Benjamin M. Kagina

Specific T Cell Frequency and Cytokine Expression Profile Do Not Correlate with Protection against Tuberculosis after Bacillus Calmette-Guérin Vaccination of Newborns

RATIONALE Immunogenicity of new tuberculosis (TB) vaccines is commonly assessed by measuring the frequency and cytokine expression profile of T cells. OBJECTIVES We tested whether this outcome correlates with protection against childhood TB disease after newborn vaccination with bacillus Calmette-Guérin (BCG). METHODS Whole blood from 10-week-old infants, routinely vaccinated with BCG at birth, was incubated with BCG for 12 hours, followed by cryopreservation for intracellular cytokine analysis. Infants were followed for 2 years to identify those who developed culture-positive TB-these infants were regarded as not protected against TB. Infants who did not develop TB disease despite exposure to TB in the household, and another group of randomly selected infants who were never evaluated for TB, were also identified-these groups were regarded as protected against TB. Cells from these groups were thawed, and CD4, CD8, and γδ T cell-specific expression of IFN-γ, TNF-α, IL-2, and IL-17 measured by flow cytometry. MEASUREMENTS AND MAIN RESULTS A total of 5,662 infants were enrolled; 29 unprotected and two groups of 55 protected infants were identified. There was no difference in frequencies of BCG-specific CD4, CD8, and γδ T cells between the three groups of infants. Although BCG induced complex patterns of intracellular cytokine expression, there were no differences between protected and unprotected infants. CONCLUSIONS The frequency and cytokine profile of mycobacteria-specific T cells did not correlate with protection against TB. Critical components of immunity against Mycobacterium tuberculosis, such as CD4 T cell IFN-γ production, may not necessarily translate into immune correlates of protection against TB disease.

Delaying BCG vaccination from birth to 10 weeks of age may result in an enhanced memory CD4 T cell response

BACKGROUND In most tuberculosis (TB) endemic countries, bacillus Calmette-Guérin (BCG) is usually given around birth to prevent severe TB in infants. The neonatal immune system is immature. Our hypothesis was that delaying BCG vaccination from birth to 10 weeks of age would enhance the vaccine-induced immune response. METHODS In a randomized clinical trial, BCG was administered intradermally either at birth (n=25) or at 10 weeks of age (n=21). Ten weeks after vaccination, and at 1 year of age, vaccine-specific CD4 and CD8 T cell responses were measured with a whole blood intracellular cytokine assay. RESULTS Infants who received delayed BCG vaccination demonstrated higher frequencies of BCG-specific CD4 T cells, particularly polyfunctional T cells co-expressing IFN-gamma, TNF-alpha and IL-2, and most strikingly at 1 year of age. CONCLUSIONS Delaying BCG vaccination from birth to 10 weeks of age enhances the quantitative and qualitative BCG-specific T cell response, when measured at 1 year of age.

First-in-human trial of the post-exposure tuberculosis vaccine H56:IC31 in Mycobacterium tuberculosis infected and non-infected healthy adults.

BACKGROUND H56:IC31 is a candidate tuberculosis vaccine comprising a fusion protein of Ag85B, ESAT-6 and Rv2660c, formulated in IC31 adjuvant. This first-in-human, open label phase I trial assessed the safety and immunogenicity of H56:IC31 in healthy adults without or with Mycobacterium tuberculosis (M.tb) infection. METHODS Low dose (15 μg H56 protein in 500 nmol IC31) or high dose (50 μg H56, 500 nmol IC31) vaccine was administered intramuscularly thrice, at 56-day intervals. Antigen-specific T cell responses were measured by intracellular cytokine staining and antibody responses by ELISA. RESULTS One hundred and twenty-six subjects were screened and 25 enrolled and vaccinated. No serious adverse events were reported. Nine subjects (36%) presented with transient cardiovascular adverse events. The H56:IC31 vaccine induced antigen-specific IgG responses and Th1 cytokine-expressing CD4(+) T cells. M.tb-infected vaccinees had higher frequencies of H56-induced CD4(+) T cells than uninfected vaccinees. Low dose vaccination induced more polyfunctional (IFN-γ(+)TNF-α(+)IL-2(+)) and higher frequencies of H56-specific CD4(+) T cells compared with high dose vaccination. A striking increase in IFN-γ-only-expressing CD4(+) T cells, displaying a CD45RA(-)CCR7(-) effector memory phenotype, emerged after the second high-dose vaccination in M.tb-infected vaccinees. TNF-α(+)IL-2(+) H56-specific memory CD4(+) T cells were detected mostly after low-dose H56 vaccination in M.tb-infected vaccinees, and predominantly expressed a CD45RA(-)CCR7(+) central memory phenotype. Our results support further clinical testing of H56:IC31.

