Safety and Immunogenicity of Boosting BCG Vaccinated Subjects with BCG: Comparison with Boosting with a New TB Vaccine, MVA85A

Objectives To investigate the safety and immunogenicity of a booster BCG vaccination delivered intradermally in healthy, BCG vaccinated subjects and to compare with a previous clinical trial where BCG vaccinated subjects were boosted with a new TB vaccine, MVA85A. Design Phase I open label observational trial, in the UK. Healthy, HIV-negative, BCG vaccinated adults were recruited and vaccinated with BCG. The primary outcome was safety; the secondary outcome was cellular immune responses to antigen 85, overlapping peptides of antigen 85A and tuberculin purified protein derivative (PPD) detected by ex vivo interferon-gamma (IFN-γ) ELISpot assay and flow cytometry. Results and Conclusions BCG revaccination (BCG-BCG) was well tolerated, and boosting of pre-existing PPD-specific T cell responses was observed. However, when these results were compared with data from a previous clinical trial, where BCG was boosted with MVA85A (BCG-MVA85A), MVA85A induced significantly higher levels (>2-fold) of antigen 85-specific CD4+ T cells (both antigen and peptide pool responses) than boosting with BCG, up to 52 weeks post-vaccination (p = 0.009). To identify antigen 85A-specific CD8+ T cells that were not detectable by ex vivo ELISpot and flow cytometry, dendritic cells (DC) were used to amplify CD8+ T cells from PBMC samples. We observed low, but detectable levels of antigen 85A-specific CD8+ T cells producing IFNγ (1.5% of total CD8 population) in the BCG primed subjects after BCG boosting in 1 (20%) of 5 subjects. In contrast, in BCG-MVA85A vaccinated subjects, high levels of antigen 85A-specific CD8+ T cells (up to 14% total CD8 population) were observed after boosting with MVA85A, in 4 (50%) of 8 subjects evaluated. In conclusion, revaccination with BCG resulted in modest boosting of pre-existing immune responses to PPD and antigen 85, but vaccination with BCG-MVA85A induced a significantly higher response to antigen 85 and generated a higher frequency of antigen 85A-specific CD8+ T cells. Trial Registration ClinicalTrials.gov NCT00654316 NCT00427830


The need for new vaccine against tuberculosis
Tuberculosis (TB) kills about three million people annually. It is estimated that one third of the world's population are latently infected with Mycobacterium tuberculosis (M.tb). Multi-drug resistant strains of M.tb, and co-infection with M.tb and HIV present major new challenges. The currently available vaccine, M. bovis BCG, is largely ineffective at protecting against adult pulmonary disease in endemic areas and it is widely agreed that a new more effective tuberculosis vaccine is a major global public health priority 1 . However, it may be unethical and impractical to test and deploy a vaccine strategy that does not include BCG, as BCG does confer worthwhile protection against TB meningitis and leprosy. An immunisation strategy that includes BCG is also attractive because the populations in which this vaccine candidate will need to be tested will already have been immunised with BCG. M.tb is an intracellular organism. CD4+ Th1-type cellular responses are essential for protection and there is increasing evidence from animal and human studies that CD8+ T cells also play a protective role 2 . However, it has generally been difficult to induce strong cellular immune responses in humans using subunit vaccines. DNA vaccines induce both CD4+ and CD8+ T cells and thus offer a potential new approach to a TB vaccine. DNA vaccines encoding various antigens from M. tuberculosis have been evaluated in the murine model, and to date no DNA vaccine alone has been shown to be superior to BCG 3,4 . A heterologous prime-boost immunisation strategy involves giving two different vaccines, each encoding the same antigen, several weeks apart. Such regimes are extremely effective at inducing a cellular immune response. Using a DNA-prime/MVA-boost immunisation strategy induces high levels of CD8+ T cells in animal models of malaria and HIV 5,6 , and high levels of both CD4+ and CD8+ T cells in animal models of TB 7 . BCG immunisation alone induces only CD4+ T cells in mice. A prime-boost strategy using BCG as the prime and a recombinant MVA encoding an antigen from M.tb that is also present in BCG (antigen 85A: 'MVA85A') as the boost, induces much higher levels of CD4+ T cells than BCG or MVA85A alone. In addition, this regime generates specific CD8+ T cells that are undetectable following immunisation with BCG alone.

