Conceived and designed the experiments: RHB PCH SAD HF CL RAA HM AVH. Performed the experiments: RHB PO HBI AH. Analyzed the data: RHB PCH PO HBI DJJ SAD HF AH HM AVH. Contributed reagents/materials/analysis tools: PCH HF HM AVH. Wrote the paper: RHB PCH PO HBI DJJ AH CL RAA HM AVH. Recruited and examined the volunteers: PCH.
RHB has a patent relating to ex vivo ELISPOT licensed through Oxford University. AH and HM are named inventors on a composition of matter patent for MVA85A filed by the University of Oxford. There are no other conflicts of interest
Vaccination with a recombinant modified vaccinia Ankara expressing antigen 85A from
We vaccinated 21 healthy adult male subjects (11 BCG scar negative and 10 BCG scar positive) with MVA85A after screening for evidence of prior exposure to mycobacteria. We monitored them over six months, observing for clinical, haematological and biochemical adverse events, together with assessment of the vaccine induced cellular immune response using ELISPOT and flow cytometry. MVA85A was well tolerated with no significant adverse events. Mild local and systemic adverse events were consistent with previous UK trials. Marked immunogenicity was found whether individuals had a previous BCG scar or not. There was not enhanced immunogenicity in those with a BCG scar, and induced T cell responses were better maintained in apparently BCG-naïve Gambians than previously studied BCG-naïve UK vaccinees. Although responses were predominantly attributable to CD4+ T cells, we also identified antigen specific CD8+ T cell responses, in subjects who were HLA B-35 and in whom enough blood was available for more detailed immunological analysis.
These data on the safety and immunogenicity of MVA85A in West Africa support its accelerated development as a promising booster vaccine for tuberculosis.
ClinicalTrials.gov
Tuberculosis (TB) causes approximately 2 million deaths annually; most of these occur in developing countries
We have developed a prime-boost strategy that seeks to overcome this limitation
We have reported the immunogenicity of MVA85A in UK volunteers
The protocols for this trial and the supporting CONSORT checklist are available as supporting information; see
Those eligible were vaccinated within 8 weeks of screening. Five were vaccinated in the BCG negative arm before enrolment into the BCG positive arm began. On the vaccination day they had a clinical evaluation and provided a blood sample. The vaccine was administered intra-dermally over the insertion of the left deltoid muscle, at a dose of 5×107 plaque forming units (pfu) of MVA85A (135 µl). A second vaccination was given 3 weeks later to the BCG scar negative vaccinees, in the right deltoid muscle. Subjects were observed for one hour following immunisation and vital signs were recorded. They were then seen on day one and two. Follow-up visits were made (
A. BCG scar negative subjects. B. BCG scar positive subjects.
We used an IFN-γ ELISPOT assay to screen volunteers at recruitment and monitor the immunogenicity of MVA85A, as previously described
To monitor Ag85A immunogenicity we used the ELISPOT response to a sum of 66 pooled peptides (7 pools of 6–10 peptides). This method will count twice a T cell that responds to any of the 10-mer overlap regions that occur in two pools with adjacent peptides. To monitor individual peptide responses we used Ag85A peptide pools in different combinations so that an individual peptide response would be clearly identifiable when two pools with the same peptide responded within a matrix. We used recombinant Ag85A protein (Leiden University), and PPD-T. ELISPOT plates were counted using an automated ELISPOT reader (AID-GmbH, Germany) with correction for artefacts. The positive control was Phytohaemaglutinin (PHA; Sigma-Aldrich, UK). All antigens were tested in duplicate wells.
An aliquot of whole blood (200 µl) was harvested after 20 h of culture at 37°C alone or stimulated with 5.0 µg/ml Ag85A peptide pool, 2.0 µg/ml PPD-T and 10 µg/ml PHA. After incubation, cells were stained for 30 min at room temperature with a monoclonal antibody cocktail mix containing anti-CD4 FITC, anti-CD69 PE, anti-CD8 PerCP and anti-CD3 APC (BD Biosciences, UK). Red blood cells were lysed and lymphocytes washed, fixed in 2% p-formaldehyde and acquired with a Cell Quest software by live gating on CD3+ T cells (50000 events) on a four-colour flow cytometer (BD FASCalibur, BD Biosciences, UK). Analyses were carried out using FlowJo software (Treestar Inc., San Carlos, CA).
