First-in-Man Open Clinical Trial of a Combined rdESAT-6 and rCFP-10 Tuberculosis Specific Skin Test Reagent

Background Tuberculin is still the only available skin test reagent for the diagnosis of mycobacterial infection. The product has a remarkable sensitivity, but poor specificity. Previous studies, including two human phase I clinical trials, have indicated that rdESAT-6 has a potential as an improved skin test reagent. Animal studies have shown that the sensitivity may be increased by inclusion of the genetically related CFP-10 antigen in the preparation without loosing specificity. Methodology In this study a Lactococcus fermented, recombinant skin test reagent consisting of a 1∶1 wt/wt of rdESAT-6 and CFP-10 was manufactured according to GMP standards and tested for the first time in 42 healthy adult volunteers. The two doses of 0.01 µg or 0.1 µg were injected intradermally by the Mantoux technique with 6 or 12 weeks interval. No serious adverse events and only mild adverse reactions were reported. The reagent elicited a positive skin test reaction after the first injection in one participant, who most likely was latently infected with M. tuberculosis as indicated by an appreciable IFN γ response just below the Quantiferon® cut-off level at the screening visit. None of the remaining participants in the four groups had any skin test reactions and sensitisation by the reagent could therefore be excluded. Conclusion The investigational skin test reagent rdESAT-6 and CFP-10 appeared safe and non-sensitising in this first-in-man clinical trial in human volunteers and can now be tested in larger clinical trials involving individuals with latent M. tuberculosis infection or active TB disease. Trial Registration ClinicalTrials.gov NCT00793702


Introduction
Tuberculosis continues to be a major cause of morbidity and mortality throughout the world. The disease is caused by infection with Mycobacterium tuberculosis (M. tuberculosis) and remains one of the most important fatal infections of human beings, with 9 million new cases every year and an estimated 2 million deaths [1].
To control the disease fast and accurate diagnosis is very important; however in many clinical situations this is not possible as the existing diagnostic methods have serious limitations [13]. The tuberculin skin test (TST) with Purified Protein Derivative (PPD) has been used world-wide for almost 100 years to support the diagnosis of tuberculosis (TB) as well as for screening in national TB programs and epidemiological studies [2,3]. The drawback of PPD is the fact that its protein components are known to be shared by many non-tuberculous/environmental mycobacterium families as well as by the BCG vaccine strains. This fact significantly decreases the specificity of the Tuberculin Skin Test (TST), since individuals exposed to non-tuberculous mycobacteria or vaccinated with BCG respond immunologically to PPD as well as those who are infected with the tuberculous families (M. tuberculosis, M. bovis, or M. africanum) [4].
Recently it has been discovered that the tuberculous bacteria contains a number of specific proteins/antigens that are not present in BCG or most environmental mycobacteria. The ESAT-6 (6 kD Early Secreted Antigen Target) protein and CFP-10 (10 kDa culture filtrate protein-10) were identified from a M. tuberculosis culture filtrate [5,28]. Both antigens, ESAT-6 and CFP-10 are expressed by the tuberculous mycobacterium families (M. tuberculosis, M. bovis, and M. africanum) but not by any of the BCG-strains and only very few of the atypical mycobacteria express the proteins (e.g., M. kansasii, M. marinum, M. szulgai) [4,28].
Based on this knowledge new in-vitro diagnostic tests have been developed in the form of T-cell based interferon γ-assays. These assays use Mycobacterium tuberculosisspecific antigens (e.g. ESAT-6, CFP-10) and show that T-cells of individuals infected with M. tuberculosis produce interferon-γ when they are exposed to these antigens [14].
The tests show very promising abilities in detecting latent infection with M. tuberculosis and were shown to be able to discriminate between patients infected by M. tuberculosis and BCG vaccinated individuals [6,7,8,9,28].
A new skin test using the rdESAT-6 + rCFP-10 antigens has been developed by SSI, Denmark and has shown to induce local delayed type hypersensitivity (DTH) skin reactions when injected intradermally to guinea pigs infected with M. tuberculosis [28,36,37]. patients at all tested dose levels. Furthermore reactions of similar size in the groups injected with 0.1 µg rdESAT-6 and 2 T.U. PPD (in separate arms) were observed. Based on these observations a dose level of 0.1 µg rdESAT-6 was selected for the next phase 1 clinical trial. [29].
Results from non clinical pharmacology studies in guinea pigs have demonstrated a risk of sensitisation upon repeated injections of rdESAT-6 + rCFP-10 visualized by a local induration and erythema at the injection site. To address this issue a phase 1b clinical trial was conducted at Rigshospitalet , Copenhagen in 2007 to assess the risk of sensitisation upon repeated injections of a fixed dose level of 0.1 µg rdESAT-6 with time intervals of 28, 56 or 112 days, respectively.
In this phase 1b clinical trial with the rdESAT-6 reagent (alone) it was documented that repeated injections of the rdESAT-6 can be given safely, but that positive reactions may occur due to sensitisation. However, the risk may be diminished by a proper time span between succeeding testing [31].
In March 2007, SSI decided to include rCFP-10 to increase the diagnostic performance of the skin test reagent reflected by an improved sensitivity without jeopardizing the specificity of the skin test reagent [28]. Non clinical pharmacology trials at SSI demonstrated in guinea pigs that rCFP-10 mixed with rdESAT-6 in the ratio 1:1 was optimal.
Due to the above change in the development plan the present clinical trial has been designed to investigate the safety and the risk of sensitisation of the improved skin test reagent in healthy adult volunteers. It will be the first in man clinical trial using a mixture of rdESAT-6 + rCFP-10. Three dose levels of 0.01, 0.1 and 1.0 µg rdESAT-6 + rCFP-10 mixed in the ratio 1:1 (w/w) will be investigated. Furthermore the possible risk of sensitisation of the rdESAT-6 + rCFP-10 skin test will be investigated by giving two test doses of 0.01 and 0.1 µg skin test reagent with time intervals of 6 or 12 weeks, respectively.
Reece et al [33] recently suggested that CFP-10 may trigger "Tuberculin shock". He found that 5/10 guinea pigs died 6-36 hours after the skin testing with CFP-10, if the test was done 6 weeks after IV infection. No deaths were observed if the skin testing was done after 4 weeks or earlier. A more likely explanation is that the testing and the deaths were coincidental as the guinea pigs after 6 weeks of infection were at a late stage of disease. In a repeat study No. F1137 [40] done at SSI with an infection period of 6 weeks before testing, 3/30 (10%) animals died or were about to die from tuberculosis even before skin testing. The same number of animals died after skin testing with rCFP-10 or PPD. It is likely that an immunological reagent at this time of disease will be able to induce a shock-like syndrome in the animals.
Non clinical toxicity studies performed in 2008 concluded that repeated subcutaneous injections of 10 µg rdESAT-6 +rCFP-10 were safe in rats.
Based on the nonclinical pharmacology and toxicity studies and two phase I clinical trials with rdESAT-6 (alone), it is expected that administration of rdESAT-6 + rCFP-10 skin test by the Mantoux injection technique in doses between 0.01 and 1.0 µg only will expose the volunteers to MINIMAL risks, such as local injection site reactions. Injection of the rdESAT-6 + rCFP-10 skin test reagent is intended to identify latent tuberculosis infection by the formation of a local erythema and induration at the injection site 48 h -96 h after the injection (positive reaction). No reaction is expected to occur in uninfected persons after a single injection. In the present study a skin reaction after the first injection will be regarded as a sign of latent infection or an adverse reaction. We will investigate if a positive skin reaction occurs after a second injection of rdESAT-6 + rCFP-10 given to healthy individuals at an interval of 6 or 12 weeks. In case such a response appears it may be regarded as a sensitisation reaction induced by the preceding injection. Such a false positive skin reaction appears similar to a positive skin reaction in a TB infected person. To the individual the response does not represent a safety issue, but it is an undesired property by the reagent.
50 male/female healthy adult volunteers will be allocated to 5 groups (A, B, C, D and E) of 10 participants. The dose levels of 0.01 and 0.1 µg rdESAT-6 + rCFP-10 will be injected twice with time intervals of 6 and 12 weeks, respectively (Group A to D). The 1.0 µg dose level will only be injected once (Group E). Details of the immunological status of each volunteer (BCG vaccination, suspected mycobacterial exposure etc.) will be recorded at study entry.