Recombinant viruses as vaccines.
Recombinant viruses used alone have for some years represented a promising vaccine delivery system, particularly for inducing cellular immune responses 8 . The recombinant virus encodes the immunising protein or peptide. Immunisation by a recombinant virus vaccine occurs when host cells take up and express the inoculated attenuated virus encoding a protective antigen 9 . The expressed protein often has the native conformation, glycosylation, and other post-translational modifications that occur during natural infection. Recombinant viral vaccines may elicit both antibody and cytotoxic T-lymphocyte responses 10 , which persist without further immunisations. Many viruses have been investigated as potential recombinant vaccines. The successful worldwide eradication of smallpox via vaccination with live vaccinia virus highlighted vaccinia as a candidate for recombinant use 11,12,13 . The recognition in recent years that non-replicating strains of poxvirus such as MVA and avipox vectors can be more immunogenic than traditional replicating vaccinia strains has enhanced the attractiveness of this approach. MVA (modified vaccinia virus Ankara) is a strain of vaccinia virus which has been passaged more than 570 times though avian cells, is replication incompetent in human cell lines and has a good safety record. It has been administered to more than 120,000 vaccinees as part of the smallpox eradication programme, with no adverse effects, despite the deliberate vaccination of high risk groups 14,15 . This safety in man is consistent with the avirulence of MVA in animal models 16 . MVA has six major genomic deletions compared to the parental genome severely compromising its ability to replicate in mammalian cells 17 . Viral replication is blocked late during infection of cells but importantly viral and recombinant protein synthesis is unimpaired even during this abortive infection 18 . Replication-deficient recombinant MVA has been seen as an exceptionally safe viral vector 19,20 . When tested in animal model studies recombinant MVAs have been shown to be avirulent, yet protectively immunogenic as vaccines against viral diseases and cancer 6,21,22,23,24 . The most useful data on the safety and efficacy of various doses of a recombinant MVA vaccine comes from clinical trial data with a recombinant MVA expressing a number of CTL epitopes from Plasmodium falciparum pre-erythrocytic antigens fused to a complete pre-erythrocytic stage antigen, Thrombospondin Related Adhesion Protein (TRAP). These trials have given a total of 169 immunisations with this recombinant MVA, to 49 UK vaccinees 38 Gambian vaccines (20 of whom were children aged 1-5). 6 doses of 1 x 10 7 pfu, 139 doses of 5 x 10 7 pfu, 6 doses of 1 x 10 8 pfu and 18 doses of 2.5 x 10 8 pfu have been administered, all without serious adverse effects.

Recombinant MVA encoding antigen 85A
Secreted antigens from M. tuberculosis are released from actively metabolising bacteria, and are important targets in protective immunity 25 . Antigen 85A is a major secreted antigen from M. tuberculosis which forms part of the antigen 85 complex (A, B and C). This complex constitutes a major portion of the secreted proteins of both M.tb and BCG. It is involved in fibronectin binding within the cell wall and has mycolyltransferase activity 26 . MVA85A induces both a CD4+ and a CD8+ epitope when used to immunise mice. When mice are primed with BCG and then given MVA85A as a boost, the levels of CD4+ and CD8+ T cells induced are higher than with either BCG or MVA85A alone. We are evaluating the safety and immunogenicity of the following 3 groups: 1. BCG alone 2. MVA85A alone 3. BCG prime-MVA85A boost BCG-BCG provides a control group for BCG-MVA85A. Many countries have a tradition of repeated BCG vaccination and the criteria for revaccination differ between countries 27 .

Study Objective
To assess the safety and immunogenicity of BCG delivered intradermally into the deltoid region in volunteers who have received BCG 10 -20 years previously.

Selection of volunteers
Volunteers for the study will be recruited through advertisements. Each volunteer will have received an information sheet concerning the study and will have agreed to participate in writing. Volunteers will be given at least 48 hours between reading the information leaflet and agreeing to participate. Female volunteers will be told of the theoretical risk of congenital anomaly should they become pregnant during the study and only those who undertake to take precautions to avoid pregnancy during the study period will be eligible. Volunteers will give signed consent for their GP's to be notified about their participation in the trial. The GP will be faxed a letter on the day of screening and asked to reply if they know of a reason why the volunteer should not take part. The signed consent form will also be faxed with the letter.

