A new tool for tuberculosis vaccine screening: Ex vivo Mycobacterial Growth Inhibition Assay indicates BCG-mediated protection in a murine model of tuberculosis.

BACKGROUND: In the absence of a validated animal model and/or an immune correlate which predict vaccine-mediated protection, large-scale clinical trials are currently the only option to prove efficacy of new tuberculosis candidate vaccines. Tools to facilitate testing of new tuberculosis (TB) vaccines are therefore urgently needed. METHODS: We present here an optimized ex vivo mycobacterial growth inhibition assay (MGIA) using a murine Mycobacterium tuberculosis infection model. This assay assesses the combined ability of host immune cells to inhibit mycobacterial growth in response to vaccination. C57BL/6 mice were immunized with Bacillus Calmette-Guérin (BCG) and growth inhibition of mycobacteria by splenocytes was assessed. Mice were also challenged with Mycobacterium tuberculosis Erdman, and bacterial burden was assessed in lungs and spleen. RESULTS: Using the growth inhibition assay, we find a reduction in BCG CFU of 0.3-0.8 log10 after co-culture with murine splenocytes from BCG vaccinated versus naïve C57BL/6 mice. BCG vaccination in our hands led to a reduction in bacterial burden after challenge with Mycobacterium tuberculosis of approx. 0.7 log10 CFU in lung and approx. 1 log10 CFU in spleen. This effect was also seen when using Mycobacterium smegmatis as the target of growth inhibition. An increase in mycobacterial numbers was found when splenocytes from interferon gamma-deficient mice were used, compared to wild type controls, indicating that immune mechanisms may also be investigated using this assay. CONCLUSIONS: We believe that the ex vivo mycobacterial growth inhibition assay could be a useful tool to help assess vaccine efficacy in future, alongside other established methods. It could also be a valuable tool for determination of underlying immune mechanisms.

Mucosal delivery of antigen-coated nanoparticles to lungs confers protective immunity against tuberculosis infection in mice.

Mucosal boosting of BCG-immunised individuals with a subunit tuberculosis (TB) vaccine would be highly desirable, considering that the lungs are the principal port of entry for Mycobacterium tuberculosis (MTB) and the site of the primary infection and reactivation. However, the main roadblock for subunit TB vaccine development is the lack of suitable adjuvants that could induce robust local and systemic immune responses. Here, we describe a novel vaccine delivery system that was designed to mimic, in part, the MTB pathogen itself. The surface of yellow carnauba wax nanoparticles was coated with the highly immunogenic Ag85B Ag of MTB and they were directed to the alveolar epithelial surfaces by the incorporation of the heparin-binding hemagglutinin adhesion (HBHA) protein. Our results showed that the i.n. immunisation of BCG-primed BALB/c mice with nanoparticles adsorbed with Ag85B-HBHA (Nano-AH vaccine) induced robust humoral and cellular immune responses and IFN-γ production, and multifunctional CD4⁺ T cells expressing IFN-γ, IL-2 and TNF-α. Mice challenged with H37Rv MTB had a significantly reduced bacterial load in their lungs when compared with controls immunised with BCG alone. We therefore conclude that this immunisation approach is an effective means of boosting the BCG-induced anti-TB immunity.

Plant-derived recombinant immune complexes as self-adjuvanting TB immunogens for mucosal boosting of BCG.

Progress with protein-based tuberculosis (TB) vaccines has been limited by poor availability of adjuvants suitable for human application. Here, we developed and tested a novel approach to molecular engineering of adjuvanticity that circumvents the need for exogenous adjuvants. Thus, we generated and expressed in transgenic tobacco plants the recombinant immune complexes (RICs) incorporating the early secreted Ag85B and the latency-associated Acr antigen of Mycobacterium tuberculosis, genetically fused as a single polypeptide to the heavy chain of a monoclonal antibody to Acr. The RICs were formed by virtue of the antibody binding to Acr from adjacent molecules, thus allowing self-polymerization of the complexes. TB-RICs were purified from the plant extracts and shown to be biologically active by demonstrating that they could bind to C1q component of the complement and also to the surface of antigen-presenting cells. Mice immunized with BCG and then boosted with two intranasal immunizations with TB-RICs developed antigen-specific serum IgG antibody responses with mean end-point titres of 1 : 8100 (Acr) and 1 : 24 300 (Ag85B) and their splenocytes responded to in vitro stimulation by producing interferon gamma. 25% of CD4+ proliferating cells simultaneously produced IFN-γ, IL-2 and TNF-α, a phenotype that has been linked with protective immune responses in TB. Importantly, mucosal boosting of BCG-immunized mice with TB-RICs led to a reduced M. tuberculosis infection in their lungs from log10 mean = 5.69 ± 0.1 to 5.04 ± 0.2, which was statistically significant. We therefore propose that the plant-expressed TB-RICs represent a novel molecular platform for developing self-adjuvanting mucosal vaccines.

Mucosal vaccination against tuberculosis using inert bioparticles.

Needle-free, mucosal immunization is a highly desirable strategy for vaccination against many pathogens, especially those entering through the respiratory mucosa, such as Mycobacterium tuberculosis. Unfortunately, mucosal vaccination against tuberculosis (TB) is impeded by a lack of suitable adjuvants and/or delivery platforms that could induce a protective immune response in humans. Here, we report on a novel biotechnological approach for mucosal vaccination against TB that overcomes some of the current limitations. This is achieved by coating protective TB antigens onto the surface of inert bacterial spores, which are then delivered to the respiratory tract. Our data showed that mice immunized nasally with coated spores developed humoral and cellular immune responses and multifunctional T cells and, most importantly, presented significantly reduced bacterial loads in their lungs and spleens following pathogenic challenge. We conclude that this new vaccine delivery platform merits further development as a mucosal vaccine for TB and possibly also other respiratory pathogens.

Expression and plasma membrane localization of the mammalian B-cell receptor complex in transgenic Nicotiana tabacum.

The B-cell antigen receptor (BCR), displayed on the plasma membrane of mature B cells of the mammalian immune system, is a multimeric complex consisting of a membrane-bound immunoglobulin (mIg) noncovalently associated with the Igα/Igß heterodimer. In this study, we engineered transgenic tobacco plants expressing all four chains of the BCR. ELISA, Western blotting and confocal microscopy demonstrated that the BCR was correctly assembled in plants, predominantly in the plasma membrane, and that the noncovalent link was detergent sensitive. This is the first example of a noncovalently assembled plasma membrane-retained heterologous receptor in plants. In B cells of the mammalian immune system, following antigen binding to mIg, BCR is internalized and tyrosine residues on Igα and Igß are phosphorylated activating a signaling cascade through interaction with protein kinases that ultimately leads to the initiation of gene expression. Expression of the BCR may therefore be an important tool for the study of plant endocytosis and the identification of previously unknown plant tyrosine kinases. The specificity and diversity of the antibody repertoire, coupled to the signal transduction capability of the Igα/Igß heterodimer, also indicates that plants expressing BCR may in future be developed as environmental biosensors.

Molecular Pharming: future targets and aspirations.

Molecular Pharming represents an unprecedented opportunity to manufacture affordable modern medicines and make these available at a global scale. The area of greatest potential is in the prevention of infectious diseases, particular in underdeveloped countries where access to medicines and vaccines has historically been limited. This is why, at St. George's, we focus on diseases such as HIV, TB and rabies, and aim to develop production strategies that are simple and potentially easy to transfer to developing countries.

Early antiretroviral therapy-treated perinatally HIV-infected seronegative children demonstrate distinct long-term persistence of HIV-specific T-cell and B-cell memory.