Primary objective
To assess the safety of three dose levels (0.01, 0.1 and 1.0 µg) of the rdESAT-6 + rCFP-10 skin test reagent when injected to healthy adults.

Secondary objective
To assess the risk of sensitisation of two dose levels (0.01 and 0.1 µg) of the rdESAT-6 + rCFP-10 skin test reagent when injected twice with time intervals of 6 or 12 weeks to healthy adults.

Primary variable
Local and systemic adverse events with onset after the FIRST (0.01, 0.1 and 1.0 µg) or SECOND (0.01 and 0.1 µg) injection.
Induration will be regarded as an effect variable and will not be categorized as a local adverse event.
Erythema ≥ 20 mm will be regarded as a local adverse reaction.

Secondary variables
-Sensitisation: The diameter of induration at the second injection site measured transversely to the long axis of the forearm 72 hours after the SECOND skin test injection (0.01 and 0.1 µg). Induration ≥ 6 mm will be regarded as a possible sensitisation reaction.
-Sensitisation: QuantiFERON ® -TB Gold in tube test results (in-vitro IFN-γ responses) measured in blood samples taken 28 days after the SECOND skin test injection (0.01 and 0.1 µg). An IFN-γ response of ≥ 0.35 IU/mL will be regarded as a possible sensitisation reaction.

Investigational Plan
This phase I clinical trial has been planned in accordance with 'Note for guidance on good clinical practice', CPMP/ICH/135/95, ICH Topic E 6 [18]. It is an open phase 1 clinical trial to investigate the safety and the risk of sensitisation by escalating doses and repeated injections of the rdESAT-6 + rCFP-10 skin test reagent following intradermal administration to healthy adults. Three dose levels of the rdESAT-6 + rCFP-10 reagent will be investigated (0.01, 0.1 and 1.0 µg). The dose levels of 0.01 and 0.1 µg will be injected twice with time intervals of 6 or 12 weeks.

Overall design
The trial is a single-centre phase 1 clinical trial and will be conducted at the Department of Infectious Diseases at Rigshospitalet, Blegdamsvej 9, 2100 København Ø, Denmark, under the responsibility of principal investigator: Åse Bengård Andersen.
The primary objective is to assess the safety of the new in-vivo rdESAT-6 + rCFP-10 reagent with three different dose levels of 0.01, 0.1 and 1.0 µg.
The potential sensitisation risk of rdESAT-6 + rCFP-10 will primarily be evaluated by measuring the diameter of induration at the injection site 72 hours after the SECOND skin test injection (i.e reading the diameter in millimetres). An induration of ≥ 6 mm will be regarded as a possible sensitisation reaction.
In addition, the in vitro interferon-γ response results from the QuantiFERON ® TB Gold in tube tests taken at the screening visit, prior to administration of the second rdESAT-6 + rCFP-10 dose and 28 days after the second rdESAT-6 + rCFP-10 dose will be compared (i.e. secondary sensitisation risk variable). An IFN-γ response of ≥ 0.35 IU/mL 28 days after the SECOND rdESAT-6 + rCFP-10 injection will be regarded as a possible sensitisation reaction.
50 healthy non-black male and/or female adult volunteers ≥ 18 and ≤ 55 years of age will be included in the trial.
Three dose levels will be investigated. The dose levels of 0.01 and 0.1 µg will be injected twice with a timeinterval of 6 and 12 weeks, respectively (Groups A, B, C and For safety reasons there will be at least 1 hour between the injections of the rdESAT-6 + rCFP-10 skin test reagent to different volunteers. Volunteers in the lowest dose groups A and B (0.01 µg rdESAT-6 + rCFP-10) will be included first and tested before proceeding to the next dose level. If no safety concerns are detected within 72 hours after the first injection in the last volunteer of group A, group C and D volunteers (0.1 µg) may be given their first injection. If no safety concerns are detected within 72 hours after the first injection in the last volunteer of group C volunteers in group E (1.0 µg) may be given their first injection.
The first dose of the rdESAT-6 + rCFP-10 reagent (0.01 or 0.1 µg) will be injected on day 0 in the RIGHT forearm of the volunteers in Groups A, B, C and D. A follow-up visit will take place 72 hours after the first dose.
The second dose will be administered on day 42 of the LEFT forearm of the volunteers in Group A (0.01 µg) and Group C (0.1 µg) or on day 84 in Group B (0.01 µg) and Group D (0.1 µg), with follow-up visits after 72 hours and after 28 days, respectively.
Volunteers allocated to Group E will only be injected with one rdESAT-6 + rCFP-10 injection with the high dose level of 1.0 µg in the RIGHT forearm. Follow up visits will take place 72 hours and 28 days after the FIRST and only rdESAT-6 + rCFP-10 injection.
For an overview of the injection schedule for all groups (A, B, C, D and E) please refer to following Volunteers with a positive sensitisation reaction or a positive QuantiFERON test result will be asked to return for a final check 6 months after the last visit in the trial to exclude an infection with tuberculosis.

Study population
Five groups of each 10 male or female QFT negative healthy adult volunteers will be included. Pregnant and/or breastfeeding female volunteers will be excluded from participation in the trial.
To obtain the optimal conditions for an accurate visual evaluation of the size of the skin test reactions (induration and/or redness) volunteers with black skin will not be included.

Recruitment
Healthy adults will mainly be recruited from the University of Copenhagen. An advertisement approved by the Ethics Committee will if needed be published in a national newspaper for further recruitment.

Inclusion criteria
The volunteer:

Predetermined reasons for discontinuation
Trial volunteers are free to withdraw from the clinical trial whenever they desire. Volunteers withdrawn from or discontinuing the trial after the FIRST injection should (if possible) for safety concerns stay in the trial for the follow up visits but will not receive the SECOND injection. These volunteers will be replaced by new volunteers. If the volunteer is withdrawn or discontinues the trial after the SECOND injection he or she will not be replaced.
No reaction is expected to occur in uninfected persons after a single injection. In the present study a skin reaction after one injection will be regarded as sign of latent infection or an adverse reaction and the volunteer should (if possible) for safety concerns stay in the trial for the follow-up visits but must NOT receive the SECOND vaccination. The volunteer should in this case be replaced by a new volunteer.
If, for any reason, a volunteer wishes to discontinue her/his participation in the trial, or if, according to principal investigator's judgement, she/he must be withdrawn from the trial, the date and reason for the withdrawal must be recorded in the CRF.
The trial can be terminated at any time if the sponsor, the principal investigator or the independent data safety monitoring board decides that the trial poses an unacceptable threat to the volunteers.

Handling of withdrawn volunteers and drop-outs
For withdrawn volunteers and for volunteers who drop out before the planned last visit, a Termination Visit will be performed. At the Termination Visit, a full medical examination will be performed, blood and urine samples will be collected, and adverse events, if any, will be assessed and recorded. If an individual has special needs, proper medical action will be taken. Such action will be at the discretion of the investigator.
All data will be included in the safety and efficacy analysis of the results, including data from withdrawn volunteers or volunteers who dropped out of the trial before the planned last visit.

Treatments administered
The rdESAT-6 + rCFP-10, is manufactured at SSI in Denmark according to Good Manufacturing Practice (GMP) standards.
The test solution is filled into 3 mL glass vials with perforable rubber stoppers and aluminium caps with white, red and blue plastic 'flip-off' tops indicating the following concentrations 0.01, 0.1 and 1.0 µg respectively. Each vial is intended for administration of 0.1 mL to one trial volunteer.

Doses and administration
The rdESAT-6 + rCFP-10 skin test is administered by the Mantoux technique by staff with training and experience in the Mantoux testing technique.
The test product should preferably be taken out of the refrigerator half an hour before each injection is performed.
Before the administration the "used by" date must be checked.
The test may only be administered if it appears as a clear and colourless solution.
Disinfection of the injection site before the injection is not necessary. If the injection site is disinfected anyway, it must be completely dry before the injection of the test product.
Use a sterile 1.0 mL disposable syringe fitted with a large-size needle to aspirate the test fluid from the vial. Replace the needle with a short-bevelled needle 26 gauge and administer 0.1 mL of the test solution by the Mantoux technique as specified by SSI (see appendix 7).
Draw up slightly more than 0.1 mL, remove air bubbles and adjust the volume to exactly 0.1 mL. The appearance of a small papule of 8-10 mm in diameter after the injection indicates correct injection technique. The papule disappears after approximately 10 minutes.
If the injection fails, i.e. does not result in a papule (as described above), this must be recorded in the CRF together with the reason. A failed injection must under no circumstances be repeated in the same volunteer. In case the FIRST injection fails in a given volunteer, he/she should (if possible) for safety concerns stay in the trial for the follow-up visits but must NOT receive the SECOND vaccination. The volunteer should in this case be replaced by a new volunteer. If the SECOND vaccination fails, the given volunteer should for safety reasons continue in the trial but should not be replaced by a new volunteer.
No reaction is expected to occur in uninfected persons after a single injection. In the present study a skin reaction after one injection will be regarded as sign of latent infection or an adverse reaction and the volunteer should (if possible) for safety concerns stay in the trial for the follow-up visits but must NOT receive the SECOND vaccination. The volunteer should in this case be replaced by a new volunteer.
The syringe and the needle must be discarded as a single unit after injection in a labelled, puncture-proof container. The vial must be kept together with the cardboard box until drug accountability documents have been completed and approved by the monitor. This will normally take place at the termination monitoring visit.

Packaging and labelling
The packaging and labelling is performed at SSI according to Good Manufacturing Practice (GMP). The vials will be packed in boxes. Each box will contain 10 vials.

Storage information
The shelf-life of the rdESAT-6 + rCFP-10 skin test is 6 months if stored in a refrigerator at + 2ºC to + 8ºC. Stability studies are ongoing at SSI and if the product shows to be stable an extension of the shelf-life may occur during the study.
To avoid exposure to light the vials should be kept in the cardboard boxes until administered. The "used by" date is printed on the vials. The test products are stored in a locked fridge at the Department of Infectious Diseases at Rigshospitalet. The refrigerator where the test products are stored is equipped with a temperature surveillance system and an alarm system. Staff at the Department of Engineering at Rigshospitalet are responsible for the monitoring of storage conditions. Principal investigator Åse Bengård Andersen is responsible for the dispensing of the trial product. The dispensed test solutions will be recorded in a dispensing log.
In case the refrigerator, where the test items are stored, breaks down, the test items should be moved to another refrigerator as soon as possible. The reason and time of when the test items are moved to another location must be registered in the temperature log of the test items.
In case of significant storage condition deviations, as judged by the principal investigator, the items subject to the deviations may not be used and the SSI clinical trial monitor should be contacted as soon as possible for advice.
The SSI clinical monitor will seek information from relevant parties at SSI, in respect to decide whether the test items, which were subject to the deviations, should be destructed or whether they can still be used in the trial.

Samples of labels for test product
Examples of the labels for the cardboard boxes and the vials will be provided as a separate document when submitting the protocol to the Competent Authorities.

Transport of study products
The test product will be transported by car under controlled temperature conditions (2-8 °C). Transport, dispatch and receipt procedures will be documented.

Randomisation procedure
By nature the trial has to be open, but it could in principle be randomised. Randomisation would, however, require that all volunteers who were found eligible at the inclusion visit would have to accept to follow any of the 5 different study schedules before the randomisation took place. This would make it difficult to recruit volunteers for the trial, so it was decided to run the trial without randomisation.

Drug accountability
All dispensed test products will be recorded in a dispensing log. All used and unused vials must be kept and returned to SSI to be accounted for at the end of the trial. Procedures in relation to the dispensing and the accountability of the test products will be the responsibility of the principal investigator Åse Bengård Andersen. The trial monitor from SSI will check the records.

Precautions and overdosing
Anaphylactic reactions are rarely seen in relation to tuberculin testing. Such reactions have not been seen in the previous phase 1a and 1b trial with the rdESAT-6 skin test; however the necessary treatment for an anaphylactic reaction must be accessible.
A vasovagal reaction to the Mantoux injection can occur.
The rdESAT-6 + rCFP-10 skin test reagent must be injected intradermally into the superficial layer of the dermis by study staff with training and experience in the Mantoux testing technique.

Concomitant medication
Concomitant medication considered necessary for the volunteer during the course of the trial should be recorded in the concomitant medication pages of the case record form (CRF) of the volunteer.
Volunteers should omit to use the following medications or live vaccines during the trial as they are all known to act as immune modulators and are therefore likely to compromise the skin test results: However if such medications are taken, they must be recorded on the concomitant medication pages of the CRF.
Intake of the above listed medications would normally not compromise the safety of the trial volunteers and would therefore not necessarily lead to withdrawal from the trial.
Decisions on withdrawal will be at the discretion of the investigator. See Section 8.4 for medications and live vaccines that are listed under exclusion criteria. Individuals who have taken these medications ≤ 3 months or have been vaccinated with a live vaccine 6 months PRIOR to entry in the trial should NOT proceed to Visit 2 -Inclusion Visit. Adverse events There are a total of 5 groups (A, B, C, D and E). In Groups A, B, C and D two injections (T1 and T2) are given. In Group E only one injection is given (T1) 2) The screening visit (Visit No. 1) takes place up to 28 days before the Inclusion visit (Visit No. 2) 3) For Groups A, B, C and D, Visit 6 is performed 28 days after T2. For Group E, Visit 6 is performed 28 days after T1 4) A termination visit should be performed for volunteers who drop out or are withdrawn from the trial 5) General medical examination Urine sample for: glucose and protein 8) Body temperature will be measured orally and must be below 38.0°C at Visit 2 and Visit 4 9) T1 = 1 st rdESAT-6 + rCFP-10 test; T2 = 2 nd rdESAT-6 + rCFP-10 test 10) Diary 1 is handed out on T1 day. Diary 2 is handed our on T2 day. The diaries are reviewed and collected at the following visits. 11) Blood sample for measuring in-vitro Interferon-γ response upon ESAT-6 stimulation with the QuantiFERON ® -TB Gold In Tube test 12) Volunteers with a positive sensitisation reaction or a positive QuantiFERON test result will be asked to return for a final check 6 months after the last visit in the trial. 13) A digital image will only be taken if there are visible signs of a reaction to the rdESAT-6 + rCFP-10 tests

Allocation of volunteers to study groups
After the volunteer has been found eligible for the trial at the inclusion visit he/she is allocated to the first (not already occupied) study group with an acceptable time schedule (in the order A, B, C, D, E).

Assessment of safety
For safety reasons there will be at least 1 hour before the next volunteer is given an injection of the rdESAT-6 + rCFP-10 skin test reagent. All adverse events will be assessed and reported by the investigator, as specified in Section 11. A detailed description of any local or systemic adverse events will be made in the CRF.
In case of local adverse events, additional images, if applicable, will be made of both the RIGHT (All Groups) and the LEFT (Groups A, B, C and D) arm's injection sites at the time of occurrence. The course of any adverse events will be documented by sequential descriptions and, if applicable, by making digital images until the events have completely disappeared. Erythema ≥ 20 mm will be regarded as a local adverse reaction.
The volunteers will be given a diary after the first (All Groups) and the second (Groups A, B, C and D) rdESAT-6 + rCFP-10 test where all adverse events (local and systemic) should be recorded. The volunteers should record all adverse events (if any) and an oral temperature daily one week after the FIRST injection (All Groups) and two weeks after the SECOND injection (Groups A, B, C and D). At the following visits the recorded adverse events will be transferred by the investigator to the CRFs.
In a previous phase 1a clinical trial conducted at Leiden University Medical College, the Netherlands, the rdESAT-6 (only) skin test was investigated at 4 different dose levels (0.01 µg, 0.1 µg, 1 µg and 10 µg) in a group of healthy volunteers and later at 3 different dose levels (0.01 µg, 0.1 µg and 1 µg) in a group of previously treated TBpatients.
No toxicity signs were seen in the 4 groups of healthy volunteers. In previous TB patients minor local adverse reactions were seen in the groups of 0.01µg and 0.1µg rdESAT-6. Increasing the dose to 1µg responses were seen in most subjects and there was a higher frequency and intensity of local adverse reactions. There were also general complaints in this group e.g headache, however, with uncertain relationship to the trial product.
In the later phase 1b clinical trial conducted at Rigshospitalet, Copenhagen to assess the sensitisation risk of repeated injections of a fixed dose of 0.1 µg rdESAT-6 (only) at time intervals of 28, 56 and 112, it was documented that the repeated injections of the rdESAT-6 skin test could be given safely.
As previously mentioned Reece et al [33] recently suggested that CFP-10 may trigger "Tuberculin shock". He found that 5/10 guinea pigs died 6-36 hours after the skin testing with CFP-10, if the test was done 6 weeks after IV infection. No  with an infection period of 6 weeks before testing, 3/30 (10%) animals died or were about to die from tuberculosis even before skin testing. The same number of animals died after skin testing with rCFP-10 or PPD. It is likely that any immunological reagent at this time of disease will be able to induce a shock-like syndrome in the animals.
Non clinical toxicity studies performed in 2008 concluded that repeated subcutaneous injections of 10µg rdESAT-6 + rCFP-10 were safe in rats.
Based on the non clinical pharmacology and toxicity studies and two phase I clinical trials with rdESAT-6 (alone), it is expected that administration of the rdESAT-6 + rCFP-10 skin test by the Mantoux injection technique in doses between 0.01 µg and 1.0 µg only will expose the volunteers to MINIMAL RISKS, such as local reversible adverse reactions at the injection sites. The rdESAT-6 + rCFP-10 test is not likely to give rise to systemic adverse reactions.