Screening
Volunteers will be asked to sign the informed consent form for screening. The following will be performed: • Medical history and examination • Laboratory evaluations -including clinical chemistry, haematology, HLA typing, anti-HBV antibodies, anti-HCV antibodies, anti-HIV antibodies • Heaf test -to exclude prior exposure to TB • Urinalysis and urine pregnancy test if female
• Normal medical history and physical examination.

Exclusion Criteria
a. Exposure to TB at any point. A positive ESAT6/CFP10 Elispot response (defined as greater than 5 spots/well above background and at least double the background response

Immunisation
On Day 0, subjects will receive a single intradermal injection of 0.1ml BCG over the deltoid muscle. Vital signs will be monitored at 30 and 60 minutes post-immunisation. Local reactions at the site of administration will be evaluated at 60 minutes.
A photograph of the injection site may be taken at 48 hours (with written consent). The injection site will be reviewed 7 days after each immunization.
Blood will be taken at the following time points: At the screening visit*, prior to the vaccination, 1 week after the first vaccination*, 2 weeks, 4 weeks, 8 weeks, 12 weeks* and 24 weeks after the vaccination. Up to 55 mls will be taken at any one time with the total being no more than 500 mls over the study period. *Samples taken on these dates will be tested for full blood count and biochemical screen. Immunological assays will be performed at all time points to determine vaccine immunogenicity. A pregnancy test will be performed prior to vaccination for female volunteers. Peripheral blood mononuclear cells will be prepared for cellular immunological assays to be performed without or following cryopreservation. Other serological measures of immune response, i.e. antibody titres, will be assayed on frozen plasma samples. All blood tests will be taken within 1-3 days of the due date as described in the schedule above.
At the 24 week timepoint, a Heaf test will be repeated in order to document the change (if any) in Heaf responsiveness after the second BCG vaccination.

Endpoints
The occurance and severity of local side-effects The occurance and severity of systemic side-effects The induction of T cell responses (as measured by an interferon-gamma Elispot assay). Proliferation assays and cytotoxic T cell assays will be performed on strong CD4+ and CD8+ responses respectively.

Adverse Events
See Appendix 1.

Definition and Grading Intensity of Adverse Events
An adverse event is defined as any unintended change in the body structure (signs) or body function (symptoms), whether or not considered related to test product. During the entire study, subjects will be instructed to report all adverse events. All adverse events, whether volunteered, elicited or noted on physical examination, will be recorded throughout the study.
The severity of adverse events will be categorized as follows: • MILD = Experience that is minor and does not cause significant discomfort to subject or change in activities of daily living (ADLs); subject is aware of symptoms but symptoms are easily tolerated. • MODERATE = Experience is an inconvenience or concern to the subject and causes interference with ADLs but the subject is able to continue with ADLs. • SEVERE = Experience significantly interferes with ADLs and the subject is incapacitated and/or unable to continue with ADLs.

Criteria for Determining Relationship to Test Product
The Investigator will make a determination of the relationship of the adverse event to the test product. The relationship to test product of all adverse events will be classified according to the following guidelines: • NOT RELATED = Data available to clearly identify an alternative cause of the reaction, e.g., hemorrhage due to mechanical injury.  Mild to moderate limitation in activity -some assistance may be needed; no or minimal medical intervention/therapy required. GRADE 3 Severe Marked limitation in activity, some assistance usually required; medical intervention/therapy required, hospitalization possible. GRADE 4 Life-threatening Extreme limitation in activity, significant assistance required; significant medical intervention/therapy required, hospitalization or hospice care probable.
SERIOUS OR LIFE-THREATENING Adverse Events ANY clinical event deemed by the clinician to be serious or life-threatening should be considered a Grade 4 Adverse Event. Clinical events considered to be serious or life-threatening include, but are not limited to: seizures, coma, tetany, diabetic ketoacidosis, disseminated intravascular coagulation, diffuse petechiae, paralysis, acute psychosis, severe depression.

MISCELLANEOUS
• When two values are used to define the criteria for each parameter, the lowest values will appear first.
• Parameters are generally grouped by body system.