The second Geneva Consensus: Recommendations for novel live TB vaccines.

Infection with Mycobacterium tuberculosis continues to be a major public health burden in most developing parts of the world and efforts to develop effective strategies for containing the disease remain a priority. It has long been evident that effective mass vaccination programmes are a cost effective and efficient approach to controlling communicable diseases in a public health setting and tuberculosis (TB) continues to be a major target. One approach with increasing acceptance is based upon on live mycobacterial vaccines, either as recombinant BCG or rationally attenuated M. tuberculosis, thus generating a new live TB vaccine. The Geneva Consensus published in March 2005 set out the opinion on priorities and requirements for developing live mycobacterial vaccines for Phase I trials. In the intervening period much progress has been made in both preclinical and clinical development of new TB vaccines and has provided the impetus for organising the second Geneva Consensus (held at WHO headquarters, April 2009) to discuss issues, including: i. Explore the regulatory requirements for live TB vaccines to enter Phase I trials, in particular those based on attenuated M. tuberculosis. Particular attention was paid to the characterisation and safety package likely to be required, including issues of attenuation, the presence of antibiotic resistance markers in live vaccines and the nature of any attenuated vaccine phenotype. ii. To identify the general criteria for further clinical development from Phase I through to Phase III. iii. Obtain a perspective of the regulatory landscape of developing countries where Phase II and III trials are to be held. iv. Review manufacturing considerations for live TB vaccines and relevance of the WHO and European Pharmacopeia guidelines and requirements for BCG vaccine. v. Consider requirements and associated issues related to the use of these new vaccines within an existing BCG vaccination programme.

Therapeutic vaccine comprising Mycobacterium HSP70.

BACKGROUND: Mycobacterium avium ssp. paratuberculosis is the causative agent of Johen's disease. This infection of the small intestine is a global problem in the livestock industry. Bacterial shedding by infected but subclinical animals, and transmission via the fecal or intrauterine route and through colostrum and/or milk, make containment and eradication of this disease highly problematic. Current vaccine strategies are ineffective and no effective therapy is available. OBJECTIVE: Within the broader scope of therapeutic uses of recombinant heat-shock protein 70 (HSP70), the present article evaluates the claim of patent WO08040691. CONCLUSION: The patent under comment here covers the ability of recombinant Mycobacterium avium ssp. paratuberculsis (MAP) HSP70, when administered in conjunction with an adjuvant, to result in a significant reduction in bacterial shedding in cattle infected with MAP. Furthermore, its administration does not mask diagnostic assays, allowing clinical diagnosis to be maintained.

Treatment of mice with IL-12 DNA constructs leads to augmented NK activity in lungs but low IFN-gamma release -- implications for Bordetella pertussis infections following aerosol challenge.

Interleukin-12 protein has been widely used experimentally in therapeutic and adjuvant settings in the treatment of different diseases including intra-cellular bacterial infections. The in vivo clearance of Bordetella pertussis infections in naive mice and in animals vaccinated with whole cell vaccine is considered to be a Th-1 dependent mechanism. Furthermore, the addition of IL-12 protein to an acellular pertussis vaccine increases the efficacy of this vaccine. Whilst the use of IL-12 protein is often beneficial, a number of problems there are associated with this cytokine including toxicities and down regulation of normal immune functions. The use of DNA constructs encoding this cytokine may be a way of achieving maximum therapeutic benefit with minimum toxicity. The aims of this study were to optimise the effects of two IL-12 DNA constructs, especially with respect to augmenting pulmonary immune responsiveness and to compare the effect of IL-12 DNA and IL-12 protein on bacterial colonisation of lungs following aerosol challenge with B. pertussis. We found that IL-12 DNA constructs augmented the activity of pulmonary NK cells but had little effect on the course of B. pertussis infections in mice. In contrast to IL-12 protein, the DNA constructs had no immunosuppressive effects on splenic lymphocyte mitogen responses.

Effect of gender on lower extremity kinematics during rapid direction changes: an integrated analysis of three sports movements.

Anterior cruciate ligament (ACL) injury is a common sports injury, particularly in females. Gender differences in knee kinematics have been observed for specific movements, but there is limited information on how these findings relate to other joints and other movements. Here we present an integrated analysis of hip, knee and ankle kinematics across three movements linked to non-contact ACL injury. It was hypothesised that there are gender differences in lower extremity kinematics, which are consistent across sports movements. Ten female and ten male NCAA basketball players had three-dimensional hip, knee and ankle kinematics quantified during the stance phase of sidestep, sidejump and shuttle-run tasks. For each joint angle, initial value at contact, peak value and between-trial variability was obtained and submitted to a two-way mixed design ANOVA (gender and movement), with movement condition treated as a repeated measure. Females had higher peak knee valgus and lower peak hip and knee flexion, with the same gender differences also existing at the beginning of stance (p<0.05). Peak valgus measures were highly correlated between movements, but not to static valgus alignment. Kinematic differences demonstrated by females for the sports movements studied, and in particular knee valgus, may explain their increased risk of ACL injury. These differences appear to stem largely from subject-specific neuromuscular mechanisms across movements, suggesting that prevention via neuromuscular training is possible.

BCG (Bacille Calmette-Guérin) HspCs (heat-shock protein-peptide complexes) induce T-helper 1 responses and protect against live challenge in a murine aerosol challenge model of pulmonary tuberculosis.

The need for an effective TB (tuberculosis) vaccine remains acute, with tuberculosis still one of the major killers worldwide and 3 million new infections annually. We report here on the immune responses elicited by HspCs (heat-shock protein-peptide complexes) isolated from BCG (Bacille Calmette-Guérin) vaccine. These HspCs elicit both the appropriate cellular and protective immune responses required to merit their further development as TB vaccine candidates.

High dose interleukin-12 exacerbates Bordetella pertussis infection and is associated with suppression of cell-mediated immunity in a murine aerosol challenge model.

The in-vivo clearance of Bordetella pertussis infections in murine models in naive mice and animals vaccinated with whole-cell vaccine is considered to be via a Th-1-dependent mechanism in which interleukin-12 (IL)-12 may play a prominent role. It has also been demonstrated clearly that the treatment of animals with macrophage-derived IL-12 administered with an acellular vaccine can increase the efficacy of this vaccine preparation to levels seen with the whole-cell vaccine. However, the effects of exogenously added IL-12 on immune responses in non-vaccinated B. pertussis-challenged mice remain unclear, with two studies giving contradictory findings. In this study we have treated mice with escalating doses of mIL-12 (0.1-10 microg/mouse) prior to challenge with B. pertussis (using an aerosol challenge model of infection). The ability of mice to clear infection was assessed in IL-12 treated and in phosphate buffered saline (PBS) control animals at days 6 and 13 post-challenge. Lymphoid cells were isolated from spleen and cell-mediated immune responses assessed at days 1, 6 and 13 post-challenge. In addition, the direct effects of high-dose IL-12 on challenged mice was assessed by checking natural killer (NK) activity from isolated lung and spleen lymphoid cells as well as interferon-gamma (IFN-gamma) generation from isolated cells and serum at day 1 post-challenge. The results from this study show that bacterial colonization of the lungs is actually enhanced following treatment with high-dose IL-12. This is associated with impaired cellular immune responses. The mechanisms associated with the immunosuppressive effects of IL-12 are discussed.

Detection and analysis of cytokine mRNA in tissues and cell lines.

Many normal and pathological processes have been shown to be influenced by the quantitative and qualitative nature of cytokine production. Cytokines have also been shown to be central in modulating host responses to invasion by pathogens. Therefore detection and analysis of cytokine production are critical in the understanding of host responses to a variety of immunological insults. The detection of cytokines by bioassay or immunoassay provides data from in vitro experiments and can be used to determine the circulating plasma levels of particular cytokines. However, many investigations require the detection of cytokines in small numbers of cells or directly from tissue samples. It is in this particular area that detection and analysis of cytokine gene expression by use of the sensitive and specific reverse transcription-polymerase chain reaction (RT-PCR) has application. The purpose of these protocols is to provide a practical guide to the detection of cytokine gene expression using RT-PCR techniques. Procedures for analysing mRNA expression in cell culture, cell suspensions and tissue samples will be described and pitfalls and caveats concerning the techniques will be discussed. These protocols should provide a starting point for the development of RT-PCR technology in laboratories with only limited molecular experience.

Modulation of the immune response to tetanus toxoid by polylactide-polyglycolide microspheres.

The cellular and antibody responses in mice to tetanus toxoid (TT), alone or adsorbed to aluminium hydroxide TT were compared to those obtained with TT incorporated in polylactide-polyglycolide (PLGA) microspheres, a candidate single dose vaccine. After subcutaneous injection, the serum IgM antibody to TT followed similar overall patterns for all preparations although the highest levels were achieved with the alum-adsorbed material. A similar pattern was observed with the overall IgG antibody responses. The isotype distribution of antibodies was broadly similar for all types of preparation although considerable qualitative differences were observed. IgG1 antibodies predominated although IgG2a and IgG2b made a substantial contribution, especially at three months in the case of the two adjuvanted preparations. The IgG3 responses to each type of vaccine were very low. Both alum and microsphere vaccines induced proliferative responses to TT in splenic lymphocytes at three months after vaccination. Each type of vaccine was much less effective in inducing proliferative responses in lymph node cells. There was evidence of induction of IL2, IL4 and interferon-gamma genes by microsphere vaccines in splenic but not lymph node cells. There were indications that PLGA microspheres alone exerted a modulating effect on cellular responses after immunization. These results suggest that TT encapsulated in microspheres induces a pattern of cellular and antibody responses qualitatively similar to those induced by conventional TT vaccines.

Monocyte antimycobacterial activity before and after Mycobacterium bovis BCG vaccination in Chingleput, India, and London, United Kingdom.

Monocytes from purified protein derivative S Mantoux-negative children and young adults inhibited intracellular growth of Mycobacterium microti more in Chingleput than in London. Mycobacterium bovis BCG vaccination did not enhance bacteriostasis with the Indians but did so with the Londoners. No evidence was found for involvement of cytokines such as macrophage-activating factor and granulocyte macrophage colony-stimulating factor in the differences.

The pattern of mycobacterial antigen recognition in sera from Mantoux-negative individuals is essentially unaffected by bacille Calmette-Guérin (BCG) vaccination in either south India or London.

Paired sera were obtained before and 8 weeks after routine BCG vaccination from 20 PPD-S Mantoux-negative individuals who were living adjacent to the Chingleput BCG vaccine trial area in Tamil Nadu and from seven Mantoux-negative school-children in London, UK. Most subjects became Mantoux-positive after vaccination. In ELISA tests against soluble extracts of BCG or Mycobacterium tuberculosis H37Rv or against PPD-S, pre-vaccination antibody titres of South Indian subjects were about twice those of British subjects but there was no increase in titre of antibodies after vaccination of either population. Western blotting showed that even before vaccination, and even in British subjects, antibodies were present that recognized numerous antigenic components in extracts of BCG and M. tuberculosis. There was no consistent difference between band patterns with South Indian and British subjects and any effect of vaccination on the patterns was minimal.

Role of Th-1 lymphocytes in the development of protective immunity against Mycobacterium leprae. Analysis of lymphocyte function by polymerase chain reaction detection of cytokine messenger RNA.

The patterns of lymphokine mRNA expression during the development of protective immunity to Mycobacterium leprae after intradermal vaccination of mice with killed M. leprae were studied. Using a polymerase chain reaction-based technique for detecting mRNA expression in small numbers of cells, we observed changes in the mRNA expression of a number of cytokine genes in the lymph nodes draining the site of vaccination. In particular, IL-1 (-alpha and -beta), IL-2, TNF (-alpha and -beta), and IFN-gamma mRNA were readily detected, whereas IL-3, IL-4, IL-5, IL-6, IL-7, and granulocyte-macrophage colony-stimulating factor mRNA were not detected, or were detectable only at very low levels. This is consistent with the selective activation of Th-1 Th cells. The effect of in vitro exposure of these cells to the immunizing Ag was also investigated; again, IL-1, IL-2, TNF, and IFN-gamma mRNA were abundant, but in addition, IL-3, IL-6, and granulocyte-macrophage colony-stimulating factor mRNA were greatly increased, suggesting an important role in the recall response.

Demonstration of increased anti-mycobacterial activity in peripheral blood monocytes after BCG vaccination in British school children.

A blood sample was taken from children aged 13-15 years immediately before BCG vaccination and 8 weeks after. The children were tuberculin skin-test negative to PPD-S before vaccination and positive after. Mononuclear cells were separated from the blood, infected with Mycobacterium microti at a low bacterium/monocyte ratio and allowed to form monolayers in microtitre wells. The infected monolayers were rinsed daily and the change in number of live bacteria in monolayers and supernatants was monitored by colony counts on agar. The cells were bacteriostatic during the first day, thereafter growth accelerated in pre-vaccination monolayers. When monolayers received pulsed exposures to autologous lymphocytes that had been incubated with whole dead tubercle bacilli the growth rates of M. microti were increased. However, growth rates in lymphocyte-pulsed monolayers were significantly lower after vaccination than before. It is proposed that this difference reflects the protective effect of vaccination.

Distinctive patterns of release of neuroendocrine peptides after nasal challenge of allergic subjects with ryegrass antigen.

The concentrations of the neuropeptides substance P, somatostatin, and calcitonin gene-related peptide in human nasal secretions were quantified by radioimmunoassays, concurrently with that of histamine, in the course of nasal challenge of allergic and control subjects with ryegrass antigen to examine contributions of neuromediation of the tissue response. Each of the neuropeptides and histamine were detected in nasal lavage fluid prior to challenge. In allergic patients, but not normal controls, antigen evoked significant increases of 3-fold in histamine at 15-60 min, 1.5- to 4-fold in calcitonin gene-related peptide at 15 min-24 hr, and more than 2-fold in somatostatin at 6 hr, without altering the concentration of substance P in nasal lavage fluid. The identity of the neuropeptides was confirmed chromatographically. Thus calcitonin gene-related peptide may mediate nasal congestion directly and somatostatin may be one of the factors regulating the late involvement of basophils and mast cells in allergic rhinitis.

Interleukin 2 synthesis in the presence of steroids: a model of steroid resistance.

In this study interleukin 2 (IL-2) synthesis by human lymphocytes in the presence and absence of prednisolone in a group of normal subjects has been assessed. An association between suppression in vitro of induced phytohaemagglutinin-blastogenesis by prednisolone and synthesis of IL-2 was found. Those subjects whose lymphocytes are identified as steroid-resistant have significantly higher IL-2 activity in the supernatants of both steroid and non-steroid treated lymphocyte cultures than steroid sensitive subjects. The addition of exogenous IL-2 was found to ablate the suppressive effects of steroids on lymphocyte blastogenesis. These results suggest that significantly greater activity of IL-2 in the culture supernatants of steroid resistant subjects may represent a mechanism for glucocorticoid resistance in vitro and help explain the relationship between increased loss of grafts and steroid resistance in renal allograft recipients.