Monalisa Martins Trentini

Immunogenicity of a Fusion Protein Containing Immunodominant Epitopes of Ag85C, MPT51, and HspX from Mycobacterium tuberculosis in Mice and Active TB Infection

Tuberculosis (TB) remains a major global health problem. The only vaccine against tuberculosis, attenuated Mycobacterium bovis Bacillus Calmette-Guerin (BCG), has demonstrated relatively low efficacy and does not provide satisfactory protection against the disease in adults. More effective vaccines and better therapies are urgently needed to reduce the global spread of TB. This study evaluated the immunogenicity of a recombinant M. tuberculosis Ag85C-MPT51-HspX fusion protein (CMX) in mice and individuals with active tuberculosis. BALB/c mice were immunized with the CMX protein liposome-encapsulated with CpG DNA or with CpGDNA liposome-encapsulated, liposome or saline as negative controls. The immunization produced high levels of anti-CMX -specific IgG1 and IgG2a antibodies and induced an increase in the relative and absolute numbers of specific TCD4 IFN-γ+ and TNF-α+ cells in the spleen. Sera from a cohort of individuals with active tuberculosis contained higher levels of IgG and IgM that recognized CMX when compared to healthy individuals. In conclusion, this protein was shown to be immunogenic both in mice and humans.

Prime–Boost with Mycobacterium smegmatis Recombinant Vaccine Improves Protection in Mice Infected with Mycobacterium tuberculosis

The development of a new vaccine as a substitute for Bacillus Calmette–Guerin or to improve its efficacy is one of the many World Health Organization goals to control tuberculosis. Mycobacterial vectors have been used successfully in the development of vaccines against tuberculosis. To enhance the potential utility of Mycobacterium smegmatis as a vaccine, it was transformed with a recombinant plasmid containing the partial sequences of the genes Ag85c, MPT51, and HspX (CMX) from M. tuberculosis. The newly generated recombinant strain mc2-CMX was tested in a murine model of infection. The recombinant vaccine induced specific IgG1 or IgG2a responses to CMX. CD4+ and CD8+ T cells from the lungs and spleen responded ex vivo to CMX, producing IFN-γ, IL17, TNF-α, and IL2. The vaccine thus induced a significant immune response in mice. Mice vaccinated with mc2-CMX and challenged with M. tuberculosis showed better protection than mice immunized with wild-type M. smegmatis or BCG. To increase the safety and immunogenicity of the CMX antigens, we used a recombinant strain of M. smegmatis, IKE (immune killing evasion), to express CMX. The recombinant vaccine IKE-CMX induced a better protective response than mc2-CMX. The data presented here suggest that the expression of CMX antigens improves the immune response and the protection induced in mice when M. smegmatis is used as vaccine against tuberculosis.

The Role of Neutrophils in the Induction of Specific Th1 and Th17 during Vaccination against Tuberculosis

Mycobacterium tuberculosis causes tuberculosis (TB), a disease that killed more than 1.5 million people worldwide in 2014, and the Bacillus Calmette Guérin (BCG) vaccine is the only currently available vaccine against TB. However, it does not protect adults. Th1 and Th17 cells are crucial for TB control, as well as the neutrophils that are directly involved in DC trafficking to the draining lymph nodes and the activation of T lymphocytes during infection. Although several studies have shown the importance of neutrophils during M. tuberculosis infection, none have shown its role in the development of a specific response to a vaccine. The vaccine mc2-CMX was shown to protect mice against M. tuberculosis challenge, mainly due to specific Th1 and Th17 cells. This study evaluated the importance of neutrophils in the generation of the Th1- and Th17-specific responses elicited by this vaccine. The vaccine injection induced a neutrophil rich lesion with a necrotic central area. The IL-17 KO mice did not generate vaccine-specific Th1 cells. The vaccinated IL-22 KO mice exhibited Th1- and Th17-specific responses. Neutrophil depletion during vaccination abrogated the induction of Th1-specific responses and prohibited the bacterial load reduction observed in the vaccinated animals. The results show, for the first time, the role of neutrophils in the generation of specific Th1 and Th17 cells in response to a tuberculosis vaccine.

Different phenotypes of CD8+ T cells associated with bacterial load in active tuberculosis.

