Ilaria Pepponi

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.

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.

Mucosal Vaccination against Tuberculosis Using Inert Bioparticles

ABSTRACT 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.

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.

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

BackgroundIn 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.MethodsWe 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.ResultsUsing 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.ConclusionsWe 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.

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

Pal R, Marwaha S, Pepponi I, Mann JFS, Paul MJ, Reljic R. Generation of self‐renewing immature dendritic cells from mouse spleen that can take up mycobacteria and present antigens to T cells. APMIS 2010; 118: 729–38.

Ex vivo mycobacterial growth inhibition assay (MGIA) for tuberculosis vaccine testing - a protocol for mouse splenocytes

The testing of vaccines for tuberculosis is costly and time-consuming, and dependent on preclinical animal challenge models and clinical trials. We have recently developed a mycobacterial growth inhibition assay (MGIA) to test vaccine efficacy ex vivo. This assay measures the summative effect of the host immune response and may serve as a novel tool to facilitate vaccine testing. It has generated much interest recently, and to facilitate technology transfer and reproducibility between laboratories, we here describe a detailed protocol for an ex vivo MGIA in mouse splenocytes.

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.

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/Igβ 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 Igβ 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/Igβ heterodimer, also indicates that plants expressing BCR may in future be developed as environmental biosensors.