Assessment of sensitisation
In the previous phase 1a and 1b clinical trials investigating the rdESAT-6 (alone) skin test reagent both redness and induration were observed as delayed type hypersensitivity reactions. The result of the classical tuberculin skin test is, however, by consensus expressed as millimetres induration only and induration (only) will therefore be regarded as the result to the new rdESAT-6 + rCFP-10 skin test reagent. Redness will in this present clinical trial be measured but will only be regarded as an exploratory value.
Delayed-type hypersensitivity (DTH) reactions will be measured independently by two experienced members of the study staff team and then compared.
In case of a discrepancy between the corresponding values of both study staff members, consensus will be sought by examining the reactions together. Only the consensus diameter will be recorded in the CRF.
On the day of the injections, the injection sites of the test substances will be marked with four dots on the direct proximal, distal arm with a black marker pen at a distance of at least 3 cm from the injection sites. This is to avoid later confusion about the precise location of the injection sites of the skin tests and to allow orientation on the digital images. The FIRST rdESAT-6 + rCFP-10 test (All Groups) will be placed on the RIGHT forearms of the volunteers and the SECOND rdESAT-6 + rCFP-10 test (Groups A, B, C and D) will be placed on the LEFT forearms of the volunteers.
Induration is measured by palpation of the injection sites from lateral to central on all sides. Because the study staff will make independent measurements of the diameters of induration, the borders will not be marked on the skin.
Redness is the visible red or pink discoloration of the skin around the injection sites. The border is assessed visually. Because the study staff will make independent measurements, the staff will not mark the borders on the skin. Next to the arm, a label will be placed indicating the subject number, visit number and, finally, if it is the FIRST or SECOND injection with the rdESAT-6 + rCFP-10 test. A ruler with millimetre indications will be placed at least 3 cm from the injection site.
Injection of the rdESAT-6 + rCFP-10 skin test reagent is intended to identify latent tuberculosis infection by the formation of a local erythema and induration at the injection site 48 h -96 h after the injection (positive reaction). No reaction is expected to occur in uninfected persons after a single injection. In the present study a skin reaction after the first injection will be regarded as a sign of latent infection or an adverse reaction. We will investigate if a positive skin reaction occurs after a second injection of rdESAT-6 + rCFP-10 given to healthy individuals at an interval of 6 or 12 weeks. In case such a response appears it may be regarded as a sensitisation reaction induced by the preceding injection. Such a false positive skin reaction appears similar to a positive skin reaction in a TB infected person. To the individual the response does not represent a safety issue, but it is an undesired property by the reagent.
The sensitisation risk of rdESAT-6 + rCFP-10 will be evaluated by assessing the diameter of induration of the injection site 72 hours after the injection of the SECOND rdESAT-6 + rCFP-10 dose (i.e reading in millimetres). An induration of ≥ 6mm will be regarded as a possible sensitisation reaction.
In addition, the sensitisation risk will be evaluated by comparing and assessing the in vitro interferon -γ response from blood samples taken at the screening visit, prior to administration of the second rdESAT-6 + rCFP-10 dose and 28 days after the second rdESAT-6 + rCFP-10 dose using the QuantiFERON ® -TB Gold In Tube test. An IFN-γ response of ≥ 0.35 IU/mL will be regarded as a possible sensitisation reaction.
Volunteers with a positive sensitisation reaction or a positive QuantiFERON test result will be asked to return for a final check 6 months after the last visit in the trial to rule out a tuberculosis infection.

Collection and handling of blood samples
Venous blood samples will be obtained by the standard techniques at Rigshospitalet using the Vacutainer ® system. All local guidelines and safety measures of Rigshospitalet with regard to blood samples will be followed. Tubes will be labelled with Rigshospitalet's 'identification stickers' with a unique number and a unique bar code identifying the volunteer from whom the sample was taken and the date the sample was taken.
Standard laboratory forms will be used and print-outs from the laboratory will be signed and initialized by investigator. In case of out of range values assessed by the investigator as not clinically significant, the investigator will write NS (or a similar abbreviation) at the individual value and date and sign the value (with initials). At each blood drawing 11 mL blood will be drawn for the safety tests, 3 mL blood for the QuantiFERON test and 7 mL blood (only at screening) for the HIV, HBV and HCV tests.
Blood samples to the Department of Clinical Chemistry will be transported according to standard procedures at Rigshospitalet. Blood samples for the QuantiFERON tests will be transported to the Mycobacteria Laboratory at Statens Serum Institut by taxi.

Laboratory assays
The routine laboratory assays will all be performed at the Department of Clinical Biochemistry at Rigshospitalet according to standard procedures.
The QuantiFERON ® -TB Gold In Tube test will be performed at the Department of Mycobacteria at Statens Serum Institut according to standard procedures.
Out of range blood test results for the routine chemistry and haematology laboratory assays will be assessed by the investigator. If the investigator assesses that the values are NOT clinically significant the volunteer is eligible for inclusion.

Time schedule
The documents to the EC and The Danish Medical Agency (Laegemiddelstyrelsen) are planned to be submitted in September 2008.
The first patient's first visit (FPFV) is planned to be in November 2008.
The last patient's last visit (LPLV) is planned to be in April 2009.

Ethical Aspects
Tuberculosis continues to be a major cause of morbidity and mortality throughout the World as being one of the most important fatal infections of human beings, with 9 million new cases every year and an estimated 2 million deaths in 2004. [1].
To control the disease fast and accurate diagnosis is very important. The new diagnostic tool to be investigated in this phase 1 clinical trial is a traditional tuberculin test skin test but with two new antigens, rdESAT-6 and rCFP-10, as the active ingredients.
Participation in this clinical trial is voluntary, and the volunteer may choose to leave the trial at any time without any specific reason.
Before entering the trial, the volunteer will be informed (verbally and in writing) that there is a risk of unexpected adverse reactions, and that this risk has to be taken into account when considering participation. Detailed information about all trial procedures and the inconveniences these might pose on the volunteer will be explained as well. The volunteer will be informed that he or she is welcome to bring a third party to receive the information. A brochure from the Central Ethical Committee ("Før du beslutter dig" -see appendix 8) giving general information on the participation in clinical trials will be given to the volunteers before participation. In 2005/2006 the rdESAT-6 (only) skin test synthesised in Lactococcus lactis, was administered to humans for the first time in a phase 1a clinical trial conducted at Leiden University Medical College, the Netherlands. The aim of this first phase 1 trial was to investigate the safety and the diagnostic potential of the rdESAT-6 skin test. The rdESAT-6 skin test was investigated at 4 different dose levels (0.01 µg, 0.1 µg, 1 µg and 10 µg) in a group of healthy volunteers and later at 3 different dose levels (0.01 µg, 0.1 µg and 1 µg) in a group of previously treated TB-patients.
No toxicity signs were seen in the 4 groups of healthy volunteers. In previous TB patients minor local adverse reactions were seen in the groups of 0.01 µg and 0.1 µg rdESAT-6. In the group of 1µg responses were seen in most subjects and there was a higher frequency and intensity of the local adverse reactions. There were general complaints in this group of e.g headache, however, with uncertain relationship to the trial product.
In the succeeding phase 1b clinical trial (TESAT-02) investigating the sensitisation risk of repeated administration of the rdESAT-6 (only) skin test reagent; it was documented that the repeated injections of the rdESAT-6 skin test could be given safely.
The trial participants in this current phase 1 trial will all be healthy volunteers ≥ 18 years and ≤ 55 years of age.
Individuals with ongoing conditions or medical histories which might make them more vulnerable to the procedures undertaken in this clinical trial or which might interfere with the performance of the skin test are excluded from participation. These are as follows: − Haemophilia or other coagulation disorder (due to the risk from repeated blood drawings) − Has been in treatment with a product which is likely to modify the immune Based on the non clinical pharmacology and toxicity studies of the rdESAT-6 + rCFP-10 and two phase I clinical trials with rdESAT-6 (alone), it is expected that the administration of the rdESAT-6 + rCFP-10 skin test by the Mantoux injection technique in doses between 0.01 and 1.0 µg only will expose the volunteers to MINIMAL RISKS, such as local reversible adverse reactions at the injection sites. The rdESAT-6 + rCFP-10 test is not likely to give rise to systemic adverse reactions.
Detailed information on the planned number and amount of blood samples will be part of the verbal and written information. In conclusion, it is anticipated that there is a reasonable balance between the risks and the inconveniences to which the volunteers will be exposed and the benefits they might obtain as a result of the increased medical attention which is part of the clinical trial, i.e. medical examinations and laboratory investigations.

