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

Resistance to First-Line Anti-TB Drugs Is Associated with Reduced Nitric Oxide Susceptibility in Mycobacterium tuberculosis

Background and objective: The relative contribution of nitric oxide (NO) to the killing of Mycobacterium tuberculosis in human tuberculosis (TB) is controversial, although this has been firmly established in rodents. Studies have demonstrated that clinical strains of M. tuberculosis differ in susceptibility to NO, but how this correlates to drug resistance and clinical outcome is not known. Methods: In this study, 50 sputum smear- and culture-positive patients with pulmonary TB in Gondar, Ethiopia were included. Clinical parameters were recorded and drug susceptibility profile and spoligotyping patterns were investigated. NO susceptibility was studied by exposing the strains to the NO donor DETA/NO. Results: Clinical isolates of M. tuberculosis showed a dose- and time-dependent response when exposed to NO. The most frequent spoligotypes found were CAS1-Delhi and T3_ETH in a total of nine known spoligotypes and four orphan patterns. There was a significant association between reduced susceptibility to NO (>10% survival after exposure to 1mM DETA/NO) and resistance against first-line anti-TB drugs, in particular isoniazid (INH). Patients infected with strains of M. tuberculosis with reduced susceptibility to NO showed no difference in cure rate or other clinical parameters, but a tendency towards lower rate of weight gain after two months of treatment. Conclusion: There is a correlation between resistance to first-line anti-TB drugs and reduced NO susceptibility in clinical strains of M. tuberculosis. Further studies including the mechanisms of reduced NO susceptibility are warranted and could identify targets for new therapeutic interventions

Nitrotyrosine formation after activation of murine macrophages with mycobacteria and mycobacterial lipoarabinomannan

Murine peritoneal macrophages, elicited with thioglycollate, were stimulated in vitro with lipopolysaccharide (LPS). The production of nitrite, superoxide anion (SOA), and the accumulation of nitrotyrosine in the cells increased after treatment, and all were inhibitable by the NO synthase inhibitor NG-monomethyl-l-arginine monoacetate (l-NMMA). This effect suggests a direct correlation between the accumulation of those metabolites and NO synthase activity. Lipoarabinomannan (LAM) purified from Mycobacterium tuberculosis was added to peritoneal macrophages in the presence of interferon-gamma (IFN-γ); the cells produced nitrite and SOA, both inhibitable by l-NMMA. There was, as well, accumulation of nitrotyrosine in the macrophage proteins. Strikingly, the amount of nitrotyrosine measured after LAM plus IFN-γ, or LAM plus the low molecular weight adjuvant glutamylmuramyl dipeptide (GMDP), increased significantly in the presence of l-NMMA. These results suggest that murine macrophages, upon LAM stimulation, might generate reactive nitrogen metabolites by a route other than NO synthase. Nitrotyrosine accumulation after infection of macrophages in vitro, with either live bacille Calmette–Guérin (BCG) or live M. tuberculosis, in the presence or absence of IFN-γ, showed no correlation with nitrite production, suggesting a low superoxide production

Influence of serum on the immune recognition of a synthetic lipopeptide mimetic of the 19-kDa lipoprotein from Mycobacterium tuberculosis

The innate immune response provides a critical first-line defense against Mycobacterium tuberculosis, an intracellular pathogen that represents a major health threat world-wide. A synthetic lipopeptide (LP) mimicking the lipid moiety of the cell-wall associated 19-kDa lipoprotein from M. tuberculosis has recently been assigned an important role in the induction of an antibacterial immune response in host macrophages. Here, we present experimental data on the biological activities and the biophysical mechanisms underlying cell activation by synthetic 19-kDa M. tuberculosis-derived lipopeptide (Mtb-LP). Investigation of the geometry of the LP (i.e. the molecular conformation and supramolecular aggregate structure) and the preference for membrane intercalation provide an explanation for the biological activities of the mycobacterial LP. Cell activation by low concentrations of Mtb-LP was enhanced by the lipopolysaccharide-binding protein and CD14. However, surprisingly, we found that activation of human macrophages to induce pro- as well as antiinflammatory mediators (tumor necrosis factor(TNF)-alpha, Interleukin(IL)-6, IL-8, and IL-10) in response to the Mtb-LP is strongly reduced in the presence of serum. This observation could be confirmed for the immune response of murine macrophages which showed a strongly enhanced TNF-alpha release in the absence of serum, suggesting that the molecular mechanisms of immune recognition of the Mtb-LP are tailored to the ambient conditions of the lung

Pesquisa de IgA contra o antígeno recombinante HspX de Mycobacterium tuberculosis no diagnóstico de tuberculose pleural Determination of levels of specific IgA to the HspX recombinant antigen of Mycobacterium tuberculosis for the diagnosis of pleural tuberculosis

OBJETIVO: Avaliar a acurácia da dosagem de IgA contra o antígeno recombinante HspX no líquido pleural e no soro de pacientes com derrame pleural para o diagnóstico de tuberculose pleural. MÉTODOS: Estudo transversal de teste diagnóstico. Amostras de líquido pleural e de soro de pacientes com derrame pleural e suspeita de tuberculose pleural foram avaliadas para a determinação da densidade óptica de IgA contra HspX utilizando ELISA indireto. RESULTADOS: Foram avaliadas amostras de líquido pleural e de soro de 132 pacientes: 97 com tuberculose pleural (grupo de estudo) e 35 com derrame pleural por outras causas (grupo controle). A dosagem de IgA em líquido pleural foi capaz de discriminar os pacientes com tuberculose pleural dos controles. A sensibilidade do teste em líquido pleural e em soro foi, respectivamente, de 69% e 30%, enquanto a especificidade foi de 83% e 84%, respectivamente. CONCLUSÕES: Os dados sugerem o potencial da utilização deste teste no diagnóstico de tuberculose pleural. Estudos com amostras maiores e em diferentes cenários epidemiológicos são necessários<br>OBJECTIVE: To evaluate the accuracy of determining specific IgA to HspX recombinant antigen in pleural fluid and serum samples for the diagnosis of pleural tuberculosis in patients with pleural effusion. METHODS: This was a cross-sectional study. Serum and pleural fluid samples of patients with pleural effusion and suspected of having pleural tuberculosis were tested with indirect ELISA in order to determine the optical density of specific IgA to HspX. RESULTS: We evaluated serum and pleural fluid samples from 132 patients: 97 diagnosed with pleural tuberculosis (study group) and 35 diagnosed with pleural effusion due to other causes (control group). The determination of IgA in pleural fluid satisfactorily discriminated between pleural tuberculosis patients and control patients. The sensitivity of the test in pleural fluid and in serum was 69% and 30%, respectively, whereas the specificity was 83% and 84%, respectively. CONCLUSIONS: Our data suggest that this test can be used in the diagnosis of pleural tuberculosis. Further studies, involving larger patient samples and different epidemiological scenarios, are warrante