Tuberculosis is an infectious disease that affects millions of people worldwide with an annual mortality rate of 1.3 million. The mechanisms contributing to the loss of balance of immune responses and progression to active tuberculosis disease are unknown. Although CD4+ and CD8+ T cells and the cytokines they produce are crucial for protection against tuberculosis they have different roles in tuberculosis immunology. The function of CD4+ T cells has been extensively studied; however, less is known about the phenotype and function of CD8+ T cells. This study evaluated the specific expression of IFN-γ, IL-17, IL-10, and TGF-β and ex vivo expression of perforin and granzyme-B by CD8+ T cells from active tuberculosis individuals compared with latent infected individuals and non-latent infected individuals. Tuberculosis responses were correlated with the baciloscopy score. We observed that the presence of IL-10 and TGF-β expression and down-expression of granzyme-B in CD8+ T cells correlated with increased sputum bacillary load in active tuberculosis individuals. These findings provide new insights into the role of CD8+ T cells in Mycobacterium tuberculosis disease.

Antimycobacterial Activity of a New Peptide Polydim-I Isolated from Neotropical Social Wasp Polybia dimorpha

Mycobacterium abscessus subsp. massiliense, a rapidly growing mycobacteria (RGM) that is becoming increasingly important among human infectious diseases, is virulent and pathogenic and presents intrinsic resistance to several antimicrobial drugs that might hamper their elimination. Therefore, the identification of new drugs to improve the current treatment or lower the risk of inducing resistance is urgently needed. Wasp venom primarily comprises peptides that are responsible for most of the biological activities in this poison. Here, a novel peptide Polydim-I, from Polybia dimorpha Neotropical wasp, was explored as an antimycobacterial agent. Polydim-I provoked cell wall disruption and exhibited non-cytotoxicity towards mammalian cells. Polydim-I treatment of macrophages infected with different M. abscessus subsp. massiliense strains reduced 40 to 50% of the bacterial load. Additionally, the Polydim-I treatment of highly susceptible mice intravenously infected with M. abscessus subsp. massiliense induced 0.8 to 1 log reduction of the bacterial load in the lungs, spleen, and liver. In conclusion, this is the first study to show the therapeutic potential of a peptide derived from wasp venom in treating mycobacteria infections. Polydim-I acts on the M. abscessus subsp. massiliense cell wall and reduce 40–90% of the bacterial load both in vitro and in vivo. The presented results encourage further studies on the use of Polydim-I as one of the components for M. abscessus subsp. massiliense treatment.

Evaluation of the antimicrobial activity of the mastoparan Polybia-MPII isolated from venom of the social wasp Pseudopolybia vespiceps testacea (Vespidae, Hymenoptera)

Mastoparans, a class of peptides found in wasp venom, have significant effects following a sting as well as useful applications in clinical practice. Among these is their potential use in the control of micro-organisms that cause infectious diseases with a significant impact on society. Thus, the present study describes the isolation and identification of a mastoparan peptide from the venom of the social wasp Pseudopolybia vespiceps and evaluated its antimicrobial profile against bacteria (Staphylococcus aureus and Mycobacterium abscessus subsp. massiliense), fungi (Candida albicans and Cryptococcus neoformans) and in vivo S. aureus infection. The membrane pore-forming ability was also assessed. The mastoparan reduced in vitro and ex vivo mycobacterial growth by 80% at 12.5 µM in infected peritoneal macrophages but did not affect the shape of bacterial cells at the dose tested (6.25 µM). The peptide also showed potent action against S. aureus in vitro (EC50 and EC90 values of 1.83 µM and 2.90 µM, respectively) and reduced the in vivo bacterial load after 6 days of topical treatment (5 mg/kg). Antifungal activity was significant, with EC50 and EC90 values of 12.9 µM and 15.3 µM, respectively, for C. albicans, and 11 µM and 22.70 µM, respectively, for C. neoformans. Peptides are currently attracting interest for their potential in the design of antimicrobial drugs, particularly due to the difficulty of micro-organisms in developing resistance to them. In this respect, Polybia-MPII proved to be highly effective, with a lower haemolysis rate compared with peptides of the same family.

Microstructured liposome subunit vaccines reduce lung inflammation and bacterial load after Mycobacterium tuberculosis infection

BACKGROUND Tuberculosis is a disease affecting millions of people throughout the world. One of the main problems in controlling the disease is the low efficacy of the Bacillus Calmette-Guérin (BCG) vaccine in protecting young adults. The development of new vaccines that induce a long-lasting immune response or that stimulate the immunity induced by BCG may improve the control of tuberculosis. METHODS The use of microstructured liposomes containing HspX, with or without MPL or CpG DNA adjuvants, as vaccines for tuberculosis was evaluated. The HspX-specific humoral and cellular immune responses to the different vaccine formulations were compared. RESULTS All vaccines containing liposome microparticles and HspX were immunogenic. Vaccines formulated with CpG DNA and HspX induced the strongest humoral and cellular immune responses, mainly by inducing interferon-γ and tumor necrosis factor-α expression by both CD4(+) and CD8(+) T cells. HspX and MPL mainly induced CD8(+) T-cell activation and specific humoral responses. When evaluated the protective efficacy of the formulations against Mycobacterium tuberculosis challenge, the microstructured liposome containing L-HspX and L-HspX-CPG DNA reduced both lung inflammatory lesions and the bacterial load. CONCLUSION We have thus demonstrated, for the first time, the use of microstructured liposomes as an adjuvant and delivery system for a vaccine formulation against tuberculosis.

The mc2-CMX vaccine induces an enhanced immune response against Mycobacterium tuberculosis compared to Bacillus Calmette-Guérin but with similar lung inflammatory effects

Although the attenuated Mycobacterium bovis Bacillus Calmette-Guérin (BCG) vaccine has been used since 1921, tuberculosis (TB) control still proceeds at a slow pace. The main reason is the variable efficacy of BCG protection against TB among adults, which ranges from 0-80%. Subsequently, the mc2-CMX vaccine was developed with promising results. Nonetheless, this recombinant vaccine needs to be compared to the standard BCG vaccine. The objective of this study was to evaluate the immune response induced by mc2-CMX and compare it to the response generated by BCG. BALB/c mice were immunised with both vaccines and challenged withMycobacterium tuberculosis (Mtb). The immune and inflammatory responses were evaluated by ELISA, flow cytometry, and histopathology. Mice vaccinated with mc2-CMX and challenged with Mtb induced an increase in the IgG1 and IgG2 levels against CMX as well as recalled specific CD4+ T-cells that produced T-helper 1 cytokines in the lungs and spleen compared with BCG vaccinated and challenged mice. Both vaccines reduced the lung inflammatory pathology induced by the Mtb infection. The mc2-CMX vaccine induces a humoral and cellular response that is superior to BCG and is efficiently recalled after challenge with Mtb, although both vaccines induced similar inflammatory reductions.

Immunogenicity of a Recombinant Mycobacterium smegmatis Vaccine Expressing the Fusion Protein CMX in Cattle from Goiás State, Brazil

ABSTRACT This study aimed to evaluate the immunogenicity of a recombinant Mycobacterium smegmatis vaccine expressing the CMX fusion protein composed of immunodominant epitopes Ag85C, MPT51 and HspX of Mycobacterium tuberculosis, which are important mycobacteria virulence factors. A group of Nelore heifers that were 10 to 12 months of age and negative for the tuberculin skin test (TST) were immunized with four doses of the recombinant vaccine mc2-CMX (M. smegmatis-Ag85C-MPT51-HspX) during a period of one year. Before each immunization, blood was collected to obtain sera for antibody analysis. Serological analysis demonstrated that mc2-CMX was able to induce a humoral response with increased levels of specific IgG antibodies against CMX, despite minimum antibody levels being detected for individual Ag85C, MPT51 or HspX recombinant antigens. However, there was no significant increase in specific CD4+ IFN-γ-positive T cells. Lymphadenomegaly was observed in superficial cervical lymph nodes adjacent to the site of vaccination among mc2-CMX-vaccinated bovines, and the histopathological analysis demonstrated follicular hyperplasia without inflammatory infiltrate or granuloma formation. Animals remained negative for the TST until the end of the experiments, showing no cross-reactivity with the recombinant vaccine and tuberculin proteins. We discuss the potential of mc2-CMX to induce an immune response in cattle.

Advax4 delta inulin combination adjuvant together with ECMX, a fusion construct of four protective mTB antigens, induces a potent Th1 immune response and protects mice against Mycobacterium tuberculosis infection

ABSTRACT Tuberculosis (TB) remains a main public health concern and 10.4 million new cases occurred in 2015 around the world. BCG is the only approved vaccine against TB, but has variable efficacy and new vaccines are needed. We developed two new mTB vaccine candidates based on the recombinant fusion proteins, rCMX and rECMX formulated with Advax4, a new combination adjuvant combining delta inulin, CpG oligonucleotide and murabutide. BALB/c mice were immunized three times intramuscularly with these vaccine formulations. Injection of Advax4 alone increased the percentage of lymphatic endothelial cells and activated macrophages (F480/CD11b+) in the draining lymph nodes consistent with a chemotactic adjuvant effect. Advax4+CMX and Advax4+ECMX induced the highest levels of IgG1 and IgG2a antibodies against rCMX and rECMX, respectively. Immunized mice challenged with Mycobacterium tuberculosis (Mtb) had increased vaccine-specific Th1 responses in the lungs together with reduced Mtb – associated alveolar damage, although only the Advax4+ECMX vaccine demonstrated significant reduction of lung bacterial load. This study confirmed Advax4+ECMX as a potential TB vaccine candidate, with potential for further optimization and clinical development.