Payment of Volunteers
Trial volunteers assigned to groups A-D are paid 1800 DKK (liable to pay taxes) at the end of the trial (i.e. on completion of Visit 6) as compensation for the number of blood samples they have given and for inconveniences caused by the intradermal administration of the skin tests. In addition the volunteer will be paid 200 DKK (liable to pay taxes) as compensation for transport expenses which will be paid (in cash) after the completion of each visit. The volunteers sign for the receipt of all payments.
A healthy volunteer assigned to groups A-D and who completes the entire trial will be paid a total of: 1800 DKK + (6 × 200 DKK) = 3000 DKK Trial volunteers assigned to group E are paid 1200 DKK (liable to pay taxes) at the end of the trial (i.e. on completion of Visit 6) as compensation for the number of blood samples they have given and for inconveniences caused by the intradermal administration of the skin tests. In addition the volunteer will be paid 200 DKK (liable to pay taxes) as compensation for transport expenses which will be paid (in cash) after the completion of each visit. The volunteers sign for the receipt of all payments.
A healthy volunteer assigned to group E and who completes the entire trial will be paid a total of:

DKK + (4 × 200 DKK) = 2000 DKK
A budget is part of the agreement signed by the Department of Infectious Diseases at Rigshospitalet and SSI.

Data Safety Monitoring Plan
The present clinical trial is expected to expose the trial volunteers to minimal risks only. The principal investigator takes the responsibility for the safety of the volunteers in the daily clinical practice. However, the principal investigator might need advice in certain critical clinical situations and, therefore, a Data Safety Monitoring Board has been established. The Data Safety Monitoring Board will, when needed, as judged by the principal investigator, evaluate critical events that occur during the trial. The independent Data Safety Monitoring Board members are all medical experts in the Netherlands and in Denmark.
When the last volunteer has completed Visit 3 in the respective groups A to D the principal investigator will go through all relevant safety information and arrive at a recommendation as to whether or not to proceed with the SECOND injections in the respective groups. Furthermore, safety information available at Visit 3 in group A (0.01 µg/0.1 mL) will be evaluated before group C (0.1 µg/mL) volunteers can be given their first injection, as well as safety information available at Visit 3 in group C (0.1 µg/mL) will be evaluated before any injection is given to group E (1.0 µg/mL) volunteers.
In a brief written statement the principal investigator will give a review of reported adverse events and she will give her recommendations in respect to giving the SECOND injections and in respect to initiating the FIRST injections at the subsequent dose level. The principal investigator will also decide if a DSMB meeting is needed.
This brief written statement will be provided by e-mail to the study director, to all investigators in the trial, and to the Data Safety Monitoring Board members by e-mail as soon as possible and no later than 7 days after the last patient's Visit 3 in groups A, B, C and D. The study director will make sure that Trine R. Nielsen (medically responsible at SSI) and Anders Mørup Jensen (statistically responsible at SSI) are kept informed at all times.
At most occasions e-mail correspondence is expected to be sufficient for discussions and for reaching conclusions. If discussions take place via e-mail, the study director, all investigators in the trial and all Data Safety Monitoring Board members should always receive copies, so that these persons are well informed at any time.

Adverse Events
This section reviews the procedures for recording and reporting of adverse events in the trial. Relevant definitions and terms are listed. Furthermore, the procedures for immediate reporting of serious adverse events to SSI and for expedited reporting to the competent authorities and to the local ethics committee are described.

Definitions and terms
All definitions in the following are in accordance with the ICH E2A guideline [21].

Adverse Event (AE)
Any untoward medical occurrence in a patient or a volunteer participating in a clinical investigation and receiving a pharmaceutical product, which does not necessarily have a causal relationship with this product.

Adverse Reaction/Adverse Drug Reaction/Suspected Adverse Reaction (AR/ADR/SAR)
Any untoward and unintended response to an investigational product related to any dose administered. The terms 'Adverse Reaction', 'Adverse Drug Reaction and 'Suspected Adverse Reaction' are the same thing (in practice), and imply that there is a suspected relationship between the event and the trial product. In practice this means that there is evidence or arguments that suggest a causal relationship, i.e. a relationship cannot be ruled out.

Unexpected adverse reaction
An adverse drug reaction, the nature or severity of which is not consistent with the applicable product information (e.g., the Investigator's Brochure).

Immediate reporting
The investigator's rapid reporting of a serious AE/AR/ADR/SAR to SSI (reporting within 72 hours of the investigator's first knowledge of the AE/AR/ADR/SAR).
Expedited reporting SSI's rapid reporting of a suspected unexpected serious adverse reaction (SUSAR) to the concerned competent authorities and the ethics committees. Only, SUSARs must be reported not SSARs. In Demark, the ethics committees only want annual update reports (i.e. no expedited reporting to the ethics committees is required in this trial).
Fatal or life-threatening, SUSARs occurring in the trial qualify for very rapid reporting, and the Danish Medicine Agency (Laegemiddelstyrelsen) should be notified (e.g., by telephone, facsimile transmission, or in writing) as soon as possible but no later than 7 calendar days after the sponsor's first knowledge of the event, followed by a complete a report within 8 additional calendar days.
SUSARs, which are not fatal or life-threatening, must be submitted as soon as possible but no later than 15 calendar days after the sponsor's first knowledge of the case meeting the criteria for expedited reporting.

Standard reporting of adverse events
The investigator is responsible for the recording of all reported adverse events in the Adverse Event Forms of the CRFs during the visits. The investigator must use the following terms: The causal relationship between an adverse event and the trial tests is assessed using the following terms: The intensity of an adverse event is assessed using the following terms: − Mild (i.e. easily tolerated) − Moderate (i.e. sufficient to interfere with daily activities) − Severe (i.e. sufficient to prevent normal activity) The outcome of an adverse event is assessed using the following terms: To ensure no confusion or misunderstanding of the difference between the terms "serious" and "severe," which are not synonymous, the following note of clarification is provided: The term "severe" is often used to describe the intensity (severity) of a specific event (as in mild, moderate, or severe myocardial infarction); the event itself, however, may be of relatively minor medical significance (such as severe headache). This is not the same as "serious," which is based on patient/event outcome or action criteria usually associated with events that pose a threat to a patient's life or functioning. Seriousness (not severity) serves as a guide for defining regulatory reporting obligations.

