Raija Ahmed

rBCG Induces Strong Antigen-Specific T Cell Responses in Rhesus Macaques in a Prime-Boost Setting with an Adenovirus 35 Tuberculosis Vaccine Vector

Background BCG vaccination, combined with adenoviral-delivered boosts, represents a reasonable strategy to augment, broaden and prolong immune protection against tuberculosis (TB). We tested BCG (SSI1331) (in 6 animals, delivered intradermally) and a recombinant (rBCG) AFRO-1 expressing perfringolysin (in 6 animals) followed by two boosts (delivered intramuscullary) with non-replicating adenovirus 35 (rAd35) expressing a fusion protein composed of Ag85A, Ag85B and TB10.4, for the capacity to induce antigen-specific cellular immune responses in rhesus macaques (Macaca mulatta). Control animals received diluent (3 animals). Methods and Findings Cellular immune responses were analyzed longitudinally (12 blood draws for each animal) using intracellular cytokine staining (TNF-alpha, IL-2 and IFN-gamma), T cell proliferation was measured in CD4+, CD8alpha/beta+, and CD8alpha/alpha+ T cell subsets and IFN-gamma production was tested in 7 day PBMC cultures (whole blood cell assay, WBA) using Ag85A, Ag85B, TB10.4 recombinant proteins, PPD or BCG as stimuli. Animals primed with AFRO-1 showed i) increased Ag85B-specific IFN-gamma production in the WBA assay (median >400 pg/ml for 6 animals) one week after the first boost with adenoviral-delivered TB-antigens as compared to animals primed with BCG (<200 pg/ml), ii) stronger T cell proliferation in the CD8alpha/alpha+ T cell subset (proliferative index 17%) as compared to BCG-primed animals (proliferative index 5% in CD8alpha/alpha+ T cells). Polyfunctional T cells, defined by IFN-gamma, TNF-alpha and IL-2 production were detected in 2/6 animals primed with AFRO-1 directed against Ag85A/b and TB10.4; 4/6 animals primed with BCG showed a Ag85A/b responses, yet only a single animal exhibited Ag85A/b and TB10.4 reactivity. Conclusion AFRO-1 induces qualitatively and quantitatively different cellular immune responses as compared with BCG in rhesus macaques. Increased IFN-gamma-responses and antigen-specific T cell proliferation in the CD8alpha/alpha+ T cell subset represents a valuable marker for vaccine-take in BCG-based TB vaccine trials

Autologous mesenchymal stromal cell infusion as adjunct treatment in patients with multidrug and extensively drug-resistant tuberculosis: an open-label phase 1 safety trial.

BACKGROUND Novel treatment options are urgently needed for multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis, which are associated with immune dysfunction and poor treatment outcomes. Mesenchymal stromal cells (MSCs) are immunomodulatory and adjunct autologous treatment with bone marrow-derived MSCs might improve clinical outcome by transforming chronic inflammation into productive immune responses. Our aim was to assess the safety of infusion of autologous MSCs as an adjunct treatment in patients with tuberculosis. METHODS 30 patients with microbiologically confirmed MDR or XDR tuberculosis were treated with single-dose autologous bone marrow-derived MSCs (aimed for 1×10(6) cells per kg), within 4 weeks of the start of antituberculosis-drug treatment in a specialist centre in Minsk, Belarus. Inclusion patients were those with pulmonary tuberculosis confirmed by sputum smear microscopy, culture, or both; MDR or XDR tuberculosis confirmed by drug-susceptibility testing to first-line and second-line drugs; age older than 21 years to 65 years or younger; and absence of lesion compatible with a malignant process or ongoing tuberculosis in organs other than the lungs and pleura. In addition to the inclusion criteria, patients were excluded if they were pregnant, coinfected with HIV, or infected with hepatitis B, C, or both. The primary endpoint was safety measured by MSC-infusion related events; any tuberculosis-related event within the 6 month observation period that related to a worsening of the underlying infectious disease, measured by conversion of Mycobacterium tuberculosis culture or microscopic examination; or any adverse event defined clinically or by changes in blood haematology and biochemistry variables, measured monthly for 6 months after MSC infusion per protocol. This study is registered with the German Clinical Trials Registry, number DRKS00000763. FINDINGS The most common (grade 1 or 2) adverse events were high cholesterol levels (14 of 30 patients), nausea (11 of 30 patients), and lymphopenia or diarrhoea (ten of 30 patients). There were no serious adverse events reported. We recorded two grade 3 events that were transitory-ie, increased plasma potassium ion concentrations in one patient and a transitory grade 3 γ-glutamyltransferase elevation in another patient. INTERPRETATION MSCs as an adjunct therapy are safe and can now be explored further for the treatment of patients with MDR or XDR tuberculosis in combination with standard drug regimens. Adjunct treatment with MSCs needs to be evaluated in controlled phase 2 trials to assess effects on immune responses and clinical and microbiological outcomes.

Beta-chemokine production in macaques vaccinated with live attenuated virus correlates with protection against simian immunodeficiency virus (SIVsm) challenge.

Simian immunodeficiency virus (SIV) uses the CCR5 chemokine receptor as the main co-receptor to enter CD4+ cells. RANTES, MIP-1alpha and MIP-1beta have been suggested as the major human immunodeficiency virus-suppressor factors produced by CD8+ T-cells. The aim of this study was to investigate the CD8+ T-cell production of anti-viral factors and of beta-chemokines in six cynomolgus macaques vaccinated with live attenuated SIVmacC8 in relation to protection against infectious intrarectal SIVsm challenge. Three of the vaccinated animals were completely protected and one was partially protected against the challenge virus. Interestingly, these monkeys showed higher in vitro anti-viral CD8+ cell suppressor activity and beta-chemokine production both before and after vaccination as compared to the infected monkeys. The results indicate that beta-chemokines may play a role in protective immunity but also that genetic and/or environmental factors may influence their production.

Prime-Boost Vaccination with rBCG/rAd35 Enhances CD8+ Cytolytic T-Cell Responses in Lesions from Mycobacterium Tuberculosis –Infected Primates

To prevent the global spread of tuberculosis (TB) infection, a novel vaccine that triggers potent and long-lived immunity is urgently required. A plasmid-based vaccine has been developed to enhance activation of major histocompatibility complex (MHC) class I-restricted CD8+ cytolytic T cells using a recombinant Bacille Calmette-Guérin (rBCG) expressing a pore-forming toxin and the Mycobacterium tuberculosis (Mtb) antigens Ag85A, 85B and TB10.4 followed by a booster with a nonreplicating adenovirus 35 (rAd35) vaccine vector encoding the same Mtb antigens. Here, the capacity of the rBCG/rAd35 vaccine to induce protective and biologically relevant CD8+ T-cell responses in a nonhuman primate model of TB was investigated. After prime/boost immunizations and challenge with virulent Mtb in rhesus macaques, quantification of immune responses at the single-cell level in cryopreserved tissue specimen from infected organs was performed using in situ computerized image analysis as a technological platform. Significantly elevated levels of CD3+ and CD8+ T cells as well as cells expressing interleukin (IL)-7, perforin and granulysin were found in TB lung lesions and spleen from rBCG/rAd35-vaccinated animals compared with BCG/rAd35-vaccinated or unvaccinated animals. The local increase in CD8+ cytolytic T cells correlated with reduced expression of the Mtb antigen MPT64 and also with prolonged survival after the challenge. Our observations suggest that a protective immune response in rBCG/rAd35-vaccinated nonhuman primates was associated with enhanced MHC class I antigen presentation and activation of CD8+ effector T-cell responses at the local site of infection in Mtb-challenged animals.

High content cellular immune profiling reveals differences between rhesus monkeys and men.

A better understanding of similarities and differences in the composition of the cellular immune system in non‐human primates (NHPs) compared with human subjects will improve the interpretation of preclinical studies. It will also aid in addressing the usefulness of NHPs as subjects for studying chronic diseases, vaccine development and immune reconstitution. We employed high content colour flow cytometry and analysed simultaneously the expression of CD3, CD4, CD8α, CD8β, CD16/CD56, CD45RA, CCR7, CD27, CD28, CD107a and the interleukin‐7 receptor α‐chain (IL‐7Rα) in peripheral blood mononuclear cells (PBMCs) of 27 rhesus macaques and 16 healthy human subjects. Regulatory T cells (Tregs) were identified using anti‐CD3, ‐CD4, ‐CD25, ‐FoxP3, and ‐IL‐7Rα monoclonal antibodies. Responsiveness to IL‐7 was gauged in a signal transducer and activation of transcription 5 (STAT‐5) phosphorylation assay. Human and NHP PBMCs showed a similar T‐cell composition pattern with some remarkable differences. Similarities: human and NHP CD4+ and CD8+ cells showed a similar STAT‐5 phosphorylation pattern in response to IL‐7. Multicolour flow cytometric analysis identified a CD4+ CD8αα+ CD8αβ+ T‐cell population in NHPs as well as in human subjects that expressed the degranulation marker CD107a and may represent a unique CD4+ T‐cell subset endowed with cytotoxic capacity. Differences: we identified in PBMCs from NHPs a higher proportion (5·16% in CD3+ T cells) of CD8αα+ T cells when compared with human donors (1·22% in CD3+ T cells). NHP CD8αα+ T cells produced tumour necrosis factor‐α / interferon‐γ (TNF‐α/IFN‐γ) or TNF‐α, whereas human CD8αα+ T cells produced simultaneously TNF‐α/IFN‐γ and IL‐2. A minor percentage of human CD8+ T cells expressed CD25bright and FoxP3 (0·01%). In contrast, 0·07% of NHP CD8+ T cells exhibited the CD25bright FoxP3+ phenotype. PBMCs from NHPs showed less IL‐7Rα‐positive events in all T‐cell subsets including CD4+ Tregs (median 5%) as compared with human (median 12%). The data visualize commonalities and differences in immune cell subsets in humans and NHPs, most of them in long‐lived memory cells and cells with suppressive functions. This provides a matrix to assess future efforts to study diseases and vaccines in NHPs.

Spontaneous production of RANTES and antigen-specific IFN-γ production in macaques vaccinated with SHIV-4 correlates with protection against SIVsm challenge

The β‐chemokines, RANTES, MIP‐1α and MIP‐1β, have been implicated as being some of the protective factors in the immune response against human immunodeficiency virus (HIV) infection. We have presented data previously indicating that these chemokines also play a role in protective immunity against HIV/SIV infection in macaques. The aim of this study was to investigate the production of β‐chemokines in eight cynomolgus macaques vaccinated with non‐pathogenic SHIV‐4 in relation to protection against pathogenic SIVsm challenge. Four control animals were also included in the study. Two of the vaccinated monkeys were completely protected and one was partially protected against the challenge virus. The monkeys that resisted infectious SIVsm virus challenge showed higher spontaneous β‐chemokine production by peripheral blood mononuclear cells and had higher numbers of antigen‐induced IFN‐γ secreting cells compared to the non‐protected animals. Our observations support our previous findings that the genetic background of the host and/or environmental factors are involved in the chemokine production and that β‐chemokines contribute to protection against HIV/SIV infection.

Role of CD8+ Cell-Produced Anti-Viral Factors in Protective Immunity in HIV-2-Exposed but Seronegative Macaques Resistant to Intrarectal SIVsm Challenge

The cell‐mediated immune response is likely to be important in controlling HIV/SIV infection. There is evidence that β‐chemokines and other, as yet unknown, anti‐viral factors play a role in host defence against HIV infection. We reported previously that HIV‐2 exposed but seronegative cynomolgus macaques developed SIV‐specific cytotoxic T lymphocytes and were resistant to mucosal SIV challenge. The aim of this study was to examine CD8+ cell‐dependent production of β‐chemokines and other anti‐viral factors in these macaques. The animals, selected from among 17 monkeys enrolled in two separate experiments, were either treated with an anti‐viral drug or immunized passively with HIV‐2 antibody‐positive serum. Three of these monkeys were protected against repeated HIV‐2 challenge and were also able to control SIV infection 3 years later. Control samples were obtained from four macaques that became SIV infected and from 39 naïve animals. The three resistant monkeys showed significantly higher production of RANTES and MIP‐1α than the 39 naïve animals. In addition, SIV infection was suppressed by CD8+ cell culture supernatants of these monkeys. However, antibodies to chemokines only partially neutralized CD8+ cell‐mediated SIV suppression indicating that the anti‐viral activity observed in these monkeys was the result of combined action of several inhibitory factors.

Pattern recognition and cellular immune responses to novel Mycobacterium tuberculosis- antigens in individuals from Belarus

BackgroundTuberculosis (TB) is an enduring health problem worldwide and the emerging threat of multidrug resistant (MDR) TB and extensively drug resistant (XDR) TB is of particular concern. A better understanding of biomarkers associated with TB will aid to guide the development of better targets for TB diagnosis and for the development of improved TB vaccines.MethodsRecombinant proteins (n = 7) and peptide pools (n = 14) from M. tuberculosis (M.tb) antigens associated with M.tb pathogenicity, modification of cell lipids or cellular metabolism, were used to compare T cell immune responses defined by IFN-γ production using a whole blood assay (WBA) from i) patients with TB, ii) individuals recovered from TB and iii) individuals exposed to TB without evidence of clinical TB infection from Minsk, Belarus.ResultsWe identified differences in M.tb target peptide recognition between the test groups, i.e. a frequent recognition of antigens associated with lipid metabolism, e.g. cyclopropane fatty acyl phospholipid synthase. The pattern of peptide recognition was broader in blood from healthy individuals and those recovered from TB as compared to individuals suffering from pulmonary TB. Detection of biologically relevant M.tb targets was confirmed by staining for intracellular cytokines (IL-2, TNF-α and IFN-γ) in T cells from non-human primates (NHPs) after BCG vaccination.ConclusionsPBMCs from healthy individuals and those recovered from TB recognized a broader spectrum of M.tb antigens as compared to patients with TB. The nature of the pattern recognition of a broad panel of M.tb antigens will devise better strategies to identify improved diagnostics gauging previous exposure to M.tb; it may also guide the development of improved TB-vaccines.

Human Leukocyte Antigens A*3001 and A*3002 Show Distinct Peptide-Binding Patterns of the Mycobacterium tuberculosis Protein TB10.4: Consequences for Immune Recognition

ABSTRACT High-tuberculosis (TB)-burden countries are located in sub-Saharan Africa. We examined the frequency of human leukocyte antigen (HLA) alleles, followed by recombinant expression of the most frequent HLA-A alleles, i.e., HLA-A*3001 and HLA-A*3002, to study differences in mycobacterial peptide presentation and CD8+ T-cell recognition. We screened a peptide library (9-mer peptides with an 8-amino-acid overlap) for binding, affinity, and off-rate of the Mycobacterium tuberculosis-associated antigen TB10.4 and identified only three TB10.4 peptides with considerable binding to HLA-A*3001. In contrast, 22 peptides bound to HLA-A*3002. This reflects a marked difference in the binding preference between the two alleles, with A*3002 tolerating a more promiscuous peptide-binding pattern and A*3001 accommodating only a very selective peptide repertoire. Subsequent analysis of the affinity and off-rate of the binding peptides revealed a strong affinity (8 nM to 7 μM) and moderate off-rate (20 min to 3 h) for both alleles. Construction of HLA-A*3001 and HLA-A*3002 tetramers containing selected binding peptides from TB10.4, including a peptide which was shared among both alleles, QIMYNYPAM (TB10.43-11), allowed us to enumerate epitope-specific T cells in HLA-A*3001- and HLA-A*3002-typed patients with active TB. HLA-A*3001 and HLA-A*3002 major histocompatibility complex-peptide complexes were recognized in individuals with active TB, irrespective of their homozygous HLA-A*3001 or HLA-A*3002 genetic background. The antigen-specific T cells exhibited the CD45RA+ CCR7+ precursor phenotype and the interleukin-7 receptor (CD127), which were different from the phenotype and receptor exhibited by the parental CD8+ T-cell population.

Differential cellular recognition pattern to M. tuberculosis targets defined by IFN-γ and IL-17 production in blood from TB + patients from Honduras as compared to health care workers: TB and immune responses in patients from Honduras

BackgroundA better understanding of the quality of cellular immune responses directed against molecularly defined targets will guide the development of TB diagnostics and identification of molecularly defined, clinically relevant M.tb vaccine candidates.MethodsRecombinant proteins (n = 8) and peptide pools (n = 14) from M. tuberculosis (M.tb) targets were used to compare cellular immune responses defined by IFN-γ and IL-17 production using a Whole Blood Assay (WBA) in a cohort of 148 individuals, i.e. patients with TB + (n = 38), TB- individuals with other pulmonary diseases (n = 81) and individuals exposed to TB without evidence of clinical TB (health care workers, n = 29).ResultsM.tb antigens Rv2958c (glycosyltransferase), Rv2962c (mycolyltransferase), Rv1886c (Ag85B), Rv3804c (Ag85A), and the PPE family member Rv3347c were frequently recognized, defined by IFN-γ production, in blood from healthy individuals exposed to M.tb (health care workers). A different recognition pattern was found for IL-17 production in blood from M.tb exposed individuals responding to TB10.4 (Rv0288), Ag85B (Rv1886c) and the PPE family members Rv0978c and Rv1917c.ConclusionsThe pattern of immune target recognition is different in regard to IFN-γ and IL-17 production to defined molecular M.tb targets in PBMCs from individuals frequently exposed to M.tb. The data represent the first mapping of cellular immune responses against M.tb targets in TB patients from Honduras.