OBJECTIVE: To investigate long-term persistence of HIV-specific lymphocyte immunity in perinatally HIV-infected children treated within the first year of life. DESIGN: Twenty perinatally HIV-infected children who received ART therapy within the first year of life (early treated) and with stable viral control (>5 years) were grouped according to their serological response to HIV. METHODS: Western blot analysis and ELISA defined 14 HIV-seropositive and six seronegative patients. Frequencies of gp140-specific T-cell and B-cell, and T-cell cytokine production were quantified by flow cytometry in both seronegatives and seropositives. Transcriptional signatures in purified gp140-specific B-cell subsets, in response to in-vitro stimulation with HIV peptides was evaluated by multiplex RT-PCR. RESULTS: Gp140-specific T cells and B cells persist at similar levels in both groups. A higher production of IL-21 in gp140-specific T cells was found in seropositives vs. seronegatives (P = 0.003). Gene expression in switched IgM-IgD- gp140-specific memory B cells after stimulation with HIV peptides in vitro demonstrated a differential expression of genes involved in signal transduction and activation after BCR/TLR triggering and B-cell activation. Genes relating to antibody production (PRDM1) and T-B cognate stimulation (CXCR4, IL21R) were differentially induced after in-vitro stimulation in seronegatives vs. seropositives suggesting a truncated process of B-cell maturation. CONCLUSION: HIV-specific memory B and T cells persist in early treated regardless their serological status. Seronegatives and seropositives are distinguished by gp140-specific T-cell function and by distinct transcriptional signatures of gp140-specific B cells after in-vitro stimulation, presumably because of a different antigen exposure. Such qualitative insights may inform future immunotherapeutic interventions.

Development of Potent Inhibitors of the Mycobacterium tuberculosis Virulence Factor Zmp1 and Evaluation of Their Effect on Mycobacterial Survival inside Macrophages.

The enzyme Zmp1 is a zinc-containing peptidase that plays a critical role in the pathogenicity of Mycobacterium tuberculosis. Herein we describe the identification of a small set of Zmp1 inhibitors based on a novel 8-hydroxyquinoline-2-hydroxamate scaffold. Among the synthesized compounds, N-(benzyloxy)-8-hydroxyquinoline-2-carboxamide (1 c) was found to be the most potent Zmp1 inhibitor known to date, and its binding mode was analyzed both by kinetics studies and molecular modeling, identifying critical interactions of 1 c with the zinc ion and residues in the active site. The effect of 1 c on intracellular Mycobacterium survival was assayed in J774 murine macrophages infected with M. tuberculosis H37Rv or M. bovis BCG and human monocyte-derived macrophages infected with M. tuberculosis H37Rv. Cytotoxicity and genotoxicity were also assessed. Overall, inhibitor 1 c displays interesting in vitro antitubercular properties worthy of further investigation.

A mycobacterial growth inhibition assay (MGIA) for bovine TB vaccine development.

Human tuberculosis remains a significant cause of mortality and morbidity throughout the world. The global economic impact of bovine TB is considerable. An effective vaccine would be the most cost-effective way to control both epidemics, particularly in emerging economies. TB vaccine research would benefit from the identification of an immune correlate of protection with which vaccines could be gated at both preclinical and clinical levels. In-vitro mycobacterial growth inhibition assays (MGIA) are functional assays that include most aspects of the complex host immune response to mycobacteria, and they may serve as functional immune correlates for vaccine development. We applied to cattle an MGIA that was developed for use with human and murine samples. Several technical difficulties were encountered while transferring it to the cattle model. However, our data demonstrate that the assay was not discriminatory in cattle and further work is needed before using it for bovine TB vaccine development.

TB biomarkers, TB correlates and human challenge models: New tools for improving assessment of new TB vaccines.

The 4th Global Forum on TB Vaccines, convened in Shanghai, China, from 21 - 24 April 2015, brought together a wide and diverse community involved in tuberculosis vaccine research and development to discuss the current status of, and future directions for this critical effort. This paper summarizes the sessions on Biomarkers and Correlates, and Human Challenge Models. Summaries of all sessions from the 4th Global Forum are compiled in a special supplement of Tuberculosis. [August 2016, Vol 99, Supp S1, S1-S30].