Expedited reporting of adverse events
The investigator is responsible for the immediate reporting (within 72 hours) to SSI of all serious adverse events (SSARs and SUSARs).
The investigator should always exercise medical and scientific judgement in deciding whether immediate reporting is appropriate.
The investigator should fill in the CIOMS form (see Appendix 1) and fax it to the Department of Regulatory & Medical Affairs at SSI within 72 hours of his/her first knowledge of a serious adverse reaction/event.
The initial report should be followed by follow-up reports (using the same form) if additional important information becomes available.
SSI will ensure that expedited reporting to the competent authorities within the required time lines.
Furthermore, SSI is responsible for submitting annual safety update reports of serious adverse events (SUSARs and SSARs) to the competent authorities and to the ethics committees (or as required by the competent authorities or ethics committees).
Post study events: Although such information is not routinely sought or collected by SSI, serious adverse events that occurred after the patient has completed the trial will possibly be reported by the investigator to the SSI. Such cases will be reported expedited to the competent authorities if they meet the criteria for expedited reporting specified above.

General considerations
The Biostatistics Unit at SSI will be responsible for data management and statistical analysis of the trial data.

Data management
Clinical data is collected in the CRFs. The CRFs are in English. The volunteers' diaries are in Danish and will be given to the volunteers on the days of skin testing. Information on adverse events noted in the diaries are transferred to the CRF by the investigator. The diaries are considered supportive for the investigators when filling in the CRFs, i.e. only relevant information from the diaries will be translated and transferred to the CRFs by the investigator. The diaries will not be sent to the sponsor's site but will remain filed at the investigator's site and filed with the investigator's copy of the CRFs.
The haematology and urinalysis data from the central lab. at Rigshospitalet will be submitted to the sponsor as paper print-outs.
The data from the QuantiFERON TB Gold in Tube test, analysed at SSI, will be submitted to the sponsor as paper print-outs.
Data from the CRFs, the central lab. at Rigshospitalet, and the QuantiFERRON results will be entered into SAS data sets and checked for consistency and plausibility by custom-made SAS programs. All ambiguous or implausible data items will be resolved by data queries to the investigators.

Clean file procedures
At the end of the trial, when the data entry from the CRFs and the laboratory measurements have been validated, the SAS data sets reach clean file status. Identical copies on CDs of the SAS data sets will then be made. One CD will be archived in the trial master file. The other CD will be archived at the Biostatistics unit.
The statistical analysis reported in the integrated clinical trial report will be based on the clean file data.

Analysis populations
All volunteers who have received at least one injection with the test substance will be included in the analysis.

Statistical methods
Because of the limited number of subjects included in the trial the statistical methods will mostly be descriptive.
All reported AEs, IFN-γ results, induration measurements and standard (clinical/chemical) laboratory measurements will be listed.
IFN-γ results, induration measurements and the standard laboratory measurements will be presented as plots.

Sample size determinations
The sample size is primarily based on practical issues and not statistical considerations.
Assume that the risk of sensitisation (defined as induration ≥ 6 mm or IFN-γ ≥ 0.35 IU/mL as measured by QuantiFERON ® TB Gold after the second injection) increases with dose and for a fixed dose of rdESAT-6 + rCFP-10 is higher if the two injections are given with a 6 week interval than with a 12 week interval.
If none of the 40 subjects in Groups A, B, C and D show evidence of sensitisation, not only the 10 subjects in that particular combination of dose and injection interval, but also those with a higher dose and those with a shorter injection interval can be taken into consideration in a conservative calculation of a one-sided upper 95% CI for the risk of sensitisation.

Good clinical practice considerations
The present phase I clinical trial is performed with the in-vivo diagnostic skin test, rdESAT-6 + rCFP-10 in humans. rdESAT-6 and rCFP-10 are both fermented in recombinant strains of Lactococcus lactis and purified separately and are, therefore, regarded as a biotechnologically-derived pharmaceutical.
The internationally recognised guidelines ICH S6 [16], ICH M3 [17] and ICH S7A [22] were used for the design of the pre-clinical study programme of rdESAT-6 + rCFP-10. However, as these guidelines are aimed at traditional pharmaceutical products, some adjustments were needed due to the special nature of rdESAT-6 + rCFP-10.
The ICH guideline, ICH Topic E 6 [18] and ICH Topic E 2A [21] were followed when designing this clinical trial. Furthermore the following documents were consulted for advice:

Declaration of Helsinki
This clinical trial will be conducted in accordance with the principles of the Declaration of Helsinki (see Appendix 2).

Volunteer information and informed consent
Healthy volunteers will mainly be recruited from the University of Copenhagen. Advertisements/posters will be objective and balanced and will be approved by the Ethics Committees, Region of Copenhagen ("De videnskabsetiske komitéer for Region Hovedstaden").
Potential volunteers are informed about the trial both verbally and in writing. The information is provided by trained staff in an objective way, describing both advantages and disadvantages of participation in the trial. The volunteer will be informed that he or she is welcome to bring a third party to receive the information.
After the verbal information, the 'Paticipant Information Sheet' (see Appendix 3) and a brochure from the Central Ethical Committee ("Før du beslutter dig") giving general information on the participation in clinical trials, will be given to the volunteers.
The informed volunteer is given reasonable time to consider whether he/she wishes to participate.
The inclusion of volunteers in the clinical trial will start AFTER the trial has been approved by the ethics committee.

Regulatory health authority submission and approval
An application file will be submitted for approval to the regulatory authorities in Denmark: the Danish Medicine Agency (Laegemiddelstyrelsen, Axel Heides Gade 1, 2300 København S). Only after approval by both the Ethics Committee ("De videnskabsetiske komiteer for Region Hovedstaden") and the Danish Medicine Agency the clinical trial will be initiated.

Volunteer data protection
The investigators are responsible for keeping a list (screening log) of all volunteers in the trial, including the subject numbers, full names and last addresses known, etc.
SSI will receive information on subject numbers, sex and date of birth only. This information will be traceable to the screening log.
Furthermore, the volunteers are informed in writing that the results will be stored and analysed by computer and that confidentiality will be maintained. Relevant Danish laws will be followed in maintaining confidentiality [38,39].
The volunteers are also informed (in writing) that authorised persons from the investigator's site, from the sponsor's site, or from the regulatory authorities may need to review his/her hospital records in relation to the clinical trial. By signing the informed consent form (see Appendix 4) the volunteer accepts these conditions.
Statens Serum Institut will according to local regulations inform "Datatilsynet" about the clinical trial.