Cryopreserved PBMC were thawed, washed and re-suspended at 2×105/well in a 96 U well microtitre plate with p23 peptide (10 µg/ml) in RPMI-1640 supplemented with 5% AB serum, L-glutamine 2mM and antibiotics. On the 3rd and 7th days IL-2 (20 IU/ml) was added. The expanded lymphocytes were harvested, washed, re-stimulated with p23 (10 µg/ml), PHA (10 µg/ml) or medium alone in separate tubes in the presence of Brefeldin A (10 µg/ml) for six hours. Cells were harvested and washed with FACs buffer (PBS, 2mM EDTA and 1%BSA) lysed with BD FACS™ Lysing Solution. Cells were then washed and permeabilized with BD FACS Permeabilizing Solution. After an additional wash, cells were divided into two tubes. Surface and intracellular staining antibodies (anti human, CD4-PE, CD69-PerCP, CD3-APC and IFN-γ -FITC) in tube 1 and anti human (CD8-PE, CD69-PerCP, CD3-APC and IFN-g-FITC) in tube 2 were added in a single staining step. Finally, the cells were washed and fixed for flow cytometric analysis.
The Ag85A HLA-B35 restricted CD8+ epitope MPVGGQSSF (ABC, Imperial College, London, UK ) was used to stimulate the cryopreserved PBMC isolated on day 7 after vaccination in an
All data were either double entered, or electronically transferred, into an ACCESS database and manually validated. ELISPOT counts were corrected for the background in the negative control well. Those beyond the upper limit of detection of the ELISPOT reader were arbitrarily given a count of 500 spots/well. All comparisons were within subject and were performed using a Wilcoxon matched pairs test.
We screened 240 subjects to identify 10 to 12 individuals for each arm of the study. Some subjects showed transient ESAT-6/CFP-10 ELISPOT positivity after enrolment, consistent with fluctuations in responses to these antigens seen in other Gambian studies
Overall, 21 volunteers were enrolled and 31 doses of MVA85A were administered. One subject in the BCG scar negative group did not receive the second vaccination because he became ESAT-6 ELISPOT positive on day 7. On subsequent assays this reverted to negative. No significant changes occurred in vital signs during the one-hour post-vaccination observation or follow-up periods. There were minimal reports of any systemic side effects; specifically no symptoms of a flu-like illness or lymphadenopathy. One subject had a mild headache and diarrhoea in the first 24 hours after vaccination, both resolved quickly. No other reports or documentation of headache, myalgia, fever, or malaise were made.
Adverse Event | Dose 1 ( |
Dose 2 ( |
4 (5–14) | 0 (0) |
|
1(3) |
||
11 | 1 |
|
2 |
||
20 | 9 |
|
7 |
||
13 | 0 |
|
7 |
The data were recorded over seven days after each vaccination
n: number of volunteers vaccinated and followed up
first vaccination site
residual change at the second vaccination site
11 BCG scar negative subjects were immunised with MVA85A (Figure1A). There was a high degree of variability between individuals in the vaccine induced immune responses; these data are shown as median ELISPOT frequencies in
A. BCG scar negative vaccines (n = 11); B. BCG scar positive vaccinees (n = 10).
ELISPOT count (Median & inter-quartile range; SPM |
||||||||||
Ag85A | PPD-T | Ag85A peptides | ||||||||
5 | 10 | 42 | 43 | 89 | 208 | 57 | 79 | 131 | ||
296 | 1625 | 1645 | 236 | 526 | 1381 | 1001 | 1704 | 3843 | ||
74 | 307 | 531 | 96 | 157 | 453 | 424 | 943 | 1559 | ||
73 | 264 | 1353 | 77 | 170 | 472 | 263 | 761 | 1704 | ||
40 | 160 | 404 | 76 | 121 | 162 | 253 | 418 | 1545 | ||
80 | 97 | 166 | 78 | 111 | 166 | 144 | 422 | 1102 | ||
33 | 61 | 116 | 49 | 74 | 121 | 123 | 281 | 502 | ||
40 | 118 | 183 | 79 | 167 | 210 | 142 | 294 | 924 | ||
3 | 7 | 26 | 28 | 79 | 94 | 26 | 53 | 89 | ||
224 | 367 | 475 | 149 | 191 | 422 | 558 | 1120 | 2049 | ||
99 | 228 | 647 | 168 | 286 | 617 | 515 | 825 | 1960 | ||
43 | 199 | 348 | 71 | 235 | 307 | 92 | 564 | 1219 | ||
35 | 64 | 264 | 50 | 155 | 191 | 228 | 276 | 661 | ||
12 | 64 | 134 | 36 | 80 | 157 | 108 | 144 | 377 | ||
44 | 67 | 151 | 88 | 116 | 183 | 151 | 282 | 449 |
SPM, Spot Forming Units/million.
PBMC, Peripheral Blood Mononuclear Cells
After the first week, the response contracted to a stable plateau frequency, higher than before vaccination, which was maintained until the end of the trial. The frequency of the Ag85A ELISPOT response to peptides at the end of the trial was significantly higher than the frequency at the start (p = 0.004). A consistent hierarchy of responses to antigens was found in a predictable order, from highest to lowest: summed pooled peptides, Ag85A, and PPD-T. It was not possible to accurately quantify differences at the peak of response, as many were beyond the upper limit of detection. However the original hierarchy was maintained as the response waned. A second MVA85A vaccination given 3 weeks after the first did not boost the ELISPOT response further, consistent with the UK trials
On the basis of the data from the BCG scar negative subjects, where there was no significant increase in the immune response to a second vaccination, it was decided that the second immunisation with MVA85A at week 3 should be omitted in the BCG scar positive group (
We stimulated the whole blood of 3 volunteers with all 66 Ag85A peptides and monitored the proportion of CD4+ and CD8+ cells activated by upregulation of expression of CD69 (
Proportion of CD69 expression | |||
Experiment number | Day 0 | Day 7 | Day 49 |
0.00 | 0.22 | 0.56 | |
ND | 0.28 | 0.02 | |
0.00 | 0.24 | 0.02 | |
0.04 | 0.27 | 1.84 | |
ND | 1.95 | 0.00 | |
0.05 | 1.09 | 0.11 |
Frequencies of positive cells are given as a percentage of the total gated lymphocytes.
To understand responding phenotypes in more detail we looked for unambiguous peptide pool responses, consistent with a single epitope being maintained throughout the entire monitoring period. After analysing all peptide pool responses we identified a single epitope in 2 subjects, which was due in both cases to the response to a single peptide (p23). Short term cell lines to p23 were generated with PBMC recovered 28 days after vaccination, and the CD4/CD8 phenotype was analysed by IFN-γ ICS. For one subject (volunteer 1), both CD4 and CD8 p23 specific responses were identified (
Short term cell lines specific for p23 were generated using PBMC recovered 28 days after vaccination and specificity demonstrated by flow cytometry. Gating on IFN-γ detection, both CD4 and CD8 responses were observed for one subject (volunteer 1) while for the other subject (volunteer 2) an exclusive CD8 response was identified. B. Volunteer 2 also showed CD4 and CD8 activation by CD69 expression after stimulation with pooled Ag85A peptides 28 days after vaccination.
An HLA-B*3501 (abbreviated to HLA-B35) class I restricted CTL epitope within Ag85A p23 sequence had previously been reported in The Gambia
We report the first trial of a new generation TB vaccine in Africa. MVA85A was administered intra-dermally to two groups of volunteers with increasing evidence of prior mycobacterial exposure. The vaccine was found to be safe in these volunteers, enabling further studies to proceed in larger numbers of individuals to fully evaluate the adverse event profile and frequency. Since safety was demonstrated in these trials in The Gambia, further trials with this vaccine in TB endemic areas in individuals more representative of the general population, including subjects latently infected with
The peak responses to vaccination with MVA85A, in the UK and The Gambia, are the highest reported for any subunit vaccine in humans, at least using ELISPOT assays
The lack of any boosting effect seen with the second MVA85A vaccination mirrors the findings of the UK trial.
Unlike the MVA85A trial in the UK,
An interesting difference is observed between the plateau level of T cell response in the Gambian vaccinees and in subjects receiving the same vaccine in the UK. While the peak responses were little different, the plateau response in BCG naïve Gambians of over 200 SPM to antigen 85A peptides was higher than that observed in the UK (median 105 SPM). Whether this relates to higher pre-vaccination levels of central memory cells in Gambians, or more frequent low level boosting by recurrent exposure to low doses of environmental mycobacteria in Africa, requires further study.
Whilst we went to significant efforts to ensure that the first volunteers were as mycobacterially naïve as possible, it is likely that most if not all individuals had been exposed to mycobacteria before
In conclusion, MVA85A appears safe and highly immunogenic in Africa. The data from this study support accelerated development of this candidate towards a large scale efficacy trial in Africa and subsequent licensure. Further phase I and II trials are now in progress in a South African population and a phase II trial in Gambian infants is also underway.
Consort Checklist.
(0.06 MB DOC)
Trial Protocol.
(0.21 MB PDF)
Trial Protocol.
(0.21 MB PDF)
We thank Loius-Marie Yindom for HLA typing. We thank the Gambian government and the National TB Control Program for their ongoing collaboration. Dr Sam McConkey was the clinical safety monitor and Mary Dowling was the quality monitor for the study. Dr Kris Huygen provided helpful comments on the manuscript and Dr Martin Ota assisted with the figures. We are particularly grateful to the hundreds of individuals in the Greater Banjul area who volunteered to take part in this study.