Higher human CD4 T cell response to novel Mycobacterium tuberculosis latency associated antigens Rv2660 and Rv2659 in latent infection compared with tuberculosis disease

One third of the worlds population is infected with Mycobacterium tuberculosis (M.tb). A vaccine that would prevent progression to TB disease will have a dramatic impact on the global TB burden. We propose that antigens of M.tb that are preferentially expressed during latent infection will be excellent candidates for post-exposure vaccination. We therefore assessed human T cell recognition of two such antigens, Rv2660 and Rv2659. Expression of these was shown to be associated with non-replicating persistence in vitro. After six days incubation of PBMC from persons with latent tuberculosis infection (LTBI) and tuberculosis (TB) disease, Rv2660 and Rv2659 induced IFN-γ production in a greater proportion of persons with LTBI, compared with TB diseased patients. Persons with LTBI also had increased numbers of viable T cells, and greater specific CD4(+) T cell proliferation and cytokine expression capacity. Persons with LTBI preferentially recognize Rv2659 and Rv2660, compared with patients with TB disease. These results suggest promise of these antigens for incorporation into post-exposure TB vaccines.

Safety and Immunogenicity of H1/IC31®, an Adjuvanted TB Subunit Vaccine, in HIV-Infected Adults with CD4+ Lymphocyte Counts Greater than 350 cells/mm3: A Phase II, Multi-Centre, Double-Blind, Randomized, Placebo-Controlled Trial

Background Novel tuberculosis vaccines should be safe, immunogenic, and effective in various population groups, including HIV-infected individuals. In this phase II multi-centre, double-blind, placebo-controlled trial, the safety and immunogenicity of the novel H1/IC31 vaccine, a fusion protein of Ag85B-ESAT-6 (H1) formulated with the adjuvant IC31, was evaluated in HIV-infected adults. Methods HIV-infected adults with CD4+ T cell counts >350/mm3 and without evidence of active tuberculosis were enrolled and followed until day 182. H1/IC31 vaccine or placebo was randomly allocated in a 5∶1 ratio. The vaccine was administered intramuscularly at day 0 and 56. Safety assessment was based on medical history, clinical examinations, and blood and urine testing. Immunogenicity was determined by a short-term whole blood intracellular cytokine staining assay. Results 47 of the 48 randomised participants completed both vaccinations. In total, 459 mild or moderate and 2 severe adverse events were reported. There were three serious adverse events in two vaccinees classified as not related to the investigational product. Local injection site reactions were more common in H1/IC31 versus placebo recipients (65.0% vs. 12.5%, p = 0.015). Solicited systemic and unsolicited adverse events were similar by study arm. The baseline CD4+ T cell count and HIV viral load were similar by study arm and remained constant over time. The H1/IC31 vaccine induced a persistent Th1-immune response with predominately TNF-α and IL-2 co-expressing CD4+ T cells, as well as polyfunctional IFN-γ, TNF-α and IL-2 expressing CD4+ T cells. Conclusion H1/IC31 was well tolerated and safe in HIV-infected adults with a CD4+ Lymphocyte count greater than 350 cells/mm3. The vaccine did not have an effect on CD4+ T cell count or HIV-1 viral load. H1/IC31 induced a specific and durable Th1 immune response. Trial registration Pan African Clinical Trials Registry (PACTR) PACTR201105000289276

The novel tuberculosis vaccine, AERAS-402, is safe in healthy infants previously vaccinated with BCG, and induces dose-dependent CD4 and CD8T cell responses

BACKGROUND Efforts to reduce risk of tuberculosis disease in children include development of effective vaccines. Our aim was to test safety and immunogenicity of the new adenovirus 35-vectored tuberculosis vaccine candidate AERAS-402 in infants, administered as a boost following a prime with the Bacille Calmette-Guerin vaccine. METHODS In a phase 1 randomised, double-blind, placebo-controlled, dose-escalation trial, BCG-vaccinated infants aged 6-9 months were sequentially assigned to four study groups, then randomized to receive an increasing dose-strength of AERAS-402, or placebo. The highest dose group received a second dose of vaccine or placebo 56 days after the first. The primary study outcome was safety. Whole blood intracellular cytokine staining assessed immunogenicity. RESULTS Forty-two infants received AERAS-402 and 15 infants received placebo. During follow-up of 182 days, an acceptable safety profile was shown with no serious adverse events or discontinuations related to the vaccine. AERAS-402 induced a specific T cell response. A single dose of AERAS-402 induced CD4T cells predominantly expressing single IFN-γ whereas two doses induced CD4T cells predominantly expressing IFN-γ, TNF-α and IL-2 together. CD8T cells were induced and were more likely to be present after 2 doses of AERAS-402. CONCLUSIONS AERAS-402 was safe and immunogenic in healthy infants previously vaccinated with BCG at birth. Administration of the highest dose twice may be the most optimal vaccination strategy, based on the induced immunity. Multiple differences in T cell responses when infants are compared with adults vaccinated with AERAS-402, in the same setting and using the same whole blood intracellular cytokine assay, suggest specific strategies may be important for vaccination for each population.

Qualification of a whole blood intracellular cytokine staining assay to measure mycobacteria-specific CD4 and CD8 T cell immunity by flow cytometry.

BACKGROUND Qualified or validated assays are essential in clinical trials. Short-term stimulation of whole blood and intracellular cytokine staining assay is commonly used to measure immunogenicity in tuberculosis vaccine clinical trials. Previously, the short-term stimulation process of whole blood with BCG was optimized. We aimed to qualify the intracellular cytokine staining process and assess the effects of long-term cryopreservation. Our hypotheses were that the assay is robust in the measurement of the mycobacteria-specific T cells, and long-term cryopreservation of fixed cells from stimulated whole blood would not compromise reliable measurement of mycobacteria induced CD4 T cell immunity. METHODS Whole blood from healthy adults was collected in sodium heparinized tubes. The blood was left unstimulated or stimulated with mycobacterial antigens or mitogens for 12h. Cells were harvested, fixed and multiple aliquots from each participant cryopreserved. Later, mycobacteria-specific CD4 and CD8 T cells expressing IFN-γ, TNF-α, IL-2 and IL-17 were quantitated by flow cytometry. Assay performance characteristics evaluated included limit of quantification and detection, reproducibility, precision, robustness, specificity and sensitivity. To assess the effects of long-term cryopreservation, fixed cells from the stimulated bloods were analysed one week post-cryopreservation and at 3-month intervals over a 3-year period. RESULTS The limit of quantification for the different cytokines was variable: 0.04% for frequencies of IFN-γ- and IL-2-expressing T cells and less than 0.01% for TNF-α- and IL-17-expressing T cells. When measurement of the mycobacteria-specific T cells was assessed at levels above the detection limit, the whole blood intracellular cytokine assay showed high precision that was operator-independent. The assay was also robust: variation in staining conditions including temperature (4 °C or 20-23 °C) and time (45, 60 or 90 min) did not markedly affect quantification of specific T cells. Finally, prolonged periods of cryopreservation also did not significantly influence quantification of mycobacteria-specific CD4 T cells. CONCLUSIONS The whole blood intracellular cytokine assay is robust and reliable in quantification of the mycobacteria-specific T cells and is not significantly affected by cryopreservation of fixed cells.

Distinct T-Cell Responses When BCG Vaccination Is Delayed From Birth to 6 Weeks of Age in Ugandan Infants

BACKGROUND In Uganda, the tuberculosis vaccine BCG is administered on the first day of life. Infants delivered at home receive BCG vaccine at their first healthcare facility visit at 6 weeks of age. Our aim was to determine the effect of this delay in BCG vaccination on the induced immune response. METHODS We assessed CD4(+) and CD8(+) T-cell responses with a 12-hour whole-blood intracellular cytokine/cytotoxic marker assay, and with a 6-day proliferation assay. RESULTS We enrolled 92 infants: 50 had received BCG vaccine at birth and 42 at 6 weeks of age. Birth vaccination was associated with (1) greater induction of CD4(+) and CD8(+) T cells expressing either interferon γ (IFN-γ) alone or IFN-γ together with perforin and (2) induction of proliferating cells that had greater capacity to produce IFN-γ, tumor necrosis factor α (TNF-α), and interleukin 2 together, compared with delayed vaccination. CONCLUSIONS Distinct patterns of T-cell induction occurred when BCG vaccine was given at birth and at 6 weeks of age. We propose that this diversity might impact protection against tuberculosis. Our results differ from those of studies of delayed BCG vaccination in South Africa and the Gambia, suggesting that geographical and population heterogeneity may affect the BCG vaccine-induced T-cell response.

A randomized clinical trial in adults and newborns in South Africa to compare the safety and immunogenicity of bacille Calmette-Guérin (BCG) vaccine administration via a disposable-syringe jet injector to conventional technique with needle and syringe

Introduction Intradermal bacille Calmette-Guérin (BCG) vaccination by needle-free, disposable-syringe jet injectors (DSJI) is an alternative to the Mantoux method using needle and syringe (NS). We compared the safety and immunogenicity of BCG administration via the DSJI and NS techniques in adults and newborn infants at the South African Tuberculosis Vaccine Initiative (SATVI) research site in South Africa. Method Thirty adults and 66 newborn infants were randomized 1:1 to receive intradermal BCG vaccine (0.1 mL in adults; 0.05 mL in infants) via DSJI or NS. Wheal diameter (mm) and skin fluid deposition at the site of injection (SOI) were measured immediately post-vaccination. Adverse events and SOI reactogenicity data were collected 30 min and 1, 2, 4, and 12 weeks after vaccination for adults and at 30 min and 4, 10, and 14 weeks for infants. Blood was collected in infants at 10 and 14 weeks to assess BCG-specific T-cell immune responses. Results More infant BCG vaccinations by DSJI deposited >5 μL fluid on the skin surface, compared to NS (49% versus 9%, p = 0.001). However, all 12 infant vaccinations that did not produce any SOI wheal occurred in the NS group (36%, p < 0.001). Median wheal diameter, in participants for which an SOI wheal formed, did not differ significantly between groups in infants (combined 3.0 mm IQR 2.0 to 4.0, p = 0.59) or in adults (combined 9.0 mm IQR 7.0 to 10.0, p = 0.13). Adverse events were similar between study arms. Proportion of participants with BCG scars after three months did not differ in adults (combined 97%, p = 0.67) or infants (combined 62%, p = 0.13). Frequencies of BCG-specific clusters of differentiation 4 (CD4) and clusters of differentiation 8 (CD8) T-cells co-expressing IFN-γ, TNF-α, IL-2, and/or IL-17 were not different in the DSJI and NS groups. Conclusion BCG vaccination of newborn infants via DSJI was more likely to deliver an appropriate intradermal wheal at the SOI as compared to NS, despite leaving more fluid on the surface of the skin. Safety, reactogenicity, and antigen-specific T-cell immune responses did not differ between DSJI and NS techniques.

H1:IC31 vaccination is safe and induces long-lived TNF-α+IL-2+CD4 T cell responses in M. tuberculosis infected and uninfected adolescents: A randomized trial

BACKGROUND Control of the tuberculosis epidemic requires a novel vaccine that is effective in preventing tuberculosis in adolescents, a key target population for vaccination against TB. METHODS Healthy adolescents, stratified by M. tuberculosis-infection status, were enrolled into this observer-blinded phase II clinical trial of the protein-subunit vaccine candidate, H1:IC31, comprising a fusion protein (H1) of Ag85B and ESAT-6, formulated with the IC31 adjuvant. Local and systemic adverse events and induced T cell responses were measured after one or two administrations of either 15μg or 50μg of the H1 protein. RESULTS Two hundred and forty participants were recruited and followed up for 224days. No notable safety events were observed regardless of H1 dose or vaccination schedule. H1:IC31 vaccination induced antigen-specific CD4 T cells, co-expressing IFN-γ, TNF-α and/or IL-2. H1:IC31 vaccination of M.tb-uninfected individuals preferentially drove the emergence of Ag85B and ESAT-6 specific TNF-α+IL-2+CD4 T cells, while H1:IC31 vaccination of M.tb-infected individuals resulted in the expansion of Ag85B-specific but not ESAT-6-specific TNF-α+IL-2+CD4 T cells. CONCLUSIONS H1:IC31 was safe and immunogenic in uninfected and M.tb-infected adolescents. Two administrations of the 15μg H1:IC31 dose induced the greatest magnitude immune response, and was considered optimal (South African National Clinical Trials Register, DoH-27-0612-3947; Pan African Clinical Trial Registry, PACTR201403000464306).