Investigator's responsibilities
The Investigator is responsible for the conduct of the clinical trial in accordance with the protocol, Good Clinical Practice (GCP) [18,19,20] and relevant Danish regulations [23,24,25]. The Investigator is, moreover, responsible for the proper reporting of all adverse events according to the procedures described in this protocol in Section 11.
If the principal investigator delegates her responsibilities to another member of the staff this must be approved by SSI and confirmed in writing.

Curricula vitae and log(s) of staff
Before initiation of the trial, current signed and dated curricula vitae in English for the principal investigator, other investigators, other significant staff at the trial site and staff at the sponsor's site (SSI) must be collected. Staff participating in the clinical trial at the trial site and at the sponsor's site will, furthermore, be listed in log(s) of staff. For trial monitors, data managers, and other persons who are allowed to make entries in the CRFs or in other trial related documents, signatures and initials must also be listed in the log(s) of staff. The log of staff will be filed in the Investigator's file at the clinical trial site and in the Trial Master File at SSI.

Indemnity statement
Prior to the inclusion of the first volunteer in the clinical trial, SSI will present a signed Indemnity Statement to all investigators participating in the clinical trial (see Appendix 5).

Training
Before the inclusion of the first volunteer in the trial, it will be ensured that all study staff is adequately qualified. The study director will if necessary perform training sessions covering general GCP issues, for example: safety procedures, reporting of Suspected Unexpected Serious Adverse Reactions (SUSARs), information of volunteers, handling of test products etc. If training is needed in relation to the Mantoux testing technique, tuberculosis, etc., the principal investigator will perform these sessions. All performed sessions will be documented in the CVs of the investigators and the staff members performing the Mantoux testing etc.

Monitoring
The clinical trial monitor from SSI will make regular visits to the investigational site at Rigshospitalet. Together with the study staff at the site, the monitor will, for example, check the following: − That the protocol is being followed

Audit and inspection
The investigator must give access to personnel from SSI for the conduct of audits.
Regulatory authorities must at all times be allowed access to conduct inspections as well. Auditors and inspectors must have access to all trial related documents, including the Investigator's File and the volunteers' personal medical records.
− Nurse notes (i.e. for data either not recorded in the CRF or for data not recorded directly in the CRF) − The CRF for data recorded directly in the CRF − Dispensing of test product and accountability information in logs − The screening log for e.g. subject number, full name and address

Archiving of essential documents
It is the responsibility of the principal investigator and SSI to maintain the essential documents as described in the ICH guidelines for at least 15 years after the termination of the trial (i.e. after the last patient's last visit).
The principal investigator is responsible for archiving (at least) of the following documents:

Agreement and financial settlement
The agreement between the Investigational institution (Rigshospitalet) and SSI must be signed prior to inclusion of the first volunteer in the clinical trial. The agreement must clearly state the rights and obligations of the parties concerned and include a detailed financial settlement.
Furthermore, a written agreement will be made between the principal investigator and the laboratory at Rigshospitalet, regarding the safety laboratory analysis.

Insurance
SSI is the sponsor and manufacturer of the test products to be administered in this clinical trial. SSI carries a product liability insurance under a worldwide liability programme written by the New Hampshire Insurance Company, through insurance brokers Marsh A/S, Teknikerbyen 25, DK-2830 Virum, Denmark, as part of the worldwide Marsh insurance broker group. The policy covers claims arising from injury/injuries caused by trial medication used in clinical trials sponsored by the company, if the trial medication has been used in accordance with the instructions given in the protocol. The insurance certificate is enclosed in Appendix 6. The insurance conditions will be explained in writing to the volunteers (see Appendix 3).

Confidentiality and disclosure
All CRFs, information and results generated by SSI, as well as information on product development, patented or not, including patent applications and manufacturing processes not previously published, are considered confidential and shall remain the sole property of SSI.
No data from the clinical trial may be published, presented or communicated, except to regulatory authorities or ethics committees, prior to the release of the internal clinical trial report, unless approved by SSI in writing. All investigators agree not to discuss externally or publish any result from the trial without the possibility of SSI to give comments for up to 90 days after receipt of the manuscript.
In the event of a publication the names of the authors and their order of appearance will be as specified in the agreement between the Investigational institution (Rigshospitalet) and SSI.

Changes to the protocol
The clinical trial procedures may be changed if the principal investigator and the study director agree to the changes. If the changes are substantial, both the Ethics Committee and Competent Authorities must approve the changes before they can be implemented. All substantial changes must be documented by protocol amendments and rewritten full protocols, if applicable.

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.

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

1.
It is the duty of the physician in medical research to protect the life, health, privacy, and dignity of the human subject.

2.
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.

3.
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.

4.
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.

5.
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.

6.
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.

7.
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.

8.
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.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.

9.
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.

10.
The subjects must be volunteers and informed participants in the research project.

11.
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.

12.
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 non-written consent must be formally documented and witnessed.

13.
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. 14.
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.

15.
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.

16.
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.  Det nye hudtestreagens er mere specifikt. Det indeholder en opløsning af proteinerne rdESAT-6 + rCFP-10, som hovedsageligt findes i tuberkulosebakterien samt i små maengder i tuberkulin testen, men hverken i BCG vaccinen eller i de fleste ufarlige mykobakterier i omgivelserne.

Indemnity Statement
Trial title: An open phase I clinical trial on the safety and the risk of sensitisation by escalating doses and repeated injections of the rdESAT-6 + rCFP-10 skin test reagent following intradermal administration to healthy adults Trial code: TESEC-01 Dear Dr. Åse Bengård Andersen, You have kindly agreed to consider undertaking the above-mentioned clinical trial as investigator, in accordance with the protocol for the trial TESEC-01.
In the event that any recruited subject in the trial should suffer any personal injury resulting from the clinical trial, SSI agrees to indemnify the institution where the clinical trial is being undertaken, Rigshospitalet, Denmark, and, through Rigshospitalet, any of its employees or agents participating in the trial, against liability imposed by law, but not assumed voluntarily, and arising from the use of the test products in the trial, PROVIDED THAT: 1) SSI shall not indemnify against, nor have any obligation whatsoever as regards liability arising from or related to any error, omission, intentional wrongful act, or other negligence on the part of said institutions or persons, such as medical malpractice; and 2) Any such institution or person seeking indemnity a) has fully complied with the protocol for the trial, and b) has promptly notified SSI of any notice of any type of claim, or the likelihood of a claim, relating to the trial, c) as regards any claim, makes no statement, takes no action, nor makes any commitment affecting SSI's interests, without SSI's prior written consent, and further, provides all reasonable and necessary assistance to SSI in the defence of any claim, allowing SSI, at its cost and in its discretion, to take over the defence of any action and to have full control in handling the claim.
Please note that this letter is not a legal contract itself, but rather summarizes the main points of SSI's liability under its agreement with Rigshospitalet.
Yours sincerely, Statens Serum Institut