Boosting BCG with Recombinant Modified Vaccinia Ankara Expressing Antigen 85A: Different Boosting Intervals and Implications for Efficacy Trials

Objectives To investigate the safety and immunogenicity of boosting BCG with modified vaccinia Ankara expressing antigen 85A (MVA85A), shortly after BCG vaccination, and to compare this first with the immunogenicity of BCG vaccination alone and second with a previous clinical trial where MVA85A was administered more than 10 years after BCG vaccination. Design There are two clinical trials reported here: a Phase I observational trial with MVA85A; and a Phase IV observational trial with BCG. These clinical trials were all conducted in the UK in healthy, HIV negative, BCG naïve adults. Subjects were vaccinated with BCG alone; or BCG and then subsequently boosted with MVA85A four weeks later (short interval). The outcome measures, safety and immunogenicity, were monitored for six months. The immunogenicity results from this short interval BCG prime–MVA85A boost trial were compared first with the BCG alone trial and second with a previous clinical trial where MVA85A vaccination was administered many years after vaccination with BCG. Results MVA85A was safe and highly immunogenic when administered to subjects who had recently received BCG vaccination. When the short interval trial data presented here were compared with the previous long interval trial data, there were no significant differences in the magnitude of immune responses generated when MVA85A was administered shortly after, or many years after BCG vaccination. Conclusions The clinical trial data presented here provides further evidence of the ability of MVA85A to boost BCG primed immune responses. This boosting potential is not influenced by the time interval between prior BCG vaccination and boosting with MVA85A. These findings have important implications for the design of efficacy trials with MVA85A. Boosting BCG induced anti-mycobacterial immunity in either infancy or adolescence are both potential applications for this vaccine, given the immunological data presented here. Trial Registration ClinicalTrials.gov NCT00427453 (short boosting interval), NCT00427830 (long boosting interval), NCT00480714 (BCG alone)


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.

Study Objective
To assess the safety and immunogenicity of MVA85A delivered intradermally into the deltoid region in volunteers who have received BCG 1-20 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, antivaccinia antibodies, 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.

Immunisation
On Day 0, subjects will receive a single intradermal injection of 5 x 10 7 pfu in 0.1ml over the deltoid muscle. Subjects will be observed for an hour after all immunisations. 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 first 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.

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.