Rationale: Administration of tuberculosis (TB) vaccines in participants with previous or current pulmonary TB may have the potential for causing harmful postvaccination immunologic (Koch-type) reactions.Objectives: To assess the safety and immunogenicity of three dose levels of the AERAS-402 live, replication-deficient adenovirus 35-vectored TB candidate vaccine, containing three mycobacterial antigens, in individuals with current or previous pulmonary TB.Methods: We performed a phase II randomized, placebo-controlled, double-blinded dose-escalation study in an HIV-negative adult South African cohort (n = 72) with active pulmonary TB (on treatment for 1-4 mo) or pulmonary TB treated at least 12 months before study entry and considered cured. Safety endpoints included clinical assessment, flow volume curves, diffusing capacity of the lung for carbon monoxide, pulse oximetry, chest radiograph, and high-resolution thoracic computerized tomography scans. Cytokine expression by CD4 and CD8 T cells, after stimulation with Ag85A, Ag85B, and TB10.4 peptide pools, was examined by intracellular cytokine staining.Measurements and Main Results: No apparent temporal or dose-related changes in clinical status (specifically acute, Koch phenomenon-like reactions), lung function, or radiology attributable to vaccine were observed. Injection site reactions were mild or moderate. Hematuria (by dipstick only) occurred in 25 (41%) of 61 AERAS-402 recipients and 3 (27%) of 11 placebo recipients, although no gross hematuria was reported. AERAS-402 induced robust CD8(+) and moderate CD4(+) T-cell responses, mainly to Ag85B in both vaccine groups.Conclusions: Administration of the AERAS-402 candidate TB vaccine to participants with current or previous pulmonary TB induced a robust immune response and is not associated with clinically significant pulmonary complications.

Intracellular cytokine staining (ICS) to detect cellular immune responses to infection or vaccination has become routine. Laboratories develop and optimize ICS assays independently and there are many variations of the assay in use today. However, little is known regarding the underlying biology that results in the variable outcomes following assay optimization. The results from the different assays can range from highly similar (slight changes in magnitude) to incredibly different, such as large changes in magnitude or a change in the responding cell types detected. These large differences prompted our investigation into the underlying reasons for the discrepancies in reported data for clinical trials. Here, we used leukapheresis samples from CMV-reactive volunteers to examine viability over time, the effect of costimulation, and response kinetics. The data show that golgi transport inhibitors diminish cell viability after 12 hours of stimulation, emphasizing the importance of viability markers particularly during long stimulations. Costimulatory antibodies increase responses but at the expense of sensitivity, given the increase in background. Interestingly, we found that CD4+ and CD8+ T cells exhibit differential kinetics in their response to CMVpp65 peptides. CD8+ T cells were largely exhausted by 6 hours following the initiation of stimulation, while CD4+ T cells continue to make cytokines well past 20 hours of stimulation. These data have implications on the design of ICS assays and the interpretation of data generated by ICS. Furthermore, the data provide a basis for understanding the differences in ICS assays and provide insight into the kinetics of CD4+ and CD8+ T cell responses.

One challenge to tuberculosis vaccine research is the lack of a correlate of immunity, which researchers are attempting to address through use of more high-throughput, high-data assays for assessing trial specimens. Multiparameter intracellular cytokine staining (ICS) assays have proven to be one of the most cost-effective tools available. Previously, we reported the development of a 13-color ICS panel for use in assessing T cell responses from human PBMCs. In an effort to better understand the metrics of the assay performance, a qualification study under GCLP conditions was executed. The parameters assessed in this performance qualification included specificity, sensitivity, selectivity, linearity, and intermediate precision. The lower limits of quantification for cytokine detection were shown to be under 0.03%. Using Fisher’s exact test to determine positivity, the false positive and false negative rates were shown to be 0% in this performance qualification. Furthermore, assessment of intermediate precision suggested that the CD3, CD4, CD8, CCR7, and CD45RO markers each showed a percent coefficient of variation (%CV) of 25% or under, while intracellular and functional markers were generally under a 30% CV as measured via responses to Staphylococcal enterotoxin B. The results of this performance qualification study illustrate the capabilities of this panel for assessing clinical trial responses and suggest it is a reliable assay for the assessment of T cell responses.

A new tuberculosis vaccine is needed to replace or enhance BCG, which induces variable protection against Mycobacterium tuberculosis pulmonary infections in adults. Development of new TB vaccine candidates is severely hampered by the lack of a correlate of immunity, unproven animal models, and limited funding opportunities. One candidate, MVA85A, recently failed to meet its efficacy endpoint goals despite promising early-phase trial data. As a result, some in the field believe we should now shift our focus away from product development and toward a research-oriented approach. Here, we outline our suggestions for this research-oriented strategy including diversification of the candidate pipeline, expanding measurements of immunity, improving pre-clinical animal models, and investing in combination pre-clinical/experimental medicine studies. As with any evolution, this change in strategy comes at a cost but may also represent an opportunity for advancing the field.

Here, we report on a first-in-man trial where the tuberculosis (TB) vaccine Ag85B-ESAT-6 (H1) was adjuvanted with escalating doses of a novel liposome adjuvant CAF01. On their own, protein antigens cannot sufficiently induce immune responses in humans, and require the addition of an adjuvant system to ensure appropriate delivery and concomitant immune activation. To date no approved adjuvants are available for induction of cellular immunity, which seems essential for a number of vaccines, including vaccines against TB. We vaccinated four groups of human volunteers: a non-adjuvanted H1 group, followed by three groups with escalating doses of CAF01-adjuvanted H1 vaccine. All subjects were vaccinated at 0 and 8 weeks and followed up for 150 weeks. Vaccination did not cause local or systemic adverse effects besides transient soreness at the injection site. Two vaccinations elicited strong antigen-specific T-cell responses which persisted after 150 weeks follow-up, indicating the induction of a long-lasting memory response in the vaccine recipients. These results show that CAF01 is a safe and tolerable, Th1-inducing adjuvant for human TB vaccination trials and for vaccination studies in general where cellular immunity is required.

Cytometry Part AVolume 85, Issue 7 p. 576-579 OMIPOpen Access OMIP-022: Comprehensive assessment of antigen-specific human T-cell functionality and memory Andrew J. Graves, Corresponding Author Andrew J. Graves Aeras, Rockville, Maryland USACorrespondence to: Andrew J. Graves. E-mail: agraves@aeras.orgSearch for more papers by this authorMarcelino G. Padilla, Marcelino G. Padilla Aeras, Rockville, Maryland USASearch for more papers by this authorDavid A. Hokey, David A. Hokey Aeras, Rockville, Maryland USASearch for more papers by this author Andrew J. Graves, Corresponding Author Andrew J. Graves Aeras, Rockville, Maryland USACorrespondence to: Andrew J. Graves. E-mail: agraves@aeras.orgSearch for more papers by this authorMarcelino G. Padilla, Marcelino G. Padilla Aeras, Rockville, Maryland USASearch for more papers by this authorDavid A. Hokey, David A. Hokey Aeras, Rockville, Maryland USASearch for more papers by this author First published: 27 May 2014 https://doi.org/10.1002/cyto.a.22478Citations: 16AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Purpose and Appropriate Sample Types This flow cytometry antibody panel was developed and optimized for the characterization of CD4+ and CD8+ T-cell memory and functional responses in adult and infant cryopreserved peripheral blood mononuclear cells (PBMC) stimulated with peptide pools to various antigens of interest (Table 1). The panel has been used to evaluate Mycobacterium tuberculosis (TB) antigen-specific responses in clinical trial specimens, and is currently undergoing assay qualification. Table 1. Summary table for application of OMIP-022 Purpose T-cell phenotyping, memory categorization, cytokine production, and function following in vitro stimulation Species Human Cell types Cryopreserved PBMC (adult and infant) Cross references OMIP-001, OMIP-008, OMIP-009, and OMIP-014 Background The lack of a correlate of immunity is a source of frustration in TB research 1. As such, it is crucial for TB vaccine researchers to "cast a wide net" when assessing immune responses to clinical trial candidates while minimizing the amount of specimen required to generate these data. This panel (Table 2) began as an incremental enhancement to OMIP-014 2, and included markers for dump (fixable viability dye, CD14, and CD19), T-cell phenotype (CD3, CD4, and CD8), Th1 cytokines (IFN-γ and TNF), a Th2 cytokine (IL-4), T-cell proliferation cytokine (IL-2), degranulation (CD107a), and activation (CD154). We have also expanded the panel to include memory markers (CCR7 and CD45RO), a Th17 cytokine (IL-17A), and an IL-10 superfamily cytokine (IL-22, used in place of IL-4). Table 2. Reagents used for OMIP-022 Specificity Clone Fluorochrome Purpose Viability Dye – AViD Dump CD14 M5E2 V500 CD19 HIB19 V500 CD3 UCHT1 ECD Phenotype CD4 RPA-T4 APC-eFluor 780 CD8 HIT8a Ax700 CCR7 G043H7 BV605 Memory CD45RO UCHL1 BV785 IFN-γ B27 V450 Th1 IL-2 MQ1–17H12 PE TNF MAb11 PE-Cy7 IL-17A BL168 PerCP-Cy5.5 Th17 IL-22 IL22JOP APC Th22 CD107a H4A3 Ax488 Degranulation CD154 TRAP1 PE-Cy5 Activation/B-Cell Help We chose to combine viability dye and dump markers on the same channel using AViD and V500 (for CD14 and CD19), thus freeing additional channels on the violet laser. For phenotyping, CD3 remains on ECD and CD4 on APC-eFluor 780. Th1 cytokines have been shown to be critical for controlling TB 3-9. To assess Th1 responses, we utilize antibodies against tumor necrosis factor (TNF) and IFN-γ. In designing this panel, we moved TNF to PE-Cy7 to improve detection of this cytokine, as fluorescein isothiocyanate (FITC) is comparatively dimmer and more prone to photobleaching. The recently introduced Brilliant Violet series of fluorochromes represents a bright and economical set of tools for detecting markers via the violet laser. Brilliant Violet 421 (BV421) was previously shown to be of similar brightness to PE 10, and was initially chosen as a replacement fluorochrome for IFN-γ. However, further investigation showed that any increase in antigen-specific response detected using the BV421 conjugate was matched with a significant increase in IFN-γ background. This increased background was observed using both the B27 (BD Biosciences, San Jose, CA) and the 4S.B3 (Biolegend, San Diego, CA) clones. When this background population was excluded from the IFN-γ gate of the BV421 conjugates, the responses detected using either the BV421 conjugates or the V450 conjugate were similar. This issue, combined with the increased cost of the reagent, led us to choose to continue to use the V450 conjugate for IFN-γ detection in this panel. Interleukin-2 (IL-2) is a cytokine responsible for T-cell proliferation and differentiation 11, 12 that also may play a role in TB immunity 13. In our panel, we use PE for bright and efficient detection of this cytokine. As M. tuberculosis is a facultative intracellular bacteria, cytolytic activity may be necessary to help control the disease. CD107a (LAMP-1) is a marker for degranulation and an indirect marker for cytolytic function 14, 15. Ideally, lytic proteins such as Perforin would be costained with the CD107a to confirm the cytotoxic potential of the CD107a+ cells. To this point, we are examining future iterations of this panel that may include a lytic marker. However, the current constraints of this panel limit our ability to add an additional marker without harming sensitivity in detecting the other markers of interest. To accommodate the change in fluorochrome for the detection of TNF, we moved CD107a to FITC initially, and then replaced FITC with the enhanced stability of Alexa Fluor 488 (Ax488). The Brilliant Violet series of fluorochromes has also allowed us to include the memory markers CCR7 and CD45RO. CCR7 is responsible for homing T-cells to lymph nodes 16 and is expressed on naïve and central memory T-cells (TCM) 17. CD45RO is the smallest isoform of CD45, and has been shown to be expressed on effector memory (TEM) and TCM 18. The inclusion of both markers allows for T-cells to be categorized as naïve, TCM, TEM, or effector T-cells. We chose Brilliant Violet 605 for CCR7 as it was the next brightest available Brilliant Violet dye and CCR7 is expressed as a continuum (not distinct populations) 19. The binding kinetics of the CCR7 antibody has shown optimal staining when incubated at 37°C (see staining protocol in Supporting Information). Brilliant Violet 785 was chosen for CD45RO based on antibody availability and minimal spectral overlap to other markers. IL-17A is secreted by Th17 T helper cells and acts to recruit innate immune effector cells to the site of inflammation 20. Recent works 21, 22 have suggested a strong role for IL-17A in tuberculosis immunity. As such, we initially replaced the MIP-1β from OMIP-014 with and anti-IL-17A antibody. In our hands, the PerCP-Cy5.5 on CD8 causes high spectral overlap into the Ax700 channel used for IL-17A. As IL-17A is lowly expressed compared to CD8 (and as PerCP-Cy5.5 is fluorescently brighter), we exchanged the fluorochromes for these two markers to minimize the impact of the high compensation. As an effect of changing CD8 to Ax700, a reduction in CD8+ events [perhaps an escapee phenomenon 23] was noted using Ax700 to detect CD8, but was mitigated by staining for CD8 intracellularly. IL-4, previously included with OMIP-014, is a representative Th2 cytokine. This interleukin is extremely difficult to detect and, in our hands, is inconsistent. We continue to include this marker as an option when assessing clinical trial specimens in which Th2 is considered a desired response. Recently, however, we have substituted IL-22 on APC instead of IL-4. IL-22 is part of the IL-10 superfamily 24, and when coexpressed with IL-17 enhances production antimicrobial peptides in the mucosa 25. Although poorly characterized to this point, recent research indicates that IL-22 plays a role in TB infection 21, 26. CD154 was maintained on this panel as emerging evidence indicates its role as a specific and sensitive marker in detecting CD4 response 27, as well as its roles in upregulating antimicrobial peptides 28 and its necessity for T-cell activation of B cells 29. As indicated in OMIP-014, the inclusion of Brefeldin A in our stimulation protocol requires intracellular staining of CD154. Figure 1 shows an example staining and analysis for adult PBMC stimulated with Staphylococcal enterotoxin B (Fig. 1A) or CMV pp65 (Fig. 1B). Figure 1Open in figure viewerPowerPoint Example staining of adult human PBMC following stimulation. (A) The first two rows demonstrate the gating hierarchy from total sample to CD4/CD8 identification. A time gate is used to exclude pressure aberrations from the cytometer that may have occurred during sample acquisition. Aggregate gates are used to exclude brightly positive events that may result from antibody or cell aggregation. The bottom row demonstrates gating for cytokines and functions from CD4+ (top half) and CD8+ (bottom half) events resulting from stimulation with Staphylococcal enterotoxin B. Note that IL-17A and CD154 were gated on the same plot to avoid mischaracterization resulting from the increased spectral overlap observed from their fluorochromes. (B) Example plots showing memory profile of CCR7 versus CD45RO in CD4+ and CD8+ populations. Using CMV pp65 peptide pool-stimulated PBMC from a CMV-reactive donor, IFN-γ+ events (blue) were overlaid onto these plots (gray), confirming localization of these events to the effector memory and effector compartments. Similarity to Published OMIPs As this panel assesses antigen-specific T-cell responses, it is similar to OMIP-001 30, OMIP-008 31, OMIP-009 32, and OMIP-014 2. Furthermore, this panel evolved from an initial desire to implement OMIP-014. Unlike these panels, however, our panel includes T-cell memory markers as well as an extensive combination of cytokines and functions associated with tuberculosis vaccine research. Our panel was developed for use in multicenter studies under good clinical laboratory practices conditions, and is currently undergoing assay qualification. Acknowledgments The authors thank Mario Roederer, Stephen P. Perfetto, Stephen C. De Rosa, and Michael R. Betts for their expertise and suggestions during the development and optimization of this panel. The authors declare no conflict of interest. Supporting Information Additional Supporting Information may be found in the online version of this article. Filename Description cytoa22478-sup-0001-suppinfo01.docx77.1 KB Supplementary Information cytoa22478-sup-0002-suppfig1.tif541.4 KB Supplementary Information Figure 1. cytoa22478-sup-0003-suppfig2.ps908.6 KB Supplementary Information Figure 3. cytoa22478-sup-0004-suppfig3.ps780.5 KB Supplementary Information Figure 3. cytoa22478-sup-0005-suppfig4.ps925.4 KB Supplementary Information Figure 4. cytoa22478-sup-0006-suppfig5.ps14.3 MB Supplementary Information Figure 5. cytoa22478-sup-0007-suppfig6.ps1.3 MB Supplementary Information Figure 6. cytoa22478-sup-0008-suppfig7.ps1.3 MB Supplementary Information Figure 7. cytoa22478-sup-0009-suppfig8.ps1.3 MB Supplementary Information Figure 8. cytoa22478-sup-0010-suppfig9.ps4.5 MB Supplementary Information Figure 9. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. Literature Cited 1 Hokey DA, Ginsberg A. The current state of tuberculosis vaccines. Hum Vaccin Immunother 2013; 9: 2142– 2146. CrossrefCASWeb of Science®Google Scholar 2 De Rosa SC, Carter DK, McElrath MJ. OMIP-014: Validated multifunctional characterization of antigen-specific human T cells by intracellular cytokine staining. Cytometry Part A 2012; 81A: 1019– 1021. Wiley Online LibraryCASWeb of Science®Google Scholar 3 Bruns H, Meinken C, Schauenberg P, Harter G, Kern P, Modlin RL, Antoni C, Stenger S. Anti-TNF immunotherapy reduces CD8+ T cell-mediated antimicrobial activity against Mycobacterium tuberculosis in humans. J Clin Invest 2009; 119: 1167– 1177. CrossrefCASPubMedWeb of Science®Google Scholar 4 Nunez Martinez O, Ripoll Noiseux C, Carneros Martin JA, Gonzalez Lara V, Gregorio Maranon HG. Reactivation tuberculosis in a patient with anti-TNF-alpha treatment. Am J Gastroenterol 2001; 96: 1665– 1666. CASPubMedGoogle Scholar 5 Keane J, Gershon S, Wise RP, Mirabile-Levens E, Kasznica J, Schwieterman WD, Siegel JN, Braun MM. Tuberculosis associated with infliximab, a tumor necrosis factor alpha-neutralizing agent. N Engl J Med 2001; 345: 1098– 1104. CrossrefCASPubMedWeb of Science®Google Scholar 6 Lammas DA, Casanova JL, Kumararatne DS. Clinical consequences of defects in the IL-12-dependent interferon-gamma (IFN-gamma) pathway. Clin Exp Immunol 2000; 121: 417– 425. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 7 Jouanguy E, Doffinger R, Dupuis S, Pallier A, Altare F, Casanova JL. IL-12 and IFN-gamma in host defense against mycobacteria and salmonella in mice and men. Curr Opin Immunol 1999; 11: 346– 351. CrossrefCASPubMedWeb of Science®Google Scholar 8 de Jong R, Altare F, Haagen IA, Elferink DG, Boer T, van Breda Vriesman PJ, Kabel PJ, Draaisma JM, van Dissel JT, Kroon FP, et al. Severe mycobacterial and Salmonella infections in interleukin-12 receptor-deficient patients. Science 1998; 280: 1435– 1438. CrossrefCASPubMedWeb of Science®Google Scholar 9 Altare F, Durandy A, Lammas D, Emile JF, Lamhamedi S, Le Deist F, Drysdale P, Jouanguy E, Doffinger R, Bernaudin F, et al. Impairment of mycobacterial immunity in human interleukin-12 receptor deficiency. Science 1998; 280: 1432– 1435. CrossrefCASPubMedWeb of Science®Google Scholar 10 Chattopadhyay PK, Gaylord B, Palmer A, Jiang N, Raven MA, Lewis G, Reuter MA, Nur-ur Rahman AK, Price DA, Betts MR, et al. Brilliant violet fluorophores: A new class of ultrabright fluorescent compounds for immunofluorescence experiments. Cytometry Part A 2012; 81A: 456– 466. Wiley Online LibraryCASWeb of Science®Google Scholar 11 Smith KA. Interleukin-2: Inception, impact, and implications. Science 1988; 240: 1169– 1176. CrossrefCASPubMedWeb of Science®Google Scholar 12 Cantrell DA, Smith KA. The interleukin-2 T-cell system: A new cell growth model. Science 1984; 224: 1312– 1316. CrossrefCASPubMedWeb of Science®Google Scholar 13 Toossi Z, Kleinhenz ME, Ellner JJ. Defective interleukin 2 production and responsiveness in human pulmonary tuberculosis. J Exp Med 1986; 163: 1162– 1172. CrossrefCASPubMedWeb of Science®Google Scholar 14 Chen JW, Murphy TL, Willingham MC, Pastan I, August JT. Identification of two lysosomal membrane glycoproteins. J Cell Biol 1985; 101: 85– 95. CrossrefCASPubMedWeb of Science®Google Scholar 15 Betts MR, Brenchley JM, Price DA, De Rosa SC, Douek DC, Roederer M, Koup RA. Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation. J Immunol Methods 2003; 281: 65– 78. CrossrefCASPubMedWeb of Science®Google Scholar 16 Ebert LM, Schaerli P, Moser B. Chemokine-mediated control of T cell traffic in lymphoid and peripheral tissues. Mol Immunol 2005; 42: 799– 809. CrossrefCASPubMedWeb of Science®Google Scholar 17 Sallusto F, Lenig D, Forster R, Lipp M, Lanzavecchia A. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 1999; 401: 708– 712. CrossrefCASPubMedWeb of Science®Google Scholar 18 Merkenschlager M, Beverley PC. Evidence for differential expression of CD45 isoforms by precursors for memory-dependent and independent cytotoxic responses: Human CD8 memory CTLp selectively express CD45RO (UCHL1). Int Immunol 1989; 1: 450– 459. CrossrefCASPubMedGoogle Scholar 19 McLaughlin BE, Baumgarth N, Bigos M, Roederer M, De Rosa SC, Altman JD, Nixon DF, Ottinger J, Oxford C, Evans TG, et al. Nine-color flow cytometry for accurate measurement of T cell subsets and cytokine responses. Part I: Panel design by an empiric approach. Cytometry Part A 2008; 73A: 400– 410. Wiley Online LibraryWeb of Science®Google Scholar 20 Weaver CT, Hatton RD, Mangan PR, Harrington LE. IL-17 family cytokines and the expanding diversity of effector T cell lineages. Annu Rev Immunol 2007; 25: 821– 852. CrossrefCASPubMedWeb of Science®Google Scholar 21 Scriba TJ, Kalsdorf B, Abrahams DA, Isaacs F, Hofmeister J, Black G, Hassan HY, Wilkinson RJ, Walzl G, Gelderbloem SJ, et al. Distinct, specific IL-17- and IL-22-producing CD4+ T cell subsets contribute to the human anti-mycobacterial immune response. J Immunol 2008; 180: 1962– 1970. CrossrefCASPubMedWeb of Science®Google Scholar 22 Khader SA, Bell GK, Pearl JE, Fountain JJ, Rangel-Moreno J, Cilley GE, Shen F, Eaton SM, Gaffen SL, Swain SL, et al. IL-23 and IL-17 in the establishment of protective pulmonary CD4+ T cell responses after vaccination and during Mycobacterium tuberculosis challenge. Nat Immunol 2007; 8: 369– 377. CrossrefCASPubMedWeb of Science®Google Scholar 23 Gratama JW, van der Linden R, van der Holt B, Bolhuis RL, van de Winkel JG. Analysis of factors contributing to the formation of mononuclear cell aggregates ("escapees") in flow cytometric immunophenotyping. Cytometry 1997; 29: 250– 260. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 24 Fickenscher H, Hor S, Kupers H, Knappe A, Wittmann S, Sticht H. The interleukin-10 family of cytokines. Trends Immunol 2002; 23: 89– 96. CrossrefCASPubMedWeb of Science®Google Scholar 25 Liang SC, Tan XY, Luxenberg DP, Karim R, Dunussi-Joannopoulos K, Collins M, Fouser LA. Interleukin (IL)−22 and IL-17 are coexpressed by Th17 cells and cooperatively enhance expression of antimicrobial peptides. J Exp Med 2006; 203: 2271– 2279. CrossrefCASPubMedWeb of Science®Google Scholar 26 Tadokera R, Wilkinson KA, Meintjes GA, Skolimowska KH, Matthews K, Seldon R, Rangaka MX, Maartens G, Wilkinson RJ. Role of the interleukin 10 family of cytokines in patients with immune reconstitution inflammatory syndrome associated with HIV infection and tuberculosis. J Infect Dis 2013; 207: 1148– 1156. CrossrefCASPubMedWeb of Science®Google Scholar 27 Frentsch M, Arbach O, Kirchhoff D, Moewes B, Worm M, Rothe M, Scheffold A, Thiel A. Direct access to CD4+ T cells specific for defined antigens according to CD154 expression. Nat Med 2005; 11: 1118– 1124. CrossrefCASPubMedWeb of Science®Google Scholar 28 Klug-Micu GM, Stenger S, Sommer A, Liu PT, Krutzik SR, Modlin RL, Fabri M. CD40 ligand and interferon-gamma induce an antimicrobial response against Mycobacterium tuberculosis in human monocytes. Immunology 2013; 139: 121– 128. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 29 Noelle RJ, Roy M, Shepherd DM, Stamenkovic I, Ledbetter JA, Aruffo A. A 39-kDa protein on activated helper T cells binds CD40 and transduces the signal for cognate activation of B cells. Proc Natl Acad Sci USA 1992; 89: 6550– 6554. CrossrefCASPubMedWeb of Science®Google Scholar 30 Mahnke YD, Roederer M. OMIP-001: Quality and phenotype of Ag-responsive human T-cells. Cytometry Part A 2010; 77A: 819– 820. Wiley Online LibraryWeb of Science®Google Scholar 31 Zuleger CL, Albertini MR. OMIP-008: Measurement of Th1 and Th2 cytokine polyfunctionality of human T cells. Cytometry Part A 2012; 81A: 450– 452. Wiley Online LibraryCASWeb of Science®Google Scholar 32 Lamoreaux L, Koup RA, Roederer M. OMIP-009: Characterization of antigen-specific human T-cells. Cytometry Part A 2012; 81A: 362– 363. Wiley Online LibraryCASWeb of Science®Google Scholar Citing Literature Volume85, Issue7July 2014Pages 576-579 This article also appears in:OMIPs Collection FiguresReferencesRelatedInformation

Abstract Development of a vaccine to prevent pulmonary tuberculosis remains a public health priority. AERAS-402 is a live, replication-deficient adenovirus 35-vectored TB vaccine expressing the mycobacterial antigens 85A, 85B, and TB10.4. While this vaccine has been shown to be safe in TB uninfected patients, little is known about the performance of this vaccine in TB-experienced populations. The safety and immunogenicity of AERAS-402 was evaluated in a Phase 2a randomized, placebo-controlled, double-blinded dose-escalation study in HIV-negative South African adults with either active or recent pulmonary TB infection. Peripheral blood mononuclear cells isolated from trial patients were assessed via intracellular cytokine staining (ICS) examining T cells for interferon-γ (IFN-γ), TNF, and IL-2 production. While modest CD4+ T cell responses were observed, the ICS data indicate the induction of a robust, dominant CD8+ T cell response not observed in placebo patients, including instances of total cytokine responsive cells approaching 6% of CD8+ T cells in some subjects. Polyfunctional analysis revealed that vaccine-specific T cells were primarily secreting IFN-γ either alone or in combination with TNF following stimulation. These data suggest that AERAS-402 is able to boost T cell responses in patients with a history of TB infection.

Abstract Developing an effective vaccine to prevent pulmonary tuberculosis represents a public health priority. AERAS-402 (adenovirus expressing a fusion of M.tuberculosis antigens 85A, 85B, and TB10.4) and MVA85A (Modified Vaccinia Anakara expressing Ag85A) are TB vaccine candidates that have been independently evaluated in adult and infant studies and shown to be safe and immunogenic. Despite robust immune responses in adults, infant trials have revealed lower than expected immunogenicity for these vaccine platforms, possibly contributing to the failure of the MVA85A vaccine in a recent phase II efficacy trial. Complimentary studies of malaria and HIV vaccines report enhanced immune responses using heterologous vaccine approaches with combinations of adenoviral and pox virus vectors. Here we present the preliminary immunogenicity data from a phase I clinical trial conducted in healthy BCG-vaccinated adults that were vaccinated with one or two doses of AERAS-402 and subsequently boosted with MVA85A. Preliminary data indicate that MVA85A boosts both CD4+ and CD8+ T cell responses. Interestingly, MVA85A, which primarily stimulates CD4+ T cell responses when given alone,robustly boosted AERAS-402 CD8+ T cell responses to Ag85A, with average responses reaching approximately 0.5% to 1% of CD8+ T cells. These data suggest that this vaccine combination is highly immunogenic in adults. Further studies are needed to assess the clinical efficacy of this vaccine combination.

Abstract Approximately one third of the world’s population is infected with Mycobacterium tuberculosis. Developing novel vaccines to protect against pulmonary tuberculosis is a public health priority. In this study, a Hybrid 1 (H1) subunit vaccine containing a recombinant fusion protein of Ag85B and ESAT-6 was paired with a two-component CAF01 liposomal adjuvant system developed and manufactured by Statens Serum Institut. A phase I clinical study was performed to evaluate H1:CAF01 in healthy non-BCG vaccinated adult male and female subjects between the ages of 18 and 55 years old. The subjects were randomized into four groups including H1 alone or H1 with 125/25µg, 313/125µg or 625/125µg CAF01 and were vaccinated on study days 0 and 56. PBMCs harvested approximately 150 weeks post vaccination were used to assess antigen specific responses by a 13-color intracellular cytokine staining assay. Vaccination with H1:CAF01 resulted in statistically significant Ag85B-specific CD4 polyfunctional CD154+ T cells compared to H1 alone. ESAT-6 stimulation resulted in detection of CD4 polyfunctional CD154+ T cells responses that were not elevated to a statistically significant extent compared to H1 alone. This is the first demonstration of the persistence of an antigen-specific cellular immune response up to 3 years after vaccination in a clinical trial using H1:CAF01 vaccination.

One-third of the world's population is estimated to be infected with TB. The WHO no longer recommends that the currently available TB vaccine, BCG, be given to infants born to HIV+ mothers due to safety concerns. An improved vaccine that is effective and safe for HIV+ infants is needed to protect this vulnerable population. AERAS-402 is a novel replication deficient Ad35 TB vaccine candidate that expresses a fusion protein of the Mtb antigens 85A, 85B, and TB10.4 and has been shown to safely induce CD8+ T cell responses in healthy adults. This study examined the safety and immunogenicity of a single IM dose of AERAS-402 in HIV+ adults. No vaccine-related serious adverse events were reported, suggesting the vaccine is safe in this population. CD4+ and CD8+ T cell responses were evaluated using ICS. Prior studies in healthy adults revealed primarily CD8+ responses to vaccination with AERAS-402. Vaccination of HIV+ adults resulted in the elicitation of both CD4+ and CD8+ responses of ~0.1% to 0.2% of the parent population largely directed to 85A and 85B. CD4+ responses were primarily polyfunctional (IFN-γ, IL-2, and TNF) and bifunctional (IL-2 and TNF) while CD8+ responses were largely bifunctional (IFN-γ and TNF) and monofunctional (IFN-γ) with lower levels of polyfunctional cells observed. Together the data suggest that vaccination with AERAS-402 in HIV+ adults is safe and immunogenic, resulting in a mixed CD4+ and CD8+ multifunctional T cell response to encoded antigens.

Despite plaguing humans for thousands of years, tuberculosis remains a widespread and lethal public health problem throughout the world today. The recent rise of multi-drug-resistant tuberculosis (MDR-TB) perpetuates the public health threat while presenting a potential bioterrorism agent. The BCG vaccine is the only available prevention against TB, yet it elicits inconsistent protection when given to infants, fails to provide consistent protection in adults against pulmonary disease, and is unsafe for use in immunocompromised patients. A new crop of TB vaccine candidates has entered into clinical trials, with a second generation following shortly. These new TB vaccines are hoped to provide a safe, efficacious replacement, or addition to, the nearly century-old BCG and provide protection against TB disease beyond childhood. This review details the status of the most promising TB vaccine candidates in development, as one of these candidates may play a key role in defending against an ominous health threat.