Philip J. Hogarth

Screening of highly expressed mycobacterial genes identifies Rv3615c as a useful differential diagnostic antigen for the Mycobacterium tuberculosis complex

ABSTRACT Tuberculous infections caused by mycobacteria, especially tuberculosis of humans and cattle, are important both clinically and economically. Human populations can be vaccinated with Mycobacterium bovis bacille Calmette-Guérin (BCG), and control measures for cattle involving vaccination are now being actively considered. However, diagnostic tests based on tuberculin cannot distinguish between genuine infection and vaccination with BCG. Therefore, identification of differential diagnostic antigens capable of making this distinction is required, and until now sequence-based approaches have been predominant. Here we explored the link between antigenicity and mRNA expression level, as well as the possibility that we may be able to detect differential antigens by analyzing quantified global transcriptional profiles. We generated a list of 14 candidate antigens that are highly expressed in Mycobacterium tuberculosis and M. bovis under a variety of growth conditions. These candidates were screened in M. bovis-infected and naïve cattle for the ability to stimulate a gamma interferon (IFN-γ) response. We identified one antigen, Rv3615c, which stimulated IFN-γ responses in a significant proportion of M. bovis-infected cattle (11 of 30 cattle [37%] [P < 0.01]) but not in naïve or BCG-vaccinated animals. Importantly, the same antigen stimulated IFN-γ responses in a significant proportion of infected cattle that did not respond to the well-characterized mycobacterial antigens ESAT-6 and CFP-10. Therefore, use of the Rv3615c epitope in combination with previously described differential tests based on ESAT-6 and CFP-10 has the potential to significantly increase diagnostic sensitivity without reducing specificity in BCG-vaccinated populations.

Systemic BCG Immunization Induces Persistent Lung Mucosal Multifunctional CD4 TEM Cells which Expand Following Virulent Mycobacterial Challenge

To more closely understand the mechanisms of how BCG vaccination confers immunity would help to rationally design improved tuberculosis vaccines that are urgently required. Given the established central role of CD4 T cells in BCG induced immunity, we sought to characterise the generation of memory CD4 T cell responses to BCG vaccination and M. bovis infection in a murine challenge model. We demonstrate that a single systemic BCG vaccination induces distinct systemic and mucosal populations of T effector memory (TEM) cells in vaccinated mice. These CD4+CD44hiCD62LloCD27− T cells concomitantly produce IFN-γ and TNF-α, or IFN-γ, IL-2 and TNF-α and have a higher cytokine median fluorescence intensity MFI or ‘quality of response’ than single cytokine producing cells. These cells are maintained for long periods (>16 months) in BCG protected mice, maintaining a vaccine–specific functionality. Following virulent mycobacterial challenge, these cells underwent significant expansion in the lungs and are, therefore, strongly associated with protection against M. bovis challenge. Our data demonstrate that a persistent mucosal population of TEM cells can be induced by parenteral immunization, a feature only previously associated with mucosal immunization routes; and that these multifunctional TEM cells are strongly associated with protection. We propose that these cells mediate protective immunity, and that vaccines designed to increase the number of relevant antigen-specific TEM in the lung may represent a new generation of TB vaccines.

Evaluation of adjuvants for protein vaccines against tuberculosis in guinea pigs

Subunit vaccines against tuberculosis show promise but require administration with adjuvants to stimulate relevant immune responses for protection. Guinea pigs are the model of choice for evaluating protective immunity to aerogenic challenge with virulent mycobacteria, but few studies have been undertaken to identify suitable adjuvants for vaccine screening in this species. Here, we compare the efficacy of several adjuvants to induce T cell responses to culture filtrate protein in guinea pigs. We report that of several adjuvants tested, the most promising was CpG ODN formulated in an aqueous emulsion. This adjuvant induced type 1 T cell responses equivalent to that of FIA, as measured by delayed-type hypersensitivity reactions (DTH), antigen-specific T cell proliferation and antigen-specific IgG1 and IgG2 responses. These data demonstrate the potential for CpG motif based adjuvants for use in TB vaccine screening in guinea pigs, and other diseases where a type 1 T cell response is required.

Transcriptional Profiling of Disease-Induced Host Responses in Bovine Tuberculosis and the Identification of Potential Diagnostic Biomarkers

Bovine tuberculosis (bTb) remains a major and economically important disease of livestock. Improved ante-mortem diagnostic tools would help to underpin novel control strategies. The definition of biomarkers correlating with disease progression could have impact on the rational design of novel diagnostic approaches for bTb. We have used a murine bTb model to identify promising candidates in the host transcriptome post-infection. RNA from in vitro-stimulated splenocytes and lung cells from BALB/c mice infected aerogenically with Mycobacterium bovis were probed with high-density microarrays to identify possible biomarkers of disease. In antigen-stimulated splenocytes we found statistically significant differential regulation of 1109 genes early (3 days) after infection and 1134 at a later time-point post-infection (14 days). 618 of these genes were modulated at both time points. In lung cells, 282 genes were significantly modulated post-infection. Amongst the most strongly up-regulated genes were: granzyme A, granzyme B, cxcl9, interleukin-22, and ccr6. The expression of 14 out of the most up-regulated genes identified in the murine studies was evaluated using in vitro with antigen-stimulated PBMC from uninfected and naturally infected cattle. We show that the expression of cxcl9, cxcl10, granzyme A and interleukin-22 was significantly increased in PBMC from infected cattle compared to naïve animals following PPD stimulation in vitro. Thus, murine transcriptome analysis can be used to predict immunological responses in cattle allowing the prioritisation of CXCLl9, CXCL10, Granzyme A and IL-22 as potential additional readout systems for the ante-mortem diagnosis of bovine tuberculosis.

Development of an Antibody to Bovine IL-2 Reveals Multifunctional CD4 TEM Cells in Cattle Naturally Infected with Bovine Tuberculosis

Gaining a better understanding of the T cell mechanisms underlying natural immunity to bovine tuberculosis would help to identify immune correlates of disease progression and facilitate the rational design of improved vaccine and diagnostic strategies. CD4 T cells play an established central role in immunity to TB, and recent interest has focussed on the potential role of multifunctional CD4 T cells expressing IFN-γ, IL-2 and TNF-α. Until now, it has not been possible to assess the contribution of these multifunctional CD4 T cells in cattle due to the lack of reagents to detect bovine IL-2 (bIL-2). Using recombinant phage display technology, we have identified an antibody that recognises biologically active bIL-2. Using this antibody, we have developed a polychromatic flow cytometric staining panel that has allowed the investigation of multifunctional CD4 T-cells responses in cattle naturally infected with M. bovis. Assessment of the frequency of antigen specific CD4 T cell subsets reveals a dominant IFN-γ+IL-2+TNF-α+ and IFN-γ+ TNF-α+ response in naturally infected cattle. These multifunctional CD4 T cells express a CD44hiCD45RO+CD62Llo T-effector memory (TEM) phenotype and display higher cytokine median fluorescence intensities than single cytokine producers, consistent with an enhanced ‘quality of response’ as reported for multifunctional cells in human and murine systems. Through our development of these novel immunological bovine tools, we provide the first description of multifunctional TEM cells in cattle. Application of these tools will improve our understanding of protective immunity in bovine TB and allow more direct comparisons of the complex T cell mediated immune responses between murine models, human clinical studies and bovine TB models in the future.

Mycobacterium bovis-BCG vaccination induces specific pulmonary transcriptome biosignatures in mice.

Background In the present study, we applied microarray technology to define biosignatures by microarray transcriptome analysis in lung and spleen samples after BCG vaccination and M. bovis infection of BALB/c mice. The aims were two-fold, namely to define biosignatures that could predict vaccine success before challenge, and biomarker patterns that correlated with anamnestic protective responses following exposure to virulent M. bovis. Further, these biosignatures should be detectable without in vitro antigenic challenge. Principal Findings After BCG vaccination, we defined a specific pulmonary gene expression signature related to the connective tissue development and function network that predicted vaccine success before M. bovis challenge. In addition, a Th17-related cytokine profile was found that correlated with vaccine-induced protective immunity following infection with virulent M. bovis in the lung as well as additional genes that were up-regulated in the spleens of vaccinated animals post-infection related to neutrophil biology and inflammation. Conclusions This study has therefore prioritized both biomarkers predicting vaccination success before challenge and bio-signatures that are potentially associated with protective immune responses that will be useful to evaluate future vaccine candidates.

Development of a Mycobacterium bovis intranasal challenge model in mice.

Tuberculosis caused by infection with Mycobacterium tuberculosis or Mycobacterium bovis remains one of the most important infectious diseases of man and animals. The current vaccine, M. bovis bacille Calmette-Guérin (BCG) demonstrates variable efficacy in humans and cattle, and so an urgent need exists for a new vaccine to replace or supplement BCG. Novel vaccine development requires the availability of a suitable animal model in which to test potential vaccine candidates. Models for tuberculosis vaccine development include mice, guinea pigs, cattle and non-human primates. Murine models provide an economical and easily manipulated tool, but the natural aerosol infection route requires extensive facilities, equipment and validation. We sought to develop a logistically simpler intranasal M. bovis infection model for use in vaccine development for bovine tuberculosis. Intranasal M. bovis infection model in mice demonstrated distinct airway associated, dose related pathology, and was strikingly more virulent than previously employed intravenous infection with M. bovis. BCG vaccination of intranasal challenged mice induced 2 logs of protection with similar kinetics as those displayed in M. tuberculosis aerosol infection models. In conclusion, we report the development of a virulent, robust, stringent, physiological and inexpensive M. bovis intranasal infection model for the screening of potential vaccine candidates against bovine tuberculosis.

Adjuvants Induce Distinct Immunological Phenotypes in a Bovine Tuberculosis Vaccine Model

ABSTRACT Tuberculosis (TB) remains one of the most important infectious diseases of humans and animals. Mycobacterium bovis BCG, the only currently available TB vaccine, demonstrates variable levels of efficacy; therefore, a replacement or supplement to BCG is required. Protein subunit vaccines have shown promise but require the use of adjuvants to enhance their immunogenicity. Using the protective mycobacterial antigen Rv3019c, we have evaluated the induction of relevant immune responses by adjuvant formulations directly in the target species for bovine TB vaccines and compared these to responses induced by BCG. We demonstrate that two classes of adjuvant induce distinct immune phenotypes in cattle, a fact not previously reported for mice. A water/oil emulsion induced both an effector cell and a central memory response. A cationic-liposome adjuvant induced a central memory response alone, similar to that induced by BCG. This suggests that water/oil emulsions may be the most promising formulations. These results demonstrate the importance of testing adjuvant formulations directly in the target species and the necessity of measuring different types of immune response when evaluating immune responses.

International Society for the Advancement of Cytometry Cell Sorter Biosafety Standards

Flow cytometric cell sorting of biological specimens has become prevalent in basic and clinical research laboratories. These specimens may contain known or unknown infectious agents, necessitating precautions to protect instrument operators and the environment from biohazards arising from the use of sorters. To this end the International Society of Analytical Cytology (ISAC) was proactive in establishing biosafety guidelines in 1997 (Schmid et al., Cytometry 1997;28:99–117) and subsequently published revised biosafety standards for cell sorting of unfixed samples in 2007 (Schmid et al., Cytometry Part A J Int Soc Anal Cytol 2007;71A:414–437). Since their publication, these documents have become recognized worldwide as the standard of practice and safety precautions for laboratories performing cell sorting experiments. However, the field of cytometry has progressed since 2007, and the document requires an update. The new Standards provides guidance: (1) for laboratory design for cell sorter laboratories; (2) for the creation of laboratory or instrument specific Standard Operating Procedures (SOP); and (3) on procedures for the safe operation of cell sorters, including personal protective equipment (PPE) and validation of aerosol containment. Published

The Duration of Antigen-Stimulation Significantly Alters the Diversity of Multifunctional CD4 T Cells Measured by Intracellular Cytokine Staining

The assessment of antigen-specific T cell responses by intracellular cytokine staining (ICS) has become a routine technique in studies of vaccination and immunity. Here, we highlight how the duration of in vitro antigen pre-stimulation, combined with the cytokine accumulation period, are critical parameters of these methods. The effect of varying these parameters upon the diversity and frequency of multifunctional CD4 T cell subsets has been investigated using a murine model of TB vaccination and in cattle naturally infected with Mycobacterium bovis. We demonstrate a substantial influence of the duration of the antigen pre-stimulation period on the repertoire of the antigen-specific CD4 T cell responses. Increasing pre-stimulation from 2 to 6 hours amplified the diversity of the seven potential multifunctional CD4 T cell subsets that secreted any combination of IFN-γ, IL-2 and TNF-α. However, increasing pre-stimulation from 6 to 16 hours markedly altered the multifunctional CD4 T cell repertoire to a dominant IFN-γ+ only response. This was observed in both murine and cattle models. Whilst these data are of particular relevance to the measurement of vaccine and infection induced immunity in TB, more generally, they demonstrate the importance of the empirical determination of the optimum duration of the individual culture steps of ICS assays for any model. We highlight the potential significance of variations in these parameters, particularly when comparing data between studies and/or models including clinical trials.