Struttura e funzione immunogenica della proteina Rv2299c, un promettente vaccino contro la Tubercolosi

Responsabili di progetto

Rita Berisio, Hwa-jung Kim

Accordo

COREA DEL SUD - NRF - National Research Foundation of Korea

Bando

CNR/NRF biennio 2018-2019 2018-2019

Dipartimento

Scienze chimiche e tecnologie dei materiali

Area tematica

Scienze chimiche e tecnologie dei materiali

Stato del progetto

Nuovo

Proposta di ricerca

Mycobacterium tuberculosis is one of the most successful human pathogens, with one-third of the world's population being infected. Because the only available vaccine, M. bovis Bacillus Calmette Guerin (BCG), is unable to provide significant protection against tuberculosis (TB) in adults, a more effective vaccine for replacing or boosting BCG is clearly needed.
In previous studies, the group of Prof. Hwa-Jung Kim of the Department of Microbiology of Chungnam National University in Daejeon, identified a protein, named as Rv2299c, as an excellent candidate for the rational design of an effective multiantigenic TB vaccine.
Rv2299c activates both dendridic cells and macrophages, but the increase in the amount of cytokine secretion was more dramatic in the dendridic cells. In addition, we showed that Rv2299c could be recognized by, and act through a Toll-like receptor (TLR) pathway. The expression of surface molecules and proinflammatory-cytokine secretion were significantly suppressed in DCs from TLR4/ mice when compared to dendridic cells from wild-type mice, indicating that Rv2299c may be an agonist of TLR4 in dendridic cells.
Despite the importance of this protein as a potential vaccine against TB, both its function and structure are hitherto unclear. Also unknown is the mode of interaction of Rv2299c with TLR4 and which regions of the protein are involved in the interaction.
Objective of this project is to analyze the functional and structural characterization of Rv2299c. For structural characterization we will adopt a plethora of biophysical techniques, including x-ray crystallography, CD and fluorescence spectroscopies, Surface Plasmon Resonance, Isothermal Titration Calorimetry and mass spectrometry. Also, we will try to gather clues on mechanism of TLR4 activation by evaluating the interaction mode of Rv2299c with TLR4 to identify which regions of the protein are involved in the interaction. The group of Prof. Hwa-Jung Kim will then evaluate the antigenic features of the selected fragments or peptides.

Both groups are internationally recognized in the study of molecular events involved in Tuberculosis infection. The group led by Prof. Hwa-Jung Kim has solid bases in the field of immunology, whereas the group led by Dr. Rita Berisio has long experience in structural biology applied to infectious diseases, by combining molecular biology, computational biology, protein crystallography, biochemistry and biophysics. These two groups already collaborate for the study of the molecular basis of elicitation of immune response by M. tuberculosis. These studies are aimed at the development of a novel vaccine against Tuberculosis.
This project will further enhance the international cooperation between the two scientific centers, both with long standing experience in the study of Tuberculosis. The bilateral collaboration project is an innovative breakthrough that combines diverse and complementary investigation methods and also facilitates the creation and/or improvement of laboratory training opportunities.

Obiettivi della ricerca

Main objectives are:
1. Analysis of the structural and functional features of Rv2299c as a heat-shock protein.
o Expression and high throughput purification of the protein
o Biophysical Studies (Circular dichroism, Spectroscopies, light scattering)
o Crystallization, X-ray crystallography
o Limited proteolysis using mass spectrometry

2. Prediction and evaluation of TLR4-binding region of Rv2299c
o Structural guided identification of functional binding region with TLR4
o Recombinant production of regions which are identified as TLR4 interaction
o Recombinant production of mutated Rv2299c protein which do not bind with TLR4
o Confirmation whether TLR4 binding region are needed to show biological activity

3. Analysis of immunogenic activity of the selected fragments ot peptides of Rv2299c
o Prediction of putative immuonogenic epitope and synthesis
o Purification of several fragments produced by limited proteolysis
o Evaluation of the immunogenic activity of the peptides or fragments
The two research teams are already collaborating to the development of this project. In this joint project, the Korean group will perform the purification of the recombinant protein, immunologic characterization of TLR4-binding region and immunodominant fragments or epitopes.
The Italian research team will perform x-ray crystallography to determine the protein structure, mass spectrometry and computational studies to identify the epitope region, as well as protein-protein interaction studies.

Ultimo aggiornamento: 19/05/2024