Introduction and Background of the Project

Introduction

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is one of the main causes of morbidity and mortality due to infectious diseases. One third of the human population is already infected with the latent form of TB, representing an enormous reservoir of the disease, as a source of new TB cases. BCG, the only vaccine against TB, is not effective in preventing infection and transmission of the disease. The development of new generation vaccines against TB is therefore urgently needed.

Most developmental approaches use the parenteral route of administration. However, TB is a disease of aerogenic transmission. Hence, the development of a mucosal vaccine would most likely provide appropriate protection at the portal of entry, protecting simultaneously against latent infection and clinical disease. Nasal administration of TB vaccine would also have the advantage of simplicity and low cost. However, the development of such vaccines has been hampered by the lack of suitable methods to ensure their stability within the harsh mucosal environment.

We propose to develop a chimeric vaccine candidate using antibody engineering, based on a well-characterized Mtb candidate antigen, Ag85B which has been previously shown to induce protective immunity against Mtb, to be administered by the intranasal route in order to elicit specific mucosal and systemic immune responses.

Project objective

a)     To express and purify a chimeric Mtb vaccine candidate containing Ag85B.

b)     To evaluate the mucosal and systemic immunogenicity of the vaccine candidate administered by the intranasal route to mice.

Project design

Using antibody engineering technology, a chimeric molecule containing Ag85B from Mtb will be obtained and used to immunized mice by the intranasal route. The local biodistribution and the specific mucosal and systemic cellular and antibody responses will be determined.

How can your partnership (project) address global health challenges?

The mucosal surfaces are the main port of entry of most infectious agents causing respiratory, digestive and sexually transmitted disease. In particular, TB is a disease of aerogenic transmission with port of entry at the respiratory mucosa, which highlights the importance of the development of a mucosal TB vaccine. Among the few mucosal vaccines for human use are the ones against poliomyelitis, cholera, typhoid and rotