interests and financial ties to regulatory authorities.

Investigator name _______________________
Investigator signature _______________________ Date: ____________ I agree: to conduct the study as outlined in the protocol and in compliance with GCPs and with applicable regulatory requirements; to provide the protocol and all investigational drug and administration mode information relating to pre-clinical and prior clinical experience, if any furnished to me by the sponsor, to all physicians responsible to me who participate in this study. I will discuss all information with them to assure that they are fully informed regarding the administration mode, drug and the conduct of the study; to appropriately direct and assist the staff under my control, who will be involved in the study; to use the trial material including drug supplies only according to the instructions of the protocol; to permit monitoring, auditing and inspection; to retain the trial-related essential documents until the Sponsor informs me that these documents are no longer needed;   Table 3: Study procedures at screening period …………………………………………31 Table 4: Study procedures during the study ………………………………………….…34

Methodology
Phase I, open label, randomized, multicenter, two cohorts, twoarm study comparing a transcutaneous mode of administration to a intramuscular administration of the Tetagrip® vaccine in healthy voluunteers (Cohort I) and in HIV-infected patients (Cohort II)

Investigational mode of administration, investigational product, and dose
Tetagrip® vaccine will be administered on two skin areas of 4x4 cm each (32 cm 2 ) on the external part of the upper left arm transcutaneously and will be compared to a conventional intramuscular injection. The selected dose is 0.5 ml that corresponds to one vaccine dose of tetanus toxin and 15 μg of two type-A (H3N3 and H1N1) and one type-B virus subunits (This information will be updated as soon as they will become available).

Number of subjects
48 in two cohorts of : The duration of the study will be approximately 3 months

Randomization
In each of the two cohorts subjects will be randomized on the mode of Tetagrip® vaccine administration to one of the two arms. Subjects fulfilling the inclusion criteria will be randomly assigned in equal numbers (12 per arm) to one of the two arms as described below : ARM Administration Volume Number of subjects mode A Transcutaneous 0.5 ml 12 B Intramuscular 0.5 ml 12 Randomization will be performed using the SAS procedure by the Data Management and Statistics Center.

Vaccine schedule
Each subject will receive one single administration 0.5 ml of Tetagrip® vaccine either transcutaneously on two sites of 4x4 cm skin area (total area of 32 cm 2 ) on the deltoid region or via conventional intramuscular injection.

Objectives
Primary Objective: • To evaluate the safety of a transcutaneous mode of tetanus/influenza vaccine administration of Tetagrip® vaccine in healthy volunteers and in HIV infected patients.

Secondary objectives:
To compare in each of the two cohorts, the two arms between baseline and day 7 (D7), day 14 (D14) and day 28 (D28) post vaccination: • Specific antibodies: Geometric titers (GMT) with 95% confidence interval, • The increase in tetanus and influenza specific antibody titers, • The pre/post vaccination ratios of specific antibodies titers, • The increase in tetanus and influenza specific T cells (CD4 and CD8), • To characterize -specific effector and memory tetanus and influenza T cells, • Proportion of participants who responded (50% increase in titers) -Clinical examination and an interview on their medical history and possible current therapies For both cohorts the same following inclusion criteria will be meet : • Age between 18 and 45 years • BMI between 21 -26 • Phototype I-IV • Absence of tetanus vaccination within the last 4 years • Absence of influenza vaccination in the past year • Absence of any psychological, familiar, sociological or geographical condition potentially hampering compliance with the study protocol schedule.
• Written informed consent

Main Exclusion Criteria
• Excessive terminal hair growth on the investigational skin area • Phototype V-VI • Any skin affection which may interfere with the trial assessment on the injection or application site • Any allergy or hypersensibility to one of the components of the Investigational Product (e.g. egg products, neomycin) • Medical history of allergy or hypersensitization to any ingredient of colorant used in the transcutaneous mode of vaccination.
• Medical history of skin cancer • Acute illness, e.g. fever, infection at Screening and/or D 0 • Any acute or chronic illness which may expose the subject or interfere with results of the trial • Use of any topical treatment on the injection or application site within the last four weeks • Continuous topical and systemic treatment that would interfere with assessment and/or investigational treatment (e.g. Immunosuppressors or any Immunomodulator agent) • Prevision of UV sessions or sun exposure 4 weeks prior to the study or during the study period • Subject being in the exclusion period of a previous clinical trial (1 month),

Study endpoints
Primary endpoint: Safety assessment Secondary endpoints: Immunogenicity assessments Safety will be assessed at each visit by recording clinical local and general tolerance : • Clinical examination, • Local lymph nodes examination, • Questioning of the patients immediately after injection or application and afterwards at each visit, • Patient diary card observations, • Reporting of any adverse events according to the grading table in appendix .
Immunogenicity will be evaluated as the specific immune response including detection of: • Tetanus and influenza specific antibody titers • Tetanus and influenza specific T cells (CD4 and CD8)

Statistical methods and Analysis
Safety will be assessed during all the study by recording any Adverse Event (AE) graded ≥ 3 according to toxicity criteria (Appendices IX, and X) .
Immunogenicity will be assessed for each subject at D7, D14 and D28. Groups will be compared by ITT and per protocol analysis using non parametric tests.

Background
Classical prophylactic vaccines aim at preventing infection by inducing in naive individuals a strong immune memory that will control pathogen dissemination at entry in the organism.
Another concept is to use vaccines as therapeutic tools during an established infection by reinforcing or broadening defenses when specific immune responses are unable to do so during the natural course of the disease and when a conventional antimicrobial therapy is not available or efficacious enough (Autran 2003). This strategy is currently being evaluated in HIV infection where therapeutic vaccines aim at limiting costs and toxicity of a lifelong antiretroviral therapy and at preventing disease progression in the absence of treatment by re-inforcing immunity to HIV.
However, although conventional vaccines have generated major successes in the control of infectious diseases, several obstacles remain in the development of vaccines against pandemic chronic diseases such as HIV against which the current candidate vaccines are still too weak to ensure protection ( McMichael 2003), or in the access to vaccines in developing countries which require safe, well tolerated and easy to use vaccines. In addition when HIV-infected patients initiate antiviral therapy at advanced stages of the HIV disease, immune reconstitution, though potent at restoring most immune defenses agianst pathogens (Autran 1997) might not be strong enough to ensure optimal responses to conventionnal vaccines (Lange 2003).Therefore progresses have to be made to improve the efficacy of these still relatively weak preventative vaccines in the development phase and to overcome the multiple obstacles opposed to the development of therapeutic immunization against HIV or of preventative vaccines in immune suppressed individuals.
Vaccines are efficient at priming strong and protective immune responses to pathogens by targeting and activating dendritic cells (DCs) which play a unique role as primers of antigenspecific immune responses. Vaccines are classically injected into muscles or subcutaneous tissues where local depots of vaccine compounds have to be captured by immature DC and promote DC maturation and migration to the draining lymph nodes where they prime the naive vaccine-specific T cells. Those tissues are however very poor in DC and vaccines generally require adjuvants to augment DC recruitment and activation and to potentiate their immunogenicity. In addition, part of the vaccine is bound unspecifically to connective tissue or undergoes degradation. Furthermore, conventional i.m. or s.c. injections raise some psychological or cultural difficulties (fear of needle), injection procedure with the risk of injury and blood transmission and, exhibit further practical disadvantages and hazards including infection caused by unsterile equipment or unsterile reconstitution, instability of the vaccine preparation and injury due to improper injection techniques (Jodar 2001).
A future goal for vaccine design is therefore to increase their efficiency by reaching the highest number of antigen-presenting cells (APCs) possible and to achieve the high local concentrations required to induce a potent immune response, as well as to use non-invasive

Transcutaneous penetration of compounds
Thus dermal or transdermal delivery of vaccines has become a great challenge. Several obstacles however prevent vaccines from attaining sufficiently high and free concentrations in these target skin DC. In intact skin, the percutaneous absorption of epicutaneously applied compounds is first limited by the stratum corneum which forms a highly structured and tight barrier on the skin surface, very effective in preventing entrance of compounds into the skin.
Under physiological conditions, active compounds in solutions or ointments spread rather homogenously on the skin surface which leads to small local concentrations of the topically applied active compound and augments the unspecific binding of active compounds to skin proteins, which further reduces their free concentration. The passage through the horny layer, , occurs via a very slow diffusion and larger molecules including proteins hardly overcome this barrier at all. Secondly, the upper living layer of the skin, the epidermis, maintains continuous renewal and sheds cells off, thereby rejecting bound active compounds to the skin surface.
Thirdly, a dense microvasculature system below the epidermis rapidly and removes active compounds from the skin to the systemic compartment.
Recent investigations by our own group and others revealed that the penetration of topically applied compounds is considerably lower in hairless skin as compared to haired skin, suggesting that hair follicles are important entry pathways for epicutaneously applied compounds skin (Hueber 1994, Tenjarla 1999, Schaefer 2001 fibres, especially vellus hairs, are removed as well (Mills 1983). The remaining stratum corneum and the viable epidermis are left intact. This technique is routinely used in our laboratory and also by other groups to improve the percutaneous penetration of topically applied compounds and to determine the amount of substance which enters the follicular reservoir (Hueber 1994, Schaefer 2001).

Study rationale
The proposed study aims to translate our current knowledge about vaccinology, immunology of the skin and on transcutaneous penetration of epicutaneously applied active compounds, into the development of more efficient and well tolerated vaccines, and to progress toward an easyto-apply patch system for transcutaneous application of vaccines.
To that purpose we aim at testing the safety and immunogenicity of a new transcutaneous route of vaccine administration. We propose to test this new route first with a well-known, safe and highly immunogenic vaccine i.e. anti-influenza and tetanus vaccine which is licensed for subcutaneous and intra-muscular routes, and to compare the vaccine-specific immune responses induced after transcutaneous administration to the conventional intramuscular (i.m) injection.
Seasonal vaccination against influenza is recommended for all individuals at risk for severe flu, including persons above 60 years of age or suffering from chronic dieases and for medical personal. It is also highly recommended at any age to prevent influenza. In addition vaccination against tetanus is mandatory in childhood and requires recall injections every 5 years to protect against the lethal disease caused by the tetanus toxin.
We hypothesize that the transcutaneous application of a licensed anti-influenza-tetanus vaccine in the commercially available standard preparation of 0.5 ml should be capable to induce at least similar antibody and CD4 and/or CD8 T cell responses to both the tetanus and the flu vaccinal antigens.
Tetagrip® vaccine is an approved and commercially available vaccine manufactured by Sanofi-Pasteur, administered in one injection for preventive vaccination of adults against tetanus and influenza. The Tetagrip® vaccine therefore represents a safe and approved test vaccine to evaluate safety and immunogenicity of the mode of administration under investigation.
The long term goal of this strategy is to improve the efficacy of vaccines that are currenly encountering major obstacles such as the HIV vaccines, and to develop a non invasive mode of vaccine administration. Results from this study will help establish a standardized study protocol for the application of HIV-vaccines in future clinical trials. Twenty-four subjects will be accrued in each cohort. Cohort I's 24 individuals will be recruited in the Berlin center. Patients from Cohort II will be accrued in Paris and Frankfurt centers (12 in each center).

STUDY DESIGN
Participants will be enrolled concurrently for both arms, and will randomly assigned to one of the two arms and will receive a single dose of 0.5 ml Tetagrip® vaccine either transcutaneously (Arm A) or via i.m. injection (Arm B) at D0 (see table 1).

Primary objective :
To evaluate the safety of a transcutaneous mode of a tetanus/influenza vaccine (Tetagrip®) administration in healthy volunteers (Cohort I) and in treated HIV-infected patients(Cohort II).
Safety will be assessed by recording adverse events (including cutaneous reactions) at each post-inclusion visit.

Secondary objectives:
To evaluate in the two arms of the two Cohorts between baseline and day 7 (D7), day 14 (D14) and day 28 (D28) post vaccination: • Protective tetanus and influenza specific antibodies Geometric titters (GMT) with 95% confidence interval, • The increase in tetanus and influenza specific antibody titters, • The pre/post vaccination ratios of specific antibodies titters, • The increase in tetanus and influenza specific peripheral blood T cells (CD4 and CD8) numbers, • The vaccine -specific effectors and memory tetanus and influenza peripheral blood T cells, • Proportion of participants who responded (50% increase in titters) • Plasma HIV RNA < 400cp/ml over the last 6 months

Inclusion criteria
• Efficient antiretroviral treatment with a minimum of three drugs since at least one year In addition Patients from both cohorts must meet the following criteria to be eligible for the study: • Age between 18 and 45 years, • BMI between 21 -26, • Phototype I to IV • Clinical examination and an interview on their medical history and possible current therapies • Subjects able to receive vaccine administration by any of the two administration routes, • Absence of tetanus vaccination within last 4 years, • Absence of influenza vaccination in the last year, • Absence of any psychological, familiar, sociological or geographical condition potentially hampering compliance with the study protocol schedule.
• Written informed consent

Exclusion criteria
In both cohorts, if any of the following apply, the subject cannot enter the study: • Excessive terminal hair growth on the two investigational skin areas used for the transcutaneous mode of vaccination.
• Phototype V-VI • Acute illness, e.g. fever, infection at screening and/or D 0 • Any acute skin affection which may interfere with the trial assessment on the injection site, • Any allergy or hypersensibility to one of the components of the Investigational Product (egg products, neomycin), • Medical history of allergy or hypersensitization to any ingredient of colorant used in the transcutaneous mode of administration, • Medical history of skin cancer, • Any acute or chronic illness which may expose the subject or interfere with results of the trial, • Use of any topical treatment on the injection site within the last four weeks, • Use, within the past 3 months, of any topical and systemic treatment that would interfere with assessment and/or investigational treatment (anti-inflammatory drugs, immunosuppressors or any immune modulator agent), • Prevision of UV sessions or sun exposure 6 weeks prior to the study or during the study period, • Subjects with current alcohol or illicit drug use which, in the opinion of the investigator, may interfere with the subject's ability to comply with the dosing schedule and protocol evaluations.
• Subject being in the exclusion period of a previous clinical trial (1 month.) In addition in each cohort:

• HIV infection
Cohort II: • Any acute infectious event within 30 days prior to enrolment • Subjects who have participated in an investigational HIV vaccine trial or any immunomodulatory trial in the last 6 months • Subjects suffering from serious medical conditions, which, in the opinion of the investigator, would compromise the safety of the subject.
• Patients having received IL2 or any other immunomodulator drug in the last 6 months

Number of subjects
A total of 48 individuals will be recruited.
24 subjects (12 per study arm) in each cohort.

Study vaccine
• The immunization will use Tetagrip® a commercially available vaccine, at the recommended dosage.
• The selected vaccine Influenza/Tetanus vaccine is registered as Tetagrip® trademark by Sanofi-Pasteur MSD, France.
-Presentation: Injectable suspension presented as 0.5 ml refilled syringe. The influenza virus was grown on chicken egg for vaccine subunits production. Other components of the vaccine are sodium chloride, sodium phosphate, potassium chloride, potassium phosphate and, injectable water.
-Shelf-life and storage conditions: The expiry date is indicated on labels on the box.
The vaccine should be stored under refrigeration (2 to 8 °C), protected from light and must not be used if coloration is observed.
-Commercial Tetagrip® vaccine will be supplied by ORVACS to the hospital pharmacist. The specific form "Drug Accountability Form" will be used in order to collect information regarding Tetagrip® vaccine itself (quantity, batches and expiry dates).

Study drug administration
Tetagrip® vaccine, 0.5 ml will be administered transcutaneously on two skin areas of 4x4 cm on the deltoid muscle and will be compared to a conventional intramuscular injection.

• Transcutaneous mode of vaccine administration (Arm A)
Tetagrip® vaccine will be applied as described in the Standard Operating Procedure, see Appendix VIII. Briefly, the two investigational skin areas of 4x4 cm each will be delimitated on the external part of the upper left arm. After shaving of the skin, cyanacrylate skin surface stripping (CSSS) will be performed. 0.25ml Tetagrip® vaccine will be applied on each investigational skin area. A silicone barrier will limit the spreading of the vaccine to the investigational skin area. At the end of the procedure, a hydrocolloid bandage will be applied to the investigational skin areas for the following 24 hrs.

• Intramuscular injection (Arm B)
Volunteers who receive Tetagrip® vaccine via intramuscular injection will receive the vaccine (0.5 ml) into the deltoid muscle according to Good Clinical Practice after careful disinfection.

Randomization of vaccine mode of administration
After verification of the eligibility criteria by the statistical department, healthy volunteers (Cohort I) or HIV-infected patients (Cohort II) will be randomly assigned to one of the two administration routes. Randomization will be based on SAS procedure plan. The randomization will be performed by the Data Management and Statistics Center. The procedure is described in After validation of the inclusion criteria, subjects will be randomized on the administration mode of Tetagrip® vaccine to one of the two study arms:

On site drug accountability and dispensing
The hospital pharmacist (Center II) or the investigator (Center I, Center III), are responsible for adequate storage of the study medication according to the manufacturer recommendations and for dispensing the treatment to the study participants.
Tetagrip® vaccine must be used in accordance to the protocol and only by the investigator.
The investigator and/or the pharmacist must maintain adequate and accurate records including batch number of Tetagrip® vaccine. For this purpose, the Drug Accountability Form should be used. This includes the patient's identification, the date of dispensing, each quantity dispensed, and the identification of the dispenser. The original will be kept by the Sponsor and the copies will be left: one in the pharmacist's file and one in the investigator's file.

CONCOMITANT MEDICATION
All treatments given in addition to the study vaccine at subject's study entry (D-7) and/or during the study period are regarded as concomitant treatments and will be documented on the appropriate pages in the CRF.

Prohibited treatments
The following medications are not allowed during the entire trial period: -Any topical treatment of the investigational site within the past 4 weeks, -Systemic treatment (drug or cosmetic), which may interfere with the trial assessment (antiinflammatory drugs, immunosuppressors or any immune modulator agent)

Present therapies
All the concomitants therapies needed by the subjects will be recorded in the CRF throughout the study.
Patients of cohort II will continue their antiretroviral treatment for the entire duration of the study.
Names, dosage and dosing intervals of the antiretroviral drugs will be recorded in the CRF throughout the study. Furthermore, any change in their antiretroviral treatment need to be recorded in the patient's CRF.

STUDY PLAN
Subjects will be informed about the study, both verbally and by reviewing the patient information sheet and consent form at day of screening. The subject must be given the opportunity to ask questions and given time to consider his participation. The investigator and the subject will both sign and personally date the consent form as confirmation of consent.

Screening Period (Day -7)
The screening time is the interval preceding registration, and includes 7 day period for performing screen assessments. Written informed consent will be obtained from the volunteer before any study specific procedure is undertaken.
Once written consent has been obtained, subjects will undergo a baseline assessment: -Verification of all inclusion/non inclusion criteria

Study entry -Randomization
All inclusion/exclusion criteria will be checked during the randomization procedure. The study monitor or study manager will notify the investigator by fax within two working days. In case of controversy on patient eligibility, or absence of any required assessment (see table 2), the investigator shall be contacted for discussion. In any case test results and assessments required to establish eligibility must be obtained prior to the administration of the vaccine.
Randomization of the subject to one of the two study arms (if all inclusion criteria are filled) will be performed by the Methodology/Data management department. .
The Methodology/Data management department will assign a registration number to the patient and allocate the administration mode of Tetagrip® vaccine. Each patient will receive a 4 digit number followed by subject initials. The first two digits represent the center and the last two digits represent the subject within the center. This subject code must be reported on all CRF pages and in any study document.
Randomization represents the starting point of the study. It is recommended to perform vaccination within no more than one week. In any case all events occurring after the registration must be recorded onto the CRF and will be taken into account in the analysis, whether the patient received the treatment or not.

Day 0 -Vaccine administration
Subjects will be definitely included in the study at D0.
Procedures corresponding to this visit are:

Evaluation during the treatment period
The study period begins with randomization and continues until 30 days after study treatment administration.
The same methods for assessment used at baseline must be used throughout the study to ensure comparability.
All adverse events which occurred between 2 visits must be assessed and documented using the toxicity guide. Subjects should be asked for recording any sign and symptom onto the patient diary card The following assessments need to be completed throughout the treatment period:

End of study treatment evaluation: D28
A clinical work-up will be performed at D28 after vaccine administration in both arms. It will include the following assessments (see also  may also, at his/her discretion; discontinue the subject from participation in this study at any time. If a subject is prematurely discontinued for any reason, the investigator must perform the assessments outlined in section 8.5 (End of study treatment evaluation).
These data should be recorded, as they comprise an essential evaluation that should be done prior to discharge of any subject from the study. When the subject is discontinued from the study due to an AE or SAE (as defined in section 10) the procedures stated in section 9 must be followed.

Subject withdrawal or Discontinuation
Subjects will be withdrawn from the trial by the investigator at any time for any of the following administrative or medical reasons: − For safety reasons: when the investigator feels that it would be detrimental for the subjects to continue the trial, for example, in case of the occurrence of a Serious Adverse Event/toxicity directly related to the investigational route and/or investigational product(s).
− For patient non-compliance reasons: poor compliance with the requirements of the study protocol (e.g. avoidance of sun exposure during the study period). − For scientific reasons: in case of any event which is known to affect the efficacy criteria: in case, for example, of systemic disease onset (without any relation to the investigational products but occurring during the trial) for which a therapy by corticoids would be requested. − Investigator non-compliance: Any significant deviation from the protocol without prior agreement of the Sponsor.
− Any other reason to be documented The primary reason for withdrawal will be clearly documented in the subject's medical records and in the CRF. A final evaluation will be completed at the time of discontinuation from the study.

Lost to follow-up
If a subject misses one visit, the investigator will to try at least twice to contact him. Without any success, a written letter will be sent to the subject. Without any response from the subject, he will be considered lost to follow-up. All of these actions will be documented in the subject's medical file as well as in the Case Report Form.

Subject replacement
To insure the availability of 10 evaluable cases per study arm we plan the inclusion of 12 cases per study arm to replace discontinuing or withdrawn subjects. The medical examination prior to the inclusion, will include a medical history interview and a clinical examination, to make sure of the eligibility of the subject .A medical examination will also be performed on study termination date.
These examinations will be performed by the investigator.

• Local clinical examination
The local clinical examination includes a careful inspection of the investigational site for local tolerance (see 7.8.4 for details) and an examination of the local (axilllary) lymph nodes.
All the clinical assessments to be performed in one subject will always have to be done by the same physician, except in case of absolute necessity. This should then be documented in the Case Report Form.

• Local tolerance
The investigator will assess local tolerance at Assessments Periods D 1 to D 28 , by direct evaluation for erythema and desquamation, and through subject's interview for pruritus and burning accordingly to appendix IX.

Assessment of the Immune Response
Evaluation of the Tetanus and Influenza vaccine-specific : • Antibody GMT: will be measured using a refernced method in two reference laboratories (Influenza: Centre National de Reference de la grippe, Institut Pasteur, Paris; Tetanus toxoid: Lab Microbiologie, Hôp Henri Mondor, Créteil, France) • Peripheral blood CD4 and CD8 T cells will be measured by ELISpot-IFNgamma and intracellular staining (IFN-g and IL-2) in the Laboratoire d'Immunologie Cellulaire in Paris, according to a Standard Operating Procedure (See Appendix VII for SOP)

Subject's Instructions
During the trial, subjects won't be allowed to : -Use any topical drug or cosmetic on the investigational sites (except usual cleaning products), -Use any topical or systemic treatment, (drug or cosmetic), which may interfere with the trial assessment (anti-inflammatory drugs or immunomodulators) -Perform intensive sport, -After the application of Tetagrip® vaccine, the volunteer will be instructed not to take a shower or bath and to avoid any activity which causes sweating or mechanical stress to the investigational site, e.g. physical exercise, during the following 24 hours.

Procedure for collection and shipping of blood samples
To be performed according to the Standard Operating procedure described in Appendix VI.

Primary Endpoints
Clinical local and systemic tolerance to Tetagrip® vaccine administration for both transcutaneous (TC) and intramusculaire (i.m) routes of administration will be used as the primary measure of safety.
This criterion will be measured as the rate of severe adverse events (grade ≥ 3) judged by the investigator to be probably or definitely related to the transcutaneous mode of vaccine administration.
Safety will be evaluated by: • Severe local toxicity at the site of injection (i.e., pain, tenderness, erythema, induration, regional lymphoadenopathy, limitation of limb movement) judged to be probably or definitely related to the mode of vaccine administration.
• Severe systemic symptoms (i.e.), fever, myalgia, fatigue, headache, anaphylaxis, hypersensitivity reactions) judged to be probably or definitely related to the administration mode of the vaccine.
• Other severe adverse reaction such as dermatologic, neurologic, gastrointestinal (nausea/vomiting, diarrhea) judged to be probably or definitely related to the administration mode of the vaccine The criteria for an adverse experience to be considered severe are described in section10)

Secondary Endpoints
Evaluation of immunogenicity of the two modes of administration will be measured in all participating subjects.
Immune response against 0.5 ml Tetagrip® vaccine administered via transcutaneous application compared to the immune response induced by conventional i.m. injection will be assessed in the peripheral blood to evaluate: • The protective tetanus and influenza-specific antibodies GMT titers.
• The increase in tetanus and influenza specific antibody titers between baseline and day 14 and day 28.
• The tetanus and influenza CD4 and CD8 peripheral blood T cells numbers.
• The characteristics of vaccine-specific CD4 and CD8 T cell differentiation.

General design issues
The primary aims of this study are to assess safety and tolerance of a transcutaneous mode of tetanus/influenza vaccine administration and to compare to the immunogenicity induced by intramuscular injection of the vaccine in healthy volunteers and HIV infected patients. The 48 participants will be randomized to two arms in two modes of vaccine administration; these groups are: Arm A) Transcutaneous administration of Tetagrip® vaccine and ArmB) Intramuscular injection of Tetagrip® vaccine.

Analysis Plan Overview
The data Analysis will consist of a comparison of safety and immunogenicity data between study arms. The primary analysis is an intent to treat analysis, i.e., it will include all participants enrolled. A per protocol analysis will also be conducted. Since enrolment is concurrent with receiving vaccination by any of the two administration modes, all participants will have received Tetagrip® vaccine, either by transcutaneous application or conventional intramuscular injection and therefore will provide some safety data.

Sample Size Calculations
Our intended group size of twelve individuals per arm in each cohort is usual for phase I trials and was not based on sample size calculations Two or more severe toxicities within an arm will indicate a safety problem. If one observe no AE (≥ grade 3) then, with this sample size (12 subjects within each arm) the true rate of occurrence of a severe toxicity event ≥ grade 3 related to the administration mode of Tetagrip® vaccine is lower than 26.5 %

Statistical Analysis
All statistical analysis will be performed using SAS or SSPS software. All randomized subjects who received one dose of the vaccine will be included in the ITT analysis.
The analysis variables consist of baselines variables, primary endpoints (safety variables), and secondary endpoints (immunogenicity variables).

Baseline
Demographic variables to be measured, include race, ethnicity, age, and sex.
Clinical variables including temperature, vital signs, and clinical symptoms.
Immunology variables will also be collected at baseline For baseline variables, descriptive analyses will be performed (%, ;median, interquartiles, ranges) by arm in each cohort.

Primary endpoints (Safety)
Primary endpoints measure safety of the candidate transcutaneous administration mode.
Toxicities for each arm are measured by local and systemic reactions to the mode of Tetagrip® vaccine administration.

Secondary endpoints ( Immunogenicity analysis)
For safety and immunogenicity the two arms will be compared using non parametric tests: Fisher exact test for qualitative variables and Mann-Whitney tests for continuous variables.

Adverse Events -Definition
An Adverse Event (AE) is any adverse change from the subject's baseline condition. This includes intercurrent signs, symptoms, illness, and significant deviations from baseline laboratory values, which may occur during the course of the clinical study, whether considered related to treatment or not.
All laboratory tests for which abnormal results are collected after study treatment initiation should be repeated until the values return to normal or stable status.
Abnormal results are defined as those falling out of the laboratory normal range that are clinically significant. The frequency with which such checks should be made will be defined by the investigator's opinion depending on the degree of the abnormality.
In all cases, the aetiology should, as much as possible, be identified and the sponsor notified.

Grading of adverse events
The severity of adverse events should be determined by using the toxicity criteria given in Appendix XI.

Reporting of adverse events
All adverse events which occurs between 2 visits must be assessed and documented using the toxicity guide. Subjects should be asked for recording any signs and symptoms; onto the patient diary card.
Any adverse or intercurrent event occurring during the study period, spontaneously reported by, or written onto the subject's diary card; or observed by others, will be recorded in the subject's Case Report Form (CRF). At each volunteer/patient visit, the investigator will inquire about any Adverse Event occurrence, by interviewing the volunteer using an open question taking care not to influence the subject's answer and, if appropriate, by directed questioning and clinical examination.
Each time a concomitant medication is reported during the study, an Adverse Event will be documented and the reason for the therapy noted.
The records will describe the nature (diagnosis, signs and symptoms) severity, date/time of onset, date/time of resolution, outcome and actions taken as well as relationship to study treatment (according to the investigator's opinion).
It will be specified whether the event is serious or not All AEs already recorded and designated as "ongoing" should be reviewed at each subsequent visit. If resolved, the details are to be recorded in the CRF. If any AE changes for the worse, in frequency, symptoms or in severity, a new record of the event must be started. Distinct reports are required for differing frequencies and/or severity of the same event to enable comprehensive safety reports and later analysis.

Serious Adverse Events -Definition
A serious adverse event (SAE) includes but is not necessarily restricted to any event which: • Results in death (whatever may be the cause) • Is life-threatening • Results in persistent or significant disability/incapacity • Requires hospitalization or prolongation of existing hospitalization • Is a congenital anomaly or birth defect • Other events including cancer, overdose, pregnancy and any additional adverse experience or abnormal laboratory values occurring during the study period defined by the protocol as serious or which the investigator considers significant enough or that suggests a significant hazard, contraindications, side effect or precaution will be handled as a serious adverse event.
The term "life threatening" in the definition of "Serious" refers to an event in which the patient was at risk of death at the time of the event; it does not refer to an event which hypothetically might have caused death if it was more severe.
Hospitalization solely for the purpose of diagnostic tests, even if related to an Adverse Event, elective hospitalization for an intervention which was already planned before the inclusion of the subject in the study, and admission to day-care facility may not themselves constitute sufficient grounds to be considered as a Serious Adverse Event.
The severity of illness and adverse events are categorized using a standard grading scale with the following severity levels: mild, moderate, severe, life threatening, and death.
Intensity will be determined, using the following definitions as guideline : • Mild: Awareness of sign or symptom, but easily tolerated, has not to be treated and is not affecting daily life, • Moderate: Discomfort enough to cause interference with daily activity, but with no risk for the subject's health and improvement after slight therapy, • Severe: Unbearable event, interfering considerably with the subject's daily activity and/or possibly leading to a disability, or a life-threatening situation.
The causal relationship of an Adverse Event to the investigational product(s) will also be determined by the investigator according to the following criteria : • Unlikely: The event, with predominant probability, is due to other reasons than the administration of the investigational product(s). sequence in respect of administering the investigational product(s) and or presents the usual response with the product(s) tested. It might also be caused by other factors such as the subject's clinical condition, therapeutic procedures or administration of concomitant medications. • Probable: There are good reasons and sufficient documentation to assume a causal relationship in the sense of plausible, conceivable, likely, but not necessarily highly probable. The event follows a plausible chronological sequence in respect of administering the investigational product(s) and/or presents an expected response of the investigational product(s) and cannot reasonably be explained by other factors such as the subject's clinical condition, therapeutic procedures or administration of concomitant medications.
• Definitively related: Should be reserved for those events which have no uncertainty in their relationship to investigational product administration : this means that arechallenge was positive.
• Unclassifiable: Causality is, for one or another reason, not assessable, e.g. because of insufficient evidence or conflicting data. The event can neither be attributed to the investigational product(s) nor to any other factor because of a lack of objective evidence.

Unexpected adverse drug reaction is defined as:
An adverse reaction, the nature or severity of which is not consistent with the applicable product information (e.g. Investigator Brochure for an unapproved investigational product or package insert / summary of product characteristics for an approve product)

Grading of serious adverse events
Safety of the transcutaneous mode of vaccination under investigation will be assessed by monitoring clinical and immunological parameters and by the occurrence of Grade 3 (severe) or Grade 4 (life-threatening) local and systemic reactions to the Tetagrip® vaccine administration by using two distinct modes of administration.
Adverse events may be temporarily incapacitating (for example, loss or cancellation of work or social activities) and could make the administration mode under investigation impractical for large scale use if they occur in more than a small proportion of cases. Separate assessments of the rates of severe systemic and local reactions will be made. All participants will be closely monitored to compare in both arms, local and systemic adverse events during the week following Tetagrip® vaccine administration, and followed for 4 weeks.
The relationship of these experiences to vaccination by using two administration routes will be assessed and recorded as one as the following: definitely related, probably related, possibly related, or not related.
Adverse events will be judged as Serious Adverse Events (SAEs) using the criteria given in appendix XI.

Reporting of serious adverse events
All serious Adverse Events occurring during treatment period and 30 days after the end of study treatment evaluation, according to the above mentioned definitions, regardless of treatment or relation to study administration route and study drug must be recorded by the investigator as soon as he/she is informed of the event.
The investigator must notify ORVACS of this event by sending within 24 hours the "Notification of Serious Adverse Event" form (initial report; Appendix XII) with all available information concerning the event to the sponsor's representatives :

Follow-up of Adverse Event
Any serious and unexpected adverse event should be medically well documented and the information should be available as soon as possible. The investigator must complete a special clinical report to describe the outcome. Subject withdrawal, investigational product discontinuation and re-introduction or permanent discontinuation will also be documented for each event.
The Serious Adverse Events must be followed-up until resolution or stabilisation or until evidence that the investigational route and/or product and/or the volunteer's participation in the trial are not responsible for the event. A Serious Adverse Event which would not be resolved at the end of the trial will have to be followed until resolution or stabilisation.
Subjects who are removed from the trial due to a SAE will be treated according to established medical practice. All pertinent information concerning the outcome of such treatment must be entered in the Case Report Form. If the Adverse Event has not been resolved at the end of the trial, a follow-up is required at appropriate intervals until recovery, unless a plausible, investigational product-unrelated explanation has been found.
Any Serious Adverse Events shall be also reported in the CRF. All data obtained in the study described in this protocol will be recorded on CRFs. The CRF for each subject will be presented in a folder. The CRF will be completed chronologically and updated regularly in order to reflect the most recent data on the subject included in the study.
Prior to the start of the study, the investigator will complete a "Delegation of significant study related duties" or "List of delegation tasks" form, showing the signatures and initials of all those who are authorized to make or change entries on the CRF.
Procedure to fill-in CRFs: Each CRF must be neatly filled in with a black-inked pen. For each page on which information is entered, the subject code must be recorded.All data generated in the course of the trial will be recorded. Incomplete entries must be substantiated by giving reasons.
• Write one digit per box. If a value has fewer digits than the number of boxes, please rightjustify.
• If a pre-defined code is given, please tick the appropriate item or complete it.
• Write data in the form of PRINTED CAPITAL LETTERS.
• If an answer cannot be provided please fill in NA or ND.
NA will be used for : not applicable, not known, not available, ND will be used for: not done.
• Errors must be corrected by drawing a single line through the incorrect entry and by writing the new value as close as possible to the original. The correction must then be initialled and dated by an authorized person.
• Photographs and any electronically issued documents (if there are) will be identified with the subject's Number and initials and filed in the CRF.

•
The registration form and the end of study form must be dated and signed by an authorized investigator Although subjects may be interviewed by a research nurse or the trained equivalent (e.g. medical student, physician assistant), the investigator must verify that all data entries are accurate and correct, including verification that the subject fulfils the criteria for entrance into the study before vaccine administration. Physical examinations have to be performed by a registered medical practitioner.

Source documents
Definition: • Source Data: All information in original records and certified copies of original records of clinical findings, observations, or other activities in a clinical trial necessary for the reconstruction and evaluation of the trial. Source data are contained in source documents. • Source Documents: Original documents, data, aand records (e.g. hospital records, clinical and office charts, laboratory notes, memoranda, records, recorded data from automated appliances, copioes or transcriptions certified after verification as being accurate copies, microfiches, photographic negatives, microfilms or magnetic media, x-rays, subject files, and records kept at the pharmacy, at the laboratories and at medico-technical separtments involved in the clinical trial).
The subject must have consented to allow their medical records to be viewed by sponsorauthorized personnel and by local and possibly foreign regulatory authorities. This information shall be included in the informed consent.

Study monitoring
A monitor will be assigned by the sponsor to monitor this study and periodically contact the site, including conducting on site visits.
Monitor's activities will include: • Site initiation visit to collect and distribute essential pre-study documents; to instruct site personnel about the protocol, study procedures and expectations; to obtain the investigator's assurance to comply with study requiorements and GCP guidelines and inform site personnel about study materials.
• Monitoring visits: According to Good Clinical Practices, the study monitor are fully instructed concerning confidentiality and able to perform any necessary control on Informed Consent and CRFs. All observations and findings should be verifiable. During monitoring visists, the sponsor's monitor will: -Check and assess the progress of the study, -Review collected study data, -Conduct Source Document verification (hospital files), -Identify any issue and address its resolution All of that will be done in order to verify that the: -Data are authentic, accurate and complete, -Safety and rights of subjects are being protected, -Study is conducted in accordance with the currently approved protocol (and any amendements), GCP and all applicable regulatory requirements.
The investigator agrees to allow sponsor's monitor direct access to all relevant documents and to allocate his/her time and the time of his/her staff to the monitor to discuss findings and any relevant issues.
• Termination visit: At study closure the monitor will also conduct the related activities.

Data entry
The study data will be entered into a data base at the end of the study. Independent double data entry will be performed by two different trained operators. The two entries will be compared in order to identify and resolve any data entry discrepancy.

Data review
Consistency checks will be performed on the data. The resulting edit queries will be transmitted to the monitoring team. Answers to these queries will be integrated into the data base.

Data coding
Adverse events will be coded according table in appendix XIV.
Serious Adverse Events will be coded according to MedDRA

Data storage
Data backup will be done on CD-Rom

Data freezing
After corrections and modifications have been performed, the data base will be locked. Data will be extracted from the data base into the data files for statistical analysis.

Ethical conditions
This study will be performed in accordance with the principles stated in the Declaration of Helsinki (appendix II) and subsequent amendments and in accordance with the Good Clinical Practice Guideline (CPMP/ICH/135/95) and in compliance with local regulatory requirements

Independent Ethics Committe and Health authorities
• Independent Ethics Committee (IEC) Before beginning the trial, the Investigator will submit to the IEC of the University Medical Center Charité, Campus Mitte, a study file. The clinical trial won't begin before obtaining the approval of the IEC on the study protocol as submitted, or after being implemented with the modifications requested by the IEC.
The study documentation will also be submitted to the ethical committee of the Goethe University according with new regulations. Any appropriate amendment to the protocol will also be submitted to the IECs.

• Health Authorities
The sponsor will submit the study protocol

Patient information and consent
The investigator will explain to the subjects the objectives and methods of the clinical trial as well as the potential risks of the trial and of the possible discomforts which might happen to them.
They will be informed that they can choose not to participate in the trial and that they are free to retrieve their consent at any time without having any disadvantage.
A written document, called "Subject Information Sheet and Consent Form", approved by the IEC, written in German in an easily understanding wording, will be handed over to the subject by the investigator. After reading, understanding and having the responses to all the questions, the subject will give his written consent twice by initialing each page and dating and signing the last page.
By signing the same form at the bottom of the page, the investigator will confirm that he/she has provided all information contained in the form prepared for this trial.
The signed consent will be obtained before engaging any trial procedure with the subject. One copy is intended for the subject, and the original for the investigator's source document file.
The Subject Information Sheet and Consent Form for each participating center is filed in Appendix I.

Confidentiality
The aim and content of this trial, its investigational products and its results must not be disclosed, other than to those directly involved in the conduct or ethical review of the trial, without written authorization by ORVACS.
All information from this study (excluding data from informed consent) will be entered into a computer by the sponsor in accordance with the French law "Loi informatique te libertés" (Art.

Insurance Policy
Insurance will be provided by ORVACS. A Copy of the Insurance certificate is filed in Appendix III.

Compensation
Healthy volunteers will receive a compensation of 250 Euros for their participation in the trial.
This compensation has been calculated on the number of visits, blood withdrawals and on the time they will spend at the Center at each visit. Additionally, public transportation costs within the city will be covered for a maximum of 40 Euros during the whole study.

Protocol amendments
Neither the investigator nor the sponsor may alter the protocol without the permission of the other parties.
All changes to the protocol will be subject to an amendment which must be dated and signed by both parties (Investigator and Sponsor) and must appear as an addendum to the protocol.
Depending on the importance of the change in the conditions of the trial, the amendment will be sent to the IEC for prior approval or for information. It will be also submitted or sent to the relevant regulatory authorities if applicable.

Source document requirement
It is the responsibility of the investigator to assure that -the trial is conducted in accordance with the protocol, -valid data are entered into the Case Report Form, To achieve this objective, the investigator will permit auditor to monitor the trial and check the Case Report Forms, Informed consent and related source documents during regular visits in order to determine that data recording and protocol adherence are satisfactory. The investigator and his staff will be expected to cooperate with the auditor providing any missing information whenever possible.
In addition, the investigator guarantees: -Prior to each monitoring visit all data should be properly record in the Case Report Forms, -At the monitoring visits source documentation should be available, (in case of electronic medical file, a dated and signed printed copy will be sorted out and filed within the CRF).

Inspections by regulatory agencies
By signing this protocol, the investigator agrees to allow any regulatory agency to have access to the study records for review. These personnel, bound by professional secrecy, will not disclose any personal identity or personal medical information. These audits involve review of source documents supporting the adequacy and accuracy of data gathered in CRFs, review of documentation required to be maintained, and checks on investigational products accountability. The investigator will have to be available to provide the auditors with all the requested documents and be available to answer to any questions.
In case of an audit, an audit certificate will be issued and joined to the final report.

Publications
Prior to publication ORVACS will receive a final report co-established by principal investigator clinical study investigator, and immunology study coordinator and the statistical data management center, in due time after the end of the study. The results will be published by the principal investigators and coworkers in a peer-reviewed journal after agreement between sponsor representative, principal investigator clinical investigator, and immunology study coordinator. Neither investigators nor the sponsor may publish without the permission of other parties.

Clinical Study Report
At completion of the data analysis, a final report will be drawn up. This report will be a clinical and statistical integrated report. This report will be signed by the Sponsor representative(s) and the clinical study investigators. If the investigator retires, relocates, or for any other reasons withdraws from the responsibilities of keeping the study records, custody must be transferred to a person who will accept the responsibility. The Sponsor must be notified in writing of the name and address of the new custodian. Fan H, Lin Q, Morrissey GR, Khavari PA: Immunization via hair follicles by topical application of naked DNA to normal skin. Nat Biotechnol 17 (9)
Mon consentement ne décharge en rien le promoteur de l'étude de ces responsabilités, je conserve tous mes droits garantis par la loi. J'accepte librement de participer à cette recherche dans les conditions précisées dans la note d'information qui m'a été remise. 2. It is the duty of the physician to promote and safeguard the health of the people. The physician's knowledge and conscience are dedicated to the fulfillment of this duty.
3. The Declaration of Geneva of the World Medical Association binds the physician with the words, "The health of my patient will be my first consideration," and the International Code of Medical Ethics declares that, "A physician shall act only in the patient's interest when providing medical care which might have the effect of weakening the physical and mental condition of the patient." 4. Medical progress is based on research which ultimately must rest in part on experimentation involving human subjects.
5. In medical research on human subjects, considerations related to the well-being of the human subject should take precedence over the interests of science and society.
6. The primary purpose of medical research involving human subjects is to improve prophylactic, diagnostic and therapeutic procedures and the understanding of the aetiology and pathogenesis of disease. Even the best proven prophylactic, diagnostic, and therapeutic methods must continuously be challenged through research for their effectiveness, efficiency, accessibility and quality.
7. In current medical practice and in medical research, most prophylactic, diagnostic and therapeutic procedures involve risks and burdens.
8. Medical research is subject to ethical standards that promote respect for all human beings and protect their health and rights. Some research populations are vulnerable and need special protection. The particular needs of the economically and medically disadvantaged must be recognized. Special attention is also required for those who cannot give or refuse consent for themselves, for those who may be subject to giving consent under duress, for those who will not benefit personally from the research and for those for whom the research is combined with care. 9. Research Investigators should be aware of the ethical, legal and regulatory requirements for research on human subjects in their own countries as well as applicable international requirements. No national ethical, legal or regulatory requirement should be allowed to reduce or eliminate any of the protections for human subjects set forth in this Declaration.

B. BASIC PRINCIPLES FOR ALL MEDICAL RESEARCH
10. It is the duty of the physician in medical research to protect the life, health, privacy, and dignity of the human subject.
11. Medical research involving human subjects must conform to generally accepted scientific principles, be based on a thorough knowledge of the scientific literature, other relevant sources of information, and on adequate laboratory and, where appropriate, animal experimentation.
12. Appropriate caution must be exercised in the conduct of research which may affect the environment, and the welfare of animals used for research must be respected.
13. The design and performance of each experimental procedure involving human subjects should be clearly formulated in an experimental protocol. This protocol should be submitted for consideration, comment, guidance, and where appropriate, approval to a specially appointed ethical review committee, which must be independent of the investigator, the sponsor or any other kind of undue influence. This independent committee should be in conformity with the laws and regulations of the country in which the research experiment is performed. The committee has the right to monitor ongoing trials. The researcher has the obligation to provide monitoring information to the committee, especially any serious adverse events. The researcher should also submit to the committee, for review, information regarding funding, sponsors, institutional affiliations, other potential conflicts of interest and incentives for subjects.
14. The research protocol should always contain a statement of the ethical considerations involved and should indicate that there is compliance with the principles enunciated in this Declaration.
15. Medical research involving human subjects should be conducted only by scientifically qualified persons and under the supervision of a clinically competent medical person. The responsibility for the human subject must always rest with a medically qualified person and never rest on the subject of the research, even though the subject has given consent.
16. Every medical research project involving human subjects should be preceded by careful assessment of predictable risks and burdens in comparison with foreseeable benefits to the subject or to others. This does not preclude the participation of healthy volunteers in medical research. The design of all studies should be publicly available.
17. Physicians should abstain from engaging in research projects involving human subjects unless they are confident that the risks involved have been adequately assessed and can be satisfactorily managed. Physicians should cease any investigation if the risks are found to outweigh the potential benefits or if there is conclusive proof of positive and beneficial results.
18. Medical research involving human subjects should only be conducted if the importance of the objective outweighs the inherent risks and burdens to the subject. This is especially important when the human subjects are healthy volunteers. 19. Medical research is only justified if there is a reasonable likelihood that the populations in which the research is carried out stand to benefit from the results of the research.
20. The subjects must be volunteers and informed participants in the research project.
21. The right of research subjects to safeguard their integrity must always be respected. Every precaution should be taken to respect the privacy of the subject, the confidentiality of the patient's information and to minimize the impact of the study on the subject's physical and mental integrity and on the personality of the subject.
22. In any research on human beings, each potential subject must be adequately informed of the aims, methods, sources of funding, any possible conflicts of interest, institutional affiliations of the researcher, the anticipated benefits and potential risks of the study and the discomfort it may entail. The subject should be informed of the right to abstain from participation in the study or to withdraw consent to participate at any time without reprisal. After ensuring that the subject has understood the information, the physician should then obtain the subject's freely-given informed consent, preferably in writing. If the consent cannot be obtained in writing, the nonwritten consent must be formally documented and witnessed.
23. When obtaining informed consent for the research project the physician should be particularly cautious if the subject is in a dependent relationship with the physician or may consent under duress. In that case the informed consent should be obtained by a well-informed physician who is not engaged in the investigation and who is completely independent of this relationship.
24. For a research subject who is legally incompetent, physically or mentally incapable of giving consent or is a legally incompetent minor, the investigator must obtain informed consent from the legally authorized representative in accordance with applicable law. These groups should not be included in research unless the research is necessary to promote the health of the population represented and this research cannot instead be performed on legally competent persons.
25. When a subject deemed legally incompetent, such as a minor child, is able to give assent to decisions about participation in research, the investigator must obtain that assent in addition to the consent of the legally authorized representative.
26. Research on individuals from whom it is not possible to obtain consent, including proxy or advance consent, should be done only if the physical/mental condition that prevents obtaining informed consent is a necessary characteristic of the research population. The specific reasons for involving research subjects with a condition that renders them unable to give informed consent should be stated in the experimental protocol for consideration and approval of the review committee. The protocol should state that consent to remain in the research should be obtained as soon as possible from the individual or a legally authorized surrogate.
27. Both authors and publishers have ethical obligations. In publication of the results of research, the investigators are obliged to preserve the accuracy of the results. Negative as well as positive results should be published or otherwise publicly available. Sources of funding, institutional affiliations and any possible conflicts of interest should be declared in the publication. Reports of experimentation not in accordance with the principles laid down in this Declaration should not be accepted for publication.

C. ADDITIONAL PRINCIPLES FOR MEDICAL RESEARCH COMBINED WITH MEDICAL CARE
28. The physician may combine medical research with medical care, only to the extent that the research is justified by its potential prophylactic, diagnostic or therapeutic value.
When medical research is combined with medical care, additional standards apply to protect the patients who are research subjects.
29. The benefits, risks, burdens and effectiveness of a new method should be tested against those of the best current prophylactic, diagnostic, and therapeutic methods. This does not exclude the use of placebo, or no treatment, in studies where no proven prophylactic, diagnostic or therapeutic method exists. (See footnote*) 30. At the conclusion of the study, every patient entered into the study should be assured of access to the best proven prophylactic, diagnostic and therapeutic methods identified by the study.
31. The physician should fully inform the patient which aspects of the care are related to the research. The refusal of a patient to participate in a study must never interfere with the patientphysician relationship.
32. In the treatment of a patient, where proven prophylactic, diagnostic and therapeutic methods do not exist or have been ineffective, the physician, with informed consent from the patient, must be free to use unproven or new prophylactic, diagnostic and therapeutic measures, if in the physician's judgement it offers hope of saving life, reestablishing health or alleviating suffering. Where possible, these measures should be made the object of research, designed to evaluate their safety and efficacy. In all cases, new information should be recorded and, where appropriate, published. The other relevant guidelines of this Declaration should be followed.

Note of Clarification on Paragraph 29 of the WMA Declaration of Helsinki
The WMA hereby reaffirms its position that extreme care must be taken in making use of a placebocontrolled trial and that in general this methodology should only be used in the absence of existing proven therapy. However, a placebo-controlled trial may be ethically acceptable, even if proven therapy is available, under the following circumstances: -Where for compelling and scientifically sound methodological reasons its use is necessary to determine the efficacy or safety of a prophylactic, diagnostic or therapeutic method; or -Where a prophylactic, diagnostic or therapeutic method is being investigated for a minor condition and the patients who receive placebo will not be subject to any additional risk of serious or irreversible harm. All other provisions of the Declaration of Helsinki must be adhered to, especially the need for appropriate ethical and scientific review.

Scientific Background
Transcutaneous vaccination strategies aim to target antigen-presenting cells of the skin in their natural environment, and various studies have shown that transcutaneously applied vaccine preparations induce specific and robust immune responses. The aim of this study ís to assess the immune response against the transcutaneously applied test vaccine Tetagrip® (0.5 ml) compared to conventional i.m. injection of 0.5 ml Tetagrip® vaccine.
The condition of the skin, e.g. the hydration state, the production of sebum, etc. is an important factor for the percutaneous penetration of topically applied compounds (Lotte 1987, Lademann 2001, Schaefer 2001. The measurement of the skin physiological parameters TEWL, SCH, Skin pH and sebum production is therefore included in the study.

Methods
Transepidermal water loss (TEWL), stratum corneum hydration, skin pH and sebum production will be assessed using a Multi Probe Adapter MPA® (Courage-Khazaka, Cologne, Germany). We use a portable system which consists of the different probes, the Multi-Probe-Adapter and a laptop which is connected to the device. The measurements will be performed in the Clinical Research Center for Hair and Skin Physiology, Department of Dermatology, Charité-Universitätsmedizin Berlin or in the HIV Treatment & Research Unit, Department of Internal Medicine II, University Frankfurt am Main, respectively. Room-temperature and air humidity will be documented. All skin measurements will be performed according to the manufacturer's recommendation (see Appendix) after an adjustment period of 30 minutes. The measurements will be performed on Day 0, Day 1, Day 7, Day 14 and Day 28 of the study. All the skin measurements performed in this study are based on non-invasive methods. The results can be obtained within seconds by placing the probes on the skin surface.
The following probes will be used on the Multi Probe Adapter:

Tewameter® TM 300
The Tewameter is used to measure the rate of water loss through the skin. Since the skin barrier regulates the rate of water loss from the body, the rate of transepidermal water loss is a measure of the condition of the skin barrier. When skin is damaged, its barrier function is impaired resulting in high water loss. The Corneometer® CM 825 confidential ORVACS 18/08/200918/08/ Final version 08/08/2005 The corneometer measures the amount of moisture in the outer layer of the skin (skin hydration) and the ability of the skin to retain moisture (skin moisture capacity The measurement of the skin moisture is based on the internationally recognized Corneometer®-method (a capacitance method).

The Skin-pH-Meter® PH 905
The skin pH will also be measured directly on the skin surface. This is possible because the excretions of the skin are almost an aqueous solution.

Sebumeter® SM 815
The sebumeter method is based on a photometric method (grease spot photometer) which allows to directly measure the sebum secretion of the skin.

Scientific Background
This study aims to assess the immune response against the epicutaneously applied test vaccine Tetagrip® (0.5 ml) compared to conventional i.m. injection of 0.5 ml Tetagrip® vaccine. Tetagrip® vaccine is approved and commercially available in France, where it is routinely used for vaccination against tetanus and influenza. Vaccination against influenza is recommended for individuals > 60 years of age, individuals who suffer from chronic dieases and for medical personal only, which means that a large population is influenza-naive. Tetagrip® vaccine therefore represents a safe and approved test vaccine for these proof of concept investigations. Kenney et al. recently demonstrated that the intradermal administration of only one fifth of the standard i.m. dose of an influenza vaccine elicited immunogenicity that was similar or better than that elicited by common i.m. injection (Kenney 2004). We therefore hypothesize that the epicutaneous application of Tetagrip® vaccine in the commercially available standard preparation of 0.5 ml should be sufficient to induce an immune response.

General information
This SOP applies to volunteers who receive Tetagrip® vaccine transcutaneously. The application will be performed at room temperature. Prior to the application the volunteer will be seated in the test room for 30 minutes to adjust to the conditions. During the incubation periods between the different steps of application the volunteer remains seated. He will have the possibility to watch TV/Video. After the application of Tetagrip® vaccine, the volunteer will be instructed not to take a shower or bath and to avoid any activity which causes sweating or mechanical stress to the investigational site, e.g. physical exercise, during the following 24 hours.

Positioning of the arm
The left arm will be abducted to 90° and positioned horizontally on a plastic pad with the external part of the upper arm facing upwards. The arm will be kept in this position for the whole duration of the application. It may only be lowered during the drying period after the application of cyanacrylate glue (see "4. CSSS").

Demarcation of the investigational site
Two squares of 4 x 4 cm will be delimitated on the external part of the left upper arm using a plastic template. Adhesive tape strips will be used to tape the template on the skin. The investigational sites will be delimitated using a permanent skin marker (skin marker H7003, Falc).

Shaving
The hair will be shaved on the investigational sites as well as on the surrounding skin (2 cm on the top and the bottom of each investigational site and 1 cm on both sides) using a dry razor (Disposable Hospital Razor, Art-Nr.: 182 H, Wilkinson Sword GmbH, Solingen, Germany).  fibres and to avoid pain during the following cyanacrylate skin surface stripping, the investigational site and the surrounding skin will be shaved.

Cyanacrylate Skin Surface Stripping (CSSS)
After the shaving, cyanacrylate glue (superglue, UHU GmbH & Co. KG, Buehl/Baden, Germany) will be applied drop-wise (9 drops per investigational site = ca. 190 mg) on each investigational site. The edge of a glass microscope slide will be used to spread the glue evenly on the investigational sites. Immediately after the spreading, adhesive tape (6x5 cm, Nr. 57176-00000, 66m x 50mm, Tesa® Beiersdorf, Beiersdorf, Germany) will be placed on the investigational site. A sheet of paper (21x7cm) will be placed on top of the tape and a rubber roll will be rolled uniformly 10 times across the investigational sites to spread the glue evenly on the skin surface and to eliminate air bubbles. During this waiting period, the left arm may be lowered. The volunteer remains seated. After 20 minutes, the arm will be repositioned as described in "1. Positioning of the arm", the tape and the adhering glue will be removed quickly from the skin surface.

Fig.2:
Cyanacrylate Skin Surface Stripping (CSSS) removes keratinized material, lipids and other cell debris from the follicular openings and approx. 30% of the stratum corneum. Occasionally hair fibres, especially vellus hairs, are removed as well (Mills 1983). The remaining stratum corneum and the viable epidermis are left intact. This technique is routinely used in our laboratory and also by other groups to improve the percutaneous penetration of topically applied compounds and to determine the amount of substance which enters the follicular reservoir (Hueber 1994, Schaefer 2001. After spreading of the cyanacrylate glue on the skin surface, adhesive tape is placed on top of the glue layer. When the glue is hardened, removal of the adhesive tape also removes the glue, the adherent stratum corneum and follicular casts (A-C). In our preliminary studies, one CSSS on the upper arm of human volunteers removed the follicular casts of all hair follicles and approx. 10% of the hair fibres. After the CSSS, the investigational sites will be deliminated with a silicone barrier Nr. 4469/ko,Wendelstein,Germany) to prevent spreading of the subsequently applied vaccine. The silicone barrier has to dry for 20 minutes. During this incubation time, the volunteer remains seated and the left arm remains in its position (see "1 Positioning of the Arm").

Application of Tetagrip® vaccine
250 µl of Tetagrip® vaccine are applied drop-wise from the original syringe as provided by the manufacturer onto the skin surface of each investigational site (16 drops per investigational site, each drop approx. 16 µl). A soft massage will be applied by moving a gloved finger tip (care & serve®) presaturated with Tetagrip® vaccine for ca. 1 minute in circles on the investigational site to spread the vaccine evenly on the skin surface and to improve the percutaneous penetration (Genina 2002, McElnay 1993). This procedure is followed by an incubation time of 20 minutes.

Application of Comfeel bandage
At the end of the application procedure a hydrocolloid bandage (Comfeel® Plus Transparent 9x 14cm Art.-Nr.: 3542, Coloplast A/S, Denmark) will be applied to protect the investigational sites and to provide occlusion which may further enhance the percutaneous penetration of Tetagrip® vaccine. The bandage remains on the skin for 24 hrs. It will be removed by the investigator on Day 1, 24 hrs after the application of Tetagrip® vaccine on Day 0. The volunteers will be instructed not to take a shower or bath and to avoid any activity which causes sweating or mechanical stress to the investigational site, e.g. physical exercise, during these 24 hours. Index 1. Material 2. Methods 3. Appendices

Methods
The purpose of this procedure is to ensure the adequate collection, shipment and retrieval of blood Before all manipulations, please pay attention, wear gloves and lab coats.
Blood samples transportation to the immunology laboratory in Paris will be done at room temperature.
Immunological determinations on fresh blood samples need to be done within 24 h maximum after drawn, therefore it is mandatory that blood samples transportation time does not exceed 22h h.

Blood Collection
Four blood samples (10 ml/tube) are planned in the protocol for immunological determinations at the following time points : D-7, D0, D7, D14 and D28.
Blood samples should be obtained as : 1 dry tube 3 A C D t u b e s ( 1 0 m l ) All tubes should be identified with patient code before blood withdrawn Blood samples should be maintained at room temperature while waiting for shipment.

Blood samples shipment
Blood samples should be shipped to the immunology laboratory in Paris.
The shipment will be carried out by the « World Courier » (WC) company located in Germany following instructions were given them by ORVACS At the scheduled times (D-7, D0, D7, D14 and D28) the fresh blood samples will be picked-up by a representative of WC. It is strongly recommended to request the samples pick-up by the transporter only once the scheduled patient is on site. This is with the aim of avoiding additional fees in case the patient could not come to have the visit. To request the pick-up of samples, the investigator will call to WC -Germany to the following phone number 0049 30 243 14 xxx, by e-mail to jstreit@worldcourier.de or tglatzer@worldcourier.de, or by fax to 0049 30 243 14 219.A preprinted fax form is included below for each participating center.
Fresh blood samples will be shipped as diagnostic specimens packed with IATA p pa ac ck ki in ng g i in ns st tr ru uc ct ti io on n 6 65 50 0. . The packaging PI650 will be supplied by WC.
A form "list of contents" (see below) should be filled at each shipment. In this form the consignee address as well as the nature of the samples will be indicated.
Note : To guarantee that all immunological determinations will be done within 24 hours after drawn, it is strongly recommended to schedule blood withdrawal of volunteers/patients between 10 and 14 hours, Monday through Thursday.
The contact address at ORVACS is :

MATERIAL
Specific marerials are specified for each procedure before technique explanation.

METHODS FOR SPECIFIC IMMUNOLOGY
This study will be performed in the core laboratory (Laboratoire d'Immunologie Cellulaire -Paris) using standard operating procedure.

A. SERUM STORAGE FOR SPECIFIC ANTIBODY MEASUREMENT
Ten ml of blood collected in a dry tube will be spun, 10 minutes at 2 000rpm, to obtain at least 2 ml of serum. Make 4 aliquots each containing 1 ml of serum, and store immediatly at -80 °C.

B. SPECIFIC ANTIBODY MEASUREMENT
Antibodies will be measured by specific sub-contractors with expertise in each field • Antibodies against Influenza will be measured at the Centre National de Référence de la Grippe -I. Pasteur, according to a standard operating procedure for evaluation of titers of neutralizing antibodies (inhibition of haemaglutination).
• Antibodies against Tetanus Toxoïd will be measured by ELISA at the Laboratoire de Microbiologie, Hôpital Henri Mondor.

C. PERIPHERAL BLOOD MONONUCLEAR CELL (PBMC) SEPARATION.
• PBMC will be isolated from fresh blood collected in ACD tubes, after centrifugation over a Ficoll-Hypaque gradient at 2200rpm for 30 minutes and washed twice in PBS before use.

a. Intended Use
The ELISPOT assay is designed to enumerate cytokine producing CD8 and/or CD4 T cells in a single cell suspension. This method has the advantage of requiring a minimum of in-vitro manipulations allowing cytokine production analysis as close as possible to in-vivo conditions. This technique is designed to determine the frequency of cytokine producing cells under a given stimulation, and the followup of such frequency during a treatment and/or a pathological state.
b. Principle of the method After cell stimulation, locally produced cytokines are captured by a specific monoclonal antibody. After cell removal, trapped cytokine molecules are revealed by a secondary biotinylated detection antibody, which is in turn recognized by streptavidin conjugated to alkaline phosphatase. PVDF-bottomed-well plates are then incubated with NBT/BCIP substrate. Colored "purple" spots indicate cytokine production by individual cells.
A response is considered positive when the number of spots obtain after deduction of the background is above 50 spot/million of cells. It is feasible on frozen cells with a good reproducibility compared to fresh cells as shown in the Manon 01 study (to be published).
-Phosphate buffered saline (PBS) 1X (If stock solution in 10X concentration prepare PBS 1X for the whole procedure). Store at +4°C.

d. Antigens
CD4 and CD8 T cell responses against tetanos and Influenza Neuraminidase will be evaluated in triplicate experiments with : • Tetanus Toxoïd antigen -Dilute capture antibody at 1/1000 in PBS. Mix strongly and dispense 100 µl into each well, cover the plate and incubate 2 hours at 37°C in a CO2 incubator or overnight at +4°C (the plate can be prepared 5 days in advance). -Wash wells three times with 100 µl of sterile PBS.
-Dispense 100 µl of culture medium into wells, cover and incubate for 30 minutes at 37°C. Empty wells by flicking the plate over a sink and tapping it on absorbent paper. -Cell preparation : dilute PBMC in culture media at 106/ml, 105/ml Do not wash wells.
-Dispense into wells 100 µl of sterile cell suspension containing the appropriate number of cells and adequate concentration of stimulator in triplicates. Leave one line with medium alone for negative control. Add the peptide in appropriate concentration or the mitogen (positive control: PHA-p = 0,5 µg/ml). Cover the plate with a standard 96-well plate plastic lid and incubate cells at 37°C in a CO2 (5%) incubator for 20 hours.
During this period do not agitate or move the plate.
-Wash again 3 times with PBS.
-This washing procedure is better performed on an automated washer alternatively it could be done manually. -Dilute reconstituted detection antibody at 1/500 into 10 ml of PBS-0,5% BSA. This step has to be prepared extemporaneously! Distribute 100 µl in wells, cover the plate and incubate 4 hours at 37°C, 5% C02.
-Wash wells three times with PBS.
At this stage it is important to remove all residual buffer. -Prepare the dye (for one plate): 10 ml Buffer Tris + 45 µl NBT + 35 µl BCIP (respect this order) extemporaneously! -Distribute 100 µl of NBT/BCIP buffer in wells.
-Let the reaction go for 10 min, no longer that 15 min, at room temperature. (Do not cover the plate) -Rinse wells three times with distilled water.
-Dry wells. Read spots the day after, or store the plate at room temperature away from direct light until lecture which can be post when plates are kept in a dark dry atmosphere.
-Read out on dried plates either immediately after assay or after storage in dark and dry condition, on an automated ELISpot reader. A response is considered positive when the number of spots obtain after deduction of the background is above 50 spot/million of cells

D-2. ICC INTRA CELLULAR CYTOKINE CYTOMETRY DETECTION
a. Intended use The nature of cell producing IFNγ upon specific antigen stimulation will be defined in a second step after results of ELISPOT on total PBMC have shown which antigens induce positive responses (above 50 SFC/million PBMC). It is chosen for practicability and accuracy of analysis to define the CD4 and CD8 cell origin by intra-cellular flow cytometry (ICC) IFNγ detection after stimulation with the positive antigen. This assay will be performed on cryopreserved cells. • Tetanus Toxoïd antigen : • Influenza Neuraminidase -Incubate 2 hours at 37°C in 5 % CO 2 -Distribute 20 µl of BFA in each well -Incubate for 4 hours at 37°C in 5 % CO 2 This preparation could be performed the same day as ICC, or the day before if the plate is stored overnight at 4°C.

E. CRYOPRESERVATION OF CELLS AND THAWING PROCEDURES
Cryopreservation: The PBMCs will be frozen in 10 % of DMSO (Dimethylsulfoxide) and 90% of FCS (Fetal calf serum) in at least 4 vials containing a minimum of 10x 10 6 PBMC, within 24 hours of blood harvest. Cells will be prepared in a sterile way on ice then immediately placed in a freezing box (Nalgene cryo 1°C freezing container), previously placed at 4°C; then immediately at -80°C for 18 hours, then transferred to nitrogen liquid for storage. The viability of the cryopreserved cells should be above 80%.
Thawing procedure : Before thawing the cells, prepare a 15 ml tube (A) containing 10 ml of RPMI-50 % FCS. Put the vial in the heat bath until obtaining an ice pea.
With 5 ml of medium of the tube A harvest the cells and rinse the vial in order to obtain the maximum of cells. Spin the tube 8 minutes at 1400 rpm, resuspend the pellet in 10 ml of RPMI-20% FCS. Spin again the tube 8 minutes at 1400 rpm, then resuspend the pellet in 4 ml of RPMI -5% FCS. Count the cells (% of alive with exclusion dye for dead cells and dead cells), and adjust at 1.106 million live cells/ml of medium. Leave the cells in a culture flask overnight in incubator. The next morning, transfer the cells in a 15 ml tube, rinse the flask with a small volume of 20% FCS and transfer in the 15ml tube. Centrifuge the tube at 1400 rpm for 8 minutes and resuspend the pellet in 4 ml 20%-FCS. Measure the volume, count the cells and adjust the volume to obtain 1 million cells/ml.  To be completed by the methodology/data management department representative responsible for the randomization of the subject:

Name of study manager: Norma Wincker
Name of the responsible for randomization: Tchadie BOMMENEL The volunteer/patient respect all the criteria of eligibility and he is definitively included in the study.

Comments (if appropriate) :
Signature of study manager: Signature of randomization responsible: The volunteer/patient will receive Tetagrip® vaccine:      Investigator's name: Signature: