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1.  Heterologous vaccination against human tuberculosis modulates antigen-specific CD4 T cell function 
European journal of immunology  2013;43(9):10.1002/eji.201343454.
Summary
Heterologous prime-boost strategies hold promise for vaccination against tuberculosis (TB). However, the T cell characteristics required for protection are not known. We proposed that boost vaccines should induce long-lived functional and phenotypic changes to T cells primed by BCG and/or natural exposure to mycobacteria.
We characterized changes among specific CD4 T cells after vaccination with the MVA85A vaccine in adults, adolescents and children.
CD4 T cells identified with Ag85A peptide-bearing HLA class II tetramers, were characterized by flow cytometry. We also measured proliferative potential and cytokine expression of Ag85A-specific CD4 T cells.
During the effector phase, MVA85A-induced specific CD4 T cells co-expressed IFN-γ and IL-2, skin homing integrins and the activation marker CD38. This was followed by contraction and a transition to predominantly IL-2-expressing, CD45RACCR7+CD27+ or CD45RA+CCR7+CD27+ specific CD4 T cells. These surface phenotypes were similar to Ag85A-specific T cells prior to MVA85A. However, functional differences were observed post-vaccination: specific proliferative capacity was markedly higher after 6-12 months than before vaccination.
Our data suggest that MVA85A vaccination may modulate Ag85A-specific CD4 T cell function, resulting in greater recall potential. Importantly, surface phenotypes commonly used as proxies for memory T cell function did not associate with functional effects of vaccination.
doi:10.1002/eji.201343454
PMCID: PMC3816254  PMID: 23737382
proliferation; MVA85A; vaccine; T cells; HLA class II tetramer
2.  Process of Assay Selection and Optimization for the Study of Case and Control Samples from a Phase IIb Efficacy Trial of a Candidate Tuberculosis Vaccine, MVA85A 
The first phase IIb safety and efficacy trial of a new tuberculosis vaccine since that for BCG was completed in October 2012. BCG-vaccinated South African infants were randomized to receive modified vaccinia virus Ankara, expressing the Mycobacterium tuberculosis antigen 85A (MVA85A), or placebo. MVA85A did not significantly boost the protective effect of BCG. Cryopreserved samples provide a unique opportunity for investigating the correlates of the risk of tuberculosis disease in this population. Due to the limited amount of sample available from each infant, preliminary work was necessary to determine which assays and conditions give the most useful information. Peripheral blood mononuclear cells (PBMC) were stimulated with antigen 85A (Ag85A) and purified protein derivative from M. tuberculosis in an ex vivo gamma interferon (IFN-γ) enzyme-linked immunosorbent spot assay (ELISpot) and a Ki67 proliferation assay. The effects of a 2-h or overnight rest of thawed PBMC on ELISpot responses and cell populations were determined. Both the ELISpot and Ki67 assays detected differences between the MVA85A and placebo groups, and the results correlated well. The cell numbers and ELISpot responses decreased significantly after an overnight rest, and surface flow cytometry showed a significant loss of CD4+ and CD8+ T cells. Of the infants tested, 50% had a positive ELISpot response to a single pool of flu, Epstein-Barr virus (EBV), and cytomegalovirus (CMV) (FEC) peptides. This pilot work has been essential in determining the assays and conditions to be used in the correlate study. Moving forward, PBMC will be rested for 2 h before assay setup. The ELISpot assay, performed in duplicate, will be selected over the Ki67 assay, and further work is needed to evaluate the effect of high FEC responses on vaccine-induced immunity and susceptibility to tuberculosis disease.
doi:10.1128/CVI.00128-14
PMCID: PMC4097435  PMID: 24828094
3.  Induction and Regulation of T-Cell Immunity by the Novel Tuberculosis Vaccine M72/AS01 in South African Adults 
Rationale: Tuberculosis (TB) is a major cause of morbidity and mortality worldwide, thus there is an urgent need for novel TB vaccines.
Objectives: We investigated a novel TB vaccine candidate, M72/AS01, in a phase IIa trial of bacille Calmette-Guérin–vaccinated, HIV-uninfected, and Mycobacterium tuberculosis (Mtb)–infected and -uninfected adults in South Africa.
Methods: Two doses of M72/AS01 were administered to healthy adults, with and without latent Mtb infection. Participants were monitored for 7 months after the first dose; cytokine production profiles, cell cycling, and regulatory phenotypes of vaccine-induced T cells were measured by flow cytometry.
Measurements and Main Results: The vaccine had a clinically acceptable safety profile, and induced robust, long-lived M72-specific T-cell and antibody responses. M72-specific CD4 T cells produced multiple combinations of Th1 cytokines. Analysis of T-cell Ki67 expression showed that most vaccination-induced T cells did not express Th1 cytokines or IL-17; these cytokine-negative Ki67+ T cells included subsets of CD4 T cells with regulatory phenotypes. PD-1, a negative regulator of activated T cells, was transiently expressed on M72-specific CD4 T cells after vaccination. Specific T-cell subsets were present at significantly higher frequencies after vaccination of Mtb-infected versus -uninfected participants.
Conclusions: M72/AS01 is clinically well tolerated in Mtb-infected and -uninfected adults, induces high frequencies of multifunctional T cells, and boosts distinct T-cell responses primed by natural Mtb infection. Moreover, these results provide important novel insights into how this immunity may be appropriately regulated after novel TB vaccination of Mtb-infected and -uninfected individuals.
Clinical trial registered with www.clinicaltrials.gov (NCT 00600782).
doi:10.1164/rccm.201208-1385OC
PMCID: PMC3778736  PMID: 23306546
tuberculosis; vaccine; T cell; cytokine; proliferation
4.  The Candidate TB Vaccine, MVA85A, Induces Highly Durable Th1 Responses 
PLoS ONE  2014;9(2):e87340.
Background
Vaccination against tuberculosis (TB) should provide long-term protective immunity against Mycobacterium tuberculosis (M.tb). The current TB vaccine, Bacille Calmette-Guerin (BCG), protects against disseminated childhood TB, but protection against lung TB in adolescents and adults is variable and mostly poor. One potential reason for the limited durability of protection may be waning of immunity through gradual attrition of BCG-induced T cells. We determined if a MVA85A viral-vector boost could enhance the durability of mycobacteria-specific T cell responses above those induced by BCG alone.
Methods
We describe a long-term follow-up study of persons previously vaccinated with MVA85A. We performed a medical history and clinical examination, a tuberculin skin test and measured vaccine-specific T cell responses in persons previously enrolled as adults, adolescents, children or infants into three different Phase II trials, between 2005 and 2011.
Results
Of 252 potential participants, 183 (72.6%) consented and completed the study visit. Vaccine-induced Ag85A-specific CD4+ T cell responses were remarkably persistent in healthy, HIV-uninfected adults, adolescents, children and infants, up to 6 years after MVA85A vaccination. Specific CD4+ T cells expressed surface markers consistent with either CD45RA−CCR7+ central memory or CD45RA−CCR7− effector memory T cells. Similarly durable Ag85A-specific CD4+ T cell responses were detected in HIV-infected persons who were on successful antiretroviral therapy when MVA85A was administered. By contrast, Ag85A-specific CD4+ T cell frequencies in untreated MVA85A-vaccinated HIV-infected persons were mostly undetectable 3–5 years after vaccination.
Conclusion
MVA85A induces remarkably durable T cell responses in immunocompetent persons. However, results from a recent phase IIb trial of MVA85A, conducted in infants from the same geographic area and study population, showed no vaccine efficacy, suggesting that these durable T cell responses do not enhance BCG-induced protection against TB in infants.
doi:10.1371/journal.pone.0087340
PMCID: PMC3911992  PMID: 24498312
5.  TB Incidence in an Adolescent Cohort in South Africa 
PLoS ONE  2013;8(3):e59652.
Background
Tuberculosis (TB) is a major public health problem globally. Little is known about TB incidence in adolescents who are a proposed target group for new TB vaccines. We conducted a study to determine the TB incidence rates and risk factors for TB disease in a cohort of school-going adolescents in a high TB burden area in South Africa.
Methods
We recruited adolescents aged 12 to 18 years from high schools in Worcester, South Africa. Demographic and clinical information was collected, a tuberculin skin test (TST) performed and blood drawn for a QuantiFERON TB Gold assay at baseline. Screening for TB cases occurred at follow up visits and by surveillance of registers at public sector TB clinics over a period of up to 3.8 years after enrolment.
Results
A total of 6,363 adolescents were enrolled (58% of the school population targeted). During follow up, 67 cases of bacteriologically confirmed TB were detected giving an overall incidence rate of 0.45 per 100 person years (95% confidence interval 0.29–0.72). Black or mixed race, maternal education of primary school or less or unknown, a positive baseline QuantiFERON assay and a positive baseline TST were significant predictors of TB disease on adjusted analysis.
Conclusion
The adolescent TB incidence found in a high burden setting will help TB vaccine developers plan clinical trials in this population. Latent TB infection and low socio-economic status were predictors of TB disease.
doi:10.1371/journal.pone.0059652
PMCID: PMC3606161  PMID: 23533639
6.  Lessons learnt from the first efficacy trial of a new infant tuberculosis vaccine since BCG 
Summary
Background
New tuberculosis (TB) vaccines are being developed to combat the global epidemic. A phase IIb trial of a candidate vaccine, MVA85A, was conducted in a high burden setting in South Africa to evaluate proof-of-concept efficacy for prevention of TB in infants.
Objective
To describe the study design and implementation lessons from an infant TB vaccine efficacy trial.
Methods
This was a randomised, controlled, double-blind clinical trial comparing the safety and efficacy of MVA85A to Candin control administered to 4–6-month-old, BCG-vaccinated, HIV-negative infants at a rural site in South Africa. Infants were followed up for 15–39 months for incident TB disease based on pre-specified endpoints.
Results
2797 infants were enrolled over 22 months. Factors adversely affecting recruitment and the solutions that were implemented are discussed. Slow case accrual led to six months extension of trial follow up.
Conclusion
The clinical, regulatory and research environment for modern efficacy trials of new TB vaccines are substantially different to that when BCG vaccine was first evaluated in infants. Future infant TB vaccine trials will need to allocate sufficient resources and optimise operational efficiency. A stringent TB case definition is necessary to maximize specificity, and TB case accrual must be monitored closely.
doi:10.1016/j.tube.2013.01.003
PMCID: PMC3608032  PMID: 23410889
BCG; Vaccine; Tuberculosis; Lessons learnt; Implementation
7.  A Phase IIa Trial of the New Tuberculosis Vaccine, MVA85A, in HIV- and/or Mycobacterium tuberculosis–infected Adults 
Rationale: Novel tuberculosis (TB) vaccines should be safe and effective in populations infected with Mycobacterium tuberculosis (M.tb) and/or HIV for effective TB control.
Objective: To determine the safety and immunogenicity of MVA85A, a novel TB vaccine, among M.tb- and/or HIV-infected persons in a setting where TB and HIV are endemic.
Methods: An open-label, phase IIa trial was conducted in 48 adults with M.tb and/or HIV infection. Safety and immunogenicity were analyzed up to 52 weeks after intradermal vaccination with 5 × 107 plaque-forming units of MVA85A. Specific T-cell responses were characterized by IFN-γ enzyme-linked immunospot and whole blood intracellular cytokine staining assays.
Measurements and Main Results: MVA85A was well tolerated and no vaccine-related serious adverse events were recorded. MVA85A induced robust and durable response of mostly polyfunctional CD4+ T cells, coexpressing IFN-γ, tumor necrosis factor-α, and IL-2. Magnitudes of pre- and postvaccination T-cell responses were lower in HIV-infected, compared with HIV-uninfected, vaccinees. No significant effect of antiretroviral therapy on immunogenicity of MVA85A was observed.
Conclusions: MVA85A was safe and immunogenic in persons with HIV and/or M.tb infection. These results support further evaluation of safety and efficacy of this vaccine for prevention of TB in these target populations.
doi:10.1164/rccm.201108-1548OC
PMCID: PMC3326425  PMID: 22281831
tuberculosis; HIV-1; vaccine; MVA85A; clinical trial
8.  The Novel Tuberculosis Vaccine, AERAS-402, Induces Robust and Polyfunctional CD4+ and CD8+ T Cells in Adults 
Rationale: AERAS-402 is a novel tuberculosis vaccine designed to boost immunity primed by bacillus Calmette-Guérin (BCG), the only licensed vaccine.
Objectives: We investigated the safety and immunogenicity of AERAS-402 in healthy Mycobacterium tuberculosis–uninfected BCG-vaccinated adults from a tuberculosis-endemic region of South Africa.
Methods: Escalating doses of AERAS-402 vaccine were administered intramuscularly to each of three groups of healthy South African BCG-vaccinated adults, and a fourth group received two injections of the maximal dose. Participants were monitored for 6 months, with all adverse effects documented. Vaccine-induced CD4+ and CD8+ T-cell immunity was characterized by an intracellular cytokine staining assay of whole blood and peripheral blood mononuclear cells.
Measurements and Main Results: AERAS-402 was well tolerated, and no vaccine-related serious adverse events were recorded. The vaccine induced a robust CD4+ T-cell response dominated by cells coexpressing IFN-γ, tumor necrosis factor-α, and IL-2 (“polyfunctional” cells). AERAS-402 also induced a potent CD8+ T-cell response, characterized by cells expressing IFN-γ and/or tumor necrosis factor-α, which persisted for the duration of the study.
Conclusions: Vaccination with AERAS-402 is safe and immunogenic in healthy adults. The immunity induced by the vaccine appears promising: polyfunctional T cells are thought to be important for protection against intracellular pathogens such as Mycobacterium tuberculosis, and evidence is accumulating that CD8+ T cells are also important. AERAS-402 induced a robust and durable CD8+ T-cell response, which appears extremely promising.
Clinical trial registered with www.sanctr.gov.za (NHREC no. 1381).
doi:10.1164/rccm.200910-1484OC
PMCID: PMC2894413  PMID: 20167847
tuberculosis; vaccine; immunity; CD4; CD8
9.  MVA85A, a novel TB vaccine, is safe in adolescents and children, and induces complex subsets of polyfunctional CD4+ T cells 
European journal of immunology  2010;40(1):279-290.
Summary
MVA85A is a new tuberculosis vaccine aimed at enhancing immunity induced by BCG. We investigated the safety and immunogenicity of MVA85A in healthy adolescents and children from a tuberculosis endemic region, who received BCG at birth.
Twelve adolescents and 24 children were vaccinated and followed up for 12 or 6 months, respectively. Adverse events were documented and vaccine-induced immune responses assessed by IFN-γ ELISpot and intracellular cytokine staining.
The vaccine was well tolerated and there were no vaccine-related serious adverse events. MVA85A induced potent and durable T cell responses. Multiple CD4+ T cell subsets, based on expression of IFN-γ, TNF-α, IL-2, IL-17 and GM-CSF, were induced. Polyfunctional CD4+ T cells co-expressing IFN-γ, TNF-α and IL-2 dominated the response in both age groups. A novel CD4+ cell subset co-expressing these three Th1 cytokines and IL-17 was induced in adolescents, while a novel CD4+ T cell subset co-expressing Th1 cytokines and GM-CSF was induced in children. Antigen-specific CD8+ T cells were not detected.
We conclude that in adolescents and children MVA85A safely induces the type of immunity thought to be important in protection against tuberculosis. This includes induction of novel Th1 cell populations which have not been previously described in humans.
doi:10.1002/eji.200939754
PMCID: PMC3044835  PMID: 20017188
MVA85A; tuberculosis; vaccine; polyfunctional; IL-17
10.  Significantly skewed memory CD8+ T cell subsets in HIV-1 infected infants during the first year of life1 
Clinical immunology (Orlando, Fla.)  2008;130(3):280-289.
HIV-1 infection causes a severe T cell compromise; however, little is known about changes in naïve, memory, effector and senescent T cell subsets during the first year of life. T cell subsets were studied over the first year of life in blood from 3 infant cohorts: untreated HIV-infected, HIV-exposed but uninfected, and HIV-unexposed. In HIV-infected infants, the frequency of CCR7+CD45RA+ naïve CD8+ T cells was significantly decreased, whilst the frequency of CCR7−CD45RA− effector memory CD8+ T cells was increased, compared with the control cohorts. A larger population of CD8+ T cells in HIV-infected infants displayed a phenotype consistent with senescence. Differences in CD4+ T cell subset frequencies were less pronounced, and no significant differences were observed between exposed and unexposed HIV-uninfected infants. We concluded that the proportion of naïve, memory, effector and senescent CD8+ T cells during the first year of life is significantly altered by HIV-1 infection.
doi:10.1016/j.clim.2008.09.006
PMCID: PMC2722743  PMID: 18996749
CD4; CD8; memory; HIV-1; infants
11.  Safety and immunogenicity of a new tuberculosis vaccine, MVA85A, in healthy adults in South Africa1 
The Journal of infectious diseases  2008;198(4):544-552.
BACKGROUND
The efficacy of BCG may be enhanced by heterologous vaccination strategies that boost the BCG-primed immune response. One leading booster vaccine, MVA85A, has shown promising safety and immunogenicity in UK human trials. We investigated the safety and immunogenicity of MVA85A in mycobacteria-exposed, but Mycobacterium tuberculosis-uninfected, healthy adults from a TB-endemic region of South Africa.
METHODS
Twenty-four adults were vaccinated with MVA85A. All subjects were followed up for one year for adverse events and for immunological assessment.
RESULTS
MVA85A vaccination was well tolerated and induced potent T cell responses, measured by IFN-γ ELISPOT assay, which exceeded pre-vaccination levels up to 364 days after vaccination. BCG-specific CD4+ T cells boosted by MVA85A comprised of multiple populations expressing combinations of IFN-γ, TNF-α, IL-2 and IL-17, as measured by polychromatic flow cytometry. IFN-γ expressing and polyfunctional IFN-γ+TNF-α+IL-2+ CD4+ T cells were boosted during the peak BCG-specific response 7 days post-vaccination.
CONCLUSION
The excellent safety profile and quantitative and qualitative immunogenicity data strongly support further trials to assess the efficacy of MVA85A as a boosting vaccine in TB endemic countries.
doi:10.1086/590185
PMCID: PMC2822902  PMID: 18582195
Vaccination; tuberculosis; T cells; MVA85A; South Africa
12.  Infant HIV-1 Infection Severely Impairs the Bacille Calmette-Guerin Vaccine-Induced Immune Response1 
The Journal of infectious diseases  2009;199(7):982-990.
Background
World-wide, most infants born to HIV-infected mothers receive BCG. Tuberculosis is a major cause of death of HIV-infected infants in sub-Saharan Africa, and should be prevented. However, BCG may itself cause disease (BCGosis) in these infants. Information regarding the immunogenicity of BCG is imperative for the risk/benefit assessment of BCG vaccination in HIV-infected infants; however, no such data exists.
Methods
We compared BCG-induced CD4 and CD8 T cell responses, assessed by flow cytometry, in HIV-infected (n = 20), HIV-exposed but uninfected (n = 25), and HIV-unexposed (n = 23) infants, over their first year of life.
Results
BCG vaccination of the 2 HIV-uninfected groups induced a robust response, characterized by IFN-γ, TNF-α, and/or IL-2-expressing CD4 T cells. In contrast, HIV-infected infants had a markedly lower response, throughout the first year of life. These infants also had significantly reduced numbers of IFN-γ, TNF-α and IL-2 co-expressing polyfunctional CD4 T cells, thought to indicate T cell quality.
Conclusions
HIV-1 infection severely impairs the BCG-specific T cell response during the first year of life. BCG may therefore provide little, if any, vaccine-induced benefit in HIV-infected infants. Considering the significant risk of BCGosis, these data strongly support not giving BCG to HIV-infected infants.
doi:10.1086/597304
PMCID: PMC2815505  PMID: 19236280
Tuberculosis; BCG; HIV; infants; T cells; immune response; polyfuntional; Th1; Th17
13.  Isolation of Non-Tuberculous Mycobacteria in Children Investigated for Pulmonary Tuberculosis 
PLoS ONE  2006;1(1):e21.
Objective
To evaluate the frequency and clinical significance of non-tuberculous mycobacteria (NTM) isolates among children investigated for pulmonary tuberculosis in a rural South African community.
Methods
Children were investigated for pulmonary tuberculosis as part of a tuberculosis vaccine surveillance program (2001–2005). The clinical features of children in whom NTM were isolated, from induced sputum or gastric lavage, were compared to those with culture-proven M. tuberculosis.
Results
Mycobacterial culture demonstrated 114 NTM isolates from 109 of the 1,732 children investigated, a crude yield of 6% (95% CI 5–7). The comparative yield of positive NTM cultures from gastric lavage was 40% (95% CI 31–50), compared to 67% (95% CI 58–76) from induced sputum. 95% of children with NTM isolates were symptomatic. Two children were HIV-infected. By contrast, M. tuberculosis was isolated in 187 children, a crude yield of 11% (95% CI 9–12). Compared to those with culture-proven M. tuberculosis, children with NTM isolates were less likely to demonstrate acid-fast bacilli on direct smear microscopy (OR 0.19; 95% 0.0–0.76). Children with NTM were older (p<0.0001), and more likely to demonstrate constitutional symptoms (p = 0.001), including fever (p = 0.003) and loss of weight or failure to gain weight (p = 0.04), but less likely to demonstrate a strongly positive tuberculin skin test (p<0.0001) or radiological features consistent with pulmonary tuberculosis (p = 0.04).
Discussion
NTM were isolated in 6% of all children investigated for pulmonary tuberculosis and in more than one third of those with a positive mycobacterial culture. NTM may complicate the diagnosis of PTB in regions that lack capacity for mycobacterial species identification. The association of NTM isolates with constitutional symptoms suggestive of host recognition requires further investigation.
doi:10.1371/journal.pone.0000021
PMCID: PMC1762386  PMID: 17183648
14.  Heterologous vaccination against human tuberculosis modulates antigen-specific CD4+ T-cell function 
European Journal of Immunology  2013;43(9):2409-2420.
Heterologous prime-boost strategies hold promise for vaccination against tuberculosis. However, the T-cell characteristics required for protection are not known. We proposed that boost vaccines should induce long-lived functional and phenotypic changes to T cells primed by Bacille Calmette Guerin (BCG) and/or natural exposure to mycobacteria. We characterized changes among specific CD4+ T cells after vaccination with the MVA85A vaccine in adults, adolescents, and children. CD4+ T cells identified with Ag85A peptide-bearing HLA class II tetramers were characterized by flow cytometry. We also measured proliferative potential and cytokine expression of Ag85A-specific CD4+ T cells. During the effector phase, MVA85A-induced specific CD4+ T cells coexpressed IFN-γ and IL-2, skin homing integrins, and the activation marker CD38. This was followed by contraction and a transition to predominantly IL-2-expressing, CD45RA−CCR7+CD27+ or CD45RA+CCR7+CD27+ specific CD4+ T cells. These surface phenotypes were similar to Ag85A-specific T cells prior to MVA85A. However, functional differences were observed postvaccination: specific proliferative capacity was markedly higher after 6–12 months than before vaccination. Our data suggest that MVA85A vaccination may modulate Ag85A-specific CD4+ T-cell function, resulting in greater recall potential. Importantly, surface phenotypes commonly used as proxies for memory T-cell function did not associate with functional effects of vaccination.
doi:10.1002/eji.201343454
PMCID: PMC3816254  PMID: 23737382
HLA class II tetramer; MVA85A; Proliferation; T cells; Vaccine

Results 1-14 (14)