Modulation of CD4 and CD8 response to QuantiFERON-TB Plus in patients with active tuberculosis and latent tuberculosis infection followed over time during treatment.

OBJECTIVE/BACKGROUND: Interferon (IFN)-γ release assays (IGRA) are designed for diagnosing tuberculosis (TB) infection. The new IGRA, QuantiFERON-TB Plus (QFT-Plus), is based on the enzyme-linked immunosorbent assay detection of IFN-γ after stimulation with Mycobacterium tuberculosis TB1 and TB2 antigens. TB1 elicits a cellular-mediated immune (CMI) response by CD4 T cells, and TB2 contains peptides recognized by both CD4 and CD8 T cells. The aim of the study is to characterize the CMI to QFT-Plus peptides in active TB and latent TB infection (LTBI) at baseline and during or after specific treatment (follow-up). METHODS: We enrolled 7 individuals with active TB and 11 individuals with LTBI at baseline and followed them during the treatment, either for active diseases or preventive therapy. Peripheral blood mononuclear cells were stimulated with QFT-Plus antigens (TB1, TB2, and mitogen). Cytokine profile (IFN-γ, tumor necrosis factor-α, interleukin-2) and phenotype (CD45RA, CD27) of CD4 and CD8 T cells were characterized by flow cytometry. RESULTS: All the individuals responded to mitogen. CD4 T-cell responses to TB1 and TB2 were similar in both individuals with active TB and those with LTBI evaluated over time. Differently, we found a higher number of TB2-associated CD8 T-cell responders in individuals with active TB than in those with LTBI. For individuals with active TB, there was no change in the specific response overtime. Differently, in individuals with LTBI, the number of CD8 responders to QFT-Plus antigens increased during preventive treatment (TB1=5/11 [45%], TB2=5/11 [45%]) compared with that at the time of enrolment (TB1=1/11 [9%], TB2=1/11 [9%]). Moreover, we analyzed the effector memory profile of T cells responding to QFT-Plus antigens. The largest component of antigen-specific CD4 T cells (65%) had a central memory (CD45RA-CD27+) phenotype at enrolment and during follow-up. In contrast, specific CD8 T cells, which were analyzed only at follow-up because they were almost absent at baseline, were characterized by a large component with naïve (CD45RA+CD27+) phenotype (40%) and a minor component with central memory (25%) features. CONCLUSION: To our knowledge, this is the first report characterizing CD4 and CD8 T-cell responses of individuals with active TB and with LTBI, followed overtime, to QFT-Plus antigens by flow cytometry. The results, although preliminary, may help in identifying better tools for monitoring therapy, especially in those with LTBI undergoing preventive treatment.

Characterization of the CD4 and CD8 T-cell response in the QuantiFERON-TB Gold Plus kit.

OBJECTIVE/BACKGROUND: QuantiFERON-TB Gold In-Tube (QFT-GIT, Qiagen, Hilden, Germany) is an interferon-γ (IFN-γ) release assay designed to detect latent tuberculosis infection (LTBI). Although QFT-GIT has several advantages (mainly that it is not affected by the Bacille Calmette-Guérin vaccination), it has a poor sensitivity in immune-compromised individuals as it involves an immune response-based detection. Recently, QuantiFERON-TB Gold Plus (QFT-Plus) assay has been proposed as a new generation of QFT-GIT. QFT-Plus includes two tubes, TB1 and TB2 with Mycobacterium tuberculosis antigens to elicit a specific immune response. TB1 contains peptides derived from the antigens 6kDa early secretory antigenic target (ESAT-6) and 10kDa culture filtrate protein (CFP-10) (TB-7.7, present in QFT-GIT, has been removed), and it is designed to induce a specific CD4 T-cell response. TB2 contains newly designed peptides stimulating IFN-γ production by both CD4 and CD8 T cells. The additional peptides for eliciting CD8 T-cell responses have been included to increase the sensitivity of the test for LTBI detection. The aim of the study was to evaluate specific CD4 and CD8 T-cell responses to the M. tuberculosis antigens contained within the QFT-Plus test by flow cytometry in individuals with active TB and LTBI. METHODS: We enrolled 23 individuals with active TB and 30 individuals with LTBI. QFT-Plus assay and intracellular staining were performed. One million of peripheral blood mononuclear cells in 1ml of complete medium (RPMI 1640) were dispensed in QFT-Plus tubes. Following 16-24h stimulation, antigen-specific T cells were characterized by flow cytometry evaluating CD4, CD8, CD3 markers, and IFN-γ production. For statistical analysis, nonparametric tests were performed. RESULTS: We found that CD4 T-cell responses were induced by both TB1 and TB2. Differently, the CD8 T-cell response was mainly induced by TB2 and was significantly higher than that induced by TB1 (p=0.01). The frequency of Mtb specific T-cells observed in individuals with active TB was significantly higher than in those with LTBI (p=0.04). Finally, TB2-specific CD8 T-cell responses in individuals with active TB were associated with high radiological severity of lung lesions and microbiological diagnosis (based on M. tuberculosis isolation in sputum culture). CONCLUSION: This is the first characterization of CD4 and CD8 T-cell responses to QFT-Plus TB1 and TB2 tubes in individuals with active TB and LTBI enrolled in a low TB-endemic country such as Italy. We demonstrated that the increased sensitivity is a consequence of the ability of TB2 to induce a CD8 T-cell response which is mainly associated with active TB. This assay has the potential to be very useful in conditions of immune depression due to CD4 T-cell impairments.

First characterization of the CD4 and CD8 T-cell responses to QuantiFERON-TB Plus.

INTRODUCTION: QuantiFERON®-TB Gold Plus (QFT-Plus) is the new generation of QuantiFERON-TB Gold In-Tube test to identify latent tuberculosis infection (LTBI). QFT-Plus includes TB1 and TB2 tubes which contain selected Mycobacterium tuberculosis (Mtb) peptides designed to stimulate both CD4 and CD8 T-cells. Aim of this study is the flow cytometric characterization of the specific CD4 and CD8 T-cell responses to Mtb antigens contained within QFT-Plus. METHODS: We enrolled 27 active tuberculosis (TB) patients and 30 LTBI individuals. Following stimulation with TB1 and TB2, antigen-specific T-cells were characterized by flow cytometry. Data were also correlated with the grade of TB severity. RESULTS: TB1 mainly elicited a CD4 T-cell response while TB2 induced both CD4 and CD8 responses. Moreover, the TB2-specific CD4 response was detected for both active TB and LTBI patients, whereas the TB2-specific CD8 response was primarily associated with active TB (p = 0.01). CONCLUSIONS: To our knowledge, we report the first characterization of the CD4 and CD8 T-cell response to QFT-Plus. CD8 T-cell response is mainly due to TB2 stimulation which is largely associated to active TB. These results provide a better knowledge on the use of this assay.

Immune-complex mimics as a molecular platform for adjuvant-free vaccine delivery.

Protein-based vaccine development faces the difficult challenge of finding robust yet non-toxic adjuvants suitable for humans. Here, using a molecular engineering approach, we have developed a molecular platform for generating self-adjuvanting immunogens that do not depend on exogenous adjuvants for induction of immune responses. These are based on the concept of Immune Complex Mimics (ICM), structures that are formed between an oligomeric antigen and a monoclonal antibody (mAb) to that antigen. In this way, the roles of antigens and antibodies within the structure of immune complexes are reversed, so that a single monoclonal antibody, rather than polyclonal sera or expensive mAb cocktails can be used. We tested this approach in the context of Mycobacterium tuberculosis (MTB) infection by linking the highly immunogenic and potentially protective Ag85B with the oligomeric Acr (alpha crystallin, HspX) antigen. When combined with an anti-Acr monoclonal antibody, the fusion protein formed ICM which bound to C1q component of the complement system and were readily taken up by antigen-presenting cells in vitro. ICM induced a strong Th1/Th2 mixed type antibody response, which was comparable to cholera toxin adjuvanted antigen, but only moderate levels of T cell proliferation and IFN-γ secretion. Unfortunately, the systemic administration of ICM did not confer statistically significant protection against intranasal MTB challenge, although a small BCG-boosting effect was observed. We conclude that ICM are capable of inducing strong humoral responses to incorporated antigens and may be a suitable vaccination approach for pathogens other than MTB, where antibody-based immunity may play a more protective role.

Exploring the vaccine potential of Dec-205 targeting in Mycobacterium tuberculosis infection in mice.

Protein subunit vaccines are an attractive mode of immunisation against infectious diseases but the approach is hampered by the lack of suitable adjuvants for human use. We investigated if antigen targeting to the endocytic cell receptor Dec-205 on dendritic cells (DCs) could induce a protective immune response to Mycobacterium tuberculosis (MTB) infection in the absence of conventional adjuvants. Dec-205 receptor expressed by several subsets of DC has been shown in previous studies to be an efficient endocytic receptor for inducing both humoral and cellular immune responses, but this immunisation approach has not been tested in an experimental model of infection. We therefore prepared chemical conjugates of an anti-mouse Dec-205 monoclonal antibody (mAb) and the highly immunogenic antigen 85B (Ag85B) of MTB and showed that they bound efficiently to bone-marrow derived DC. Moreover, DC stimulated in vitro with Dec-205 conjugates could induce proliferation of splenocytes from Ag85B-immunised mice, while the negative control conjugates failed to do so. Following immunisation of mice with the anti-Dec-205-Ag85B conjugates administered together with a co-stimulatory anti-CD40 mAb, antigen-specific humoral and cellular responses were detected. Although the conjugates induced a strong Ag85B-specific humoral response, T cell proliferation and interferon-γ production were observed only when the conjugates were used to boost BCG vaccine. Importantly though, the conjugate vaccine did not offer significant protection against MTB challenge when used on its own or as a boost to BCG. Therefore, we conclude that Ag85B-based vaccine targeting to Dec-205 alone is not a sufficiently robust vaccination strategy for tuberculosis, although this approach might be more successful with other antigens or infections.

Generation of self-renewing immature dendritic cells from mouse spleen that can take up mycobacteria and present antigens to T cells.

Dendritic cells (DC) play a key role in driving the adaptive immune response to Mycobacterium tuberculosis (MTB), the causative pathogen of tuberculosis (TB). However, studying these important yet very sparse immune cells in the context of MTB pathogenesis is severely restricted by the lack of suitable cell lines and the complexity of culturing of DC progenitors, usually obtained from the bone marrow. However, significant advances have been made towards generating long-term DC cultures from various lymphoid tissues. Here, we report the evidence for generating a long-term, self-renewing DC culture from the Balb/c mouse spleen. We demonstrate that these cells, termed IDC-3, have a myeloid DC origin, i.e. they are CD11c(+) CD11b(++) CD8-α(-) F4/80(+/-) and that they also display a phenotype MHC-II(+) CD16/32(++) CD80(+/-) CD86(+) , indicating that they are immature DC. Following incubation with Mycobacterium bovis BCG (Bacillus Calmette Guerin), the IDC-3 efficiently took up bacteria and acquired the morphology of mature DC. Importantly though, when IDC-3 were pre-stimulated with a mycobacterial antigen in vitro, they were able to induce proliferation of T lymphocytes from mice immunized with the same antigen. The T-cell stimulatory potential of IDC-3 was further enhanced when the cells were co-stimulated with an anti-CD40 mAb. We therefore suggest that the IDC-3 culture system could be a useful tool for studying the interaction of DC with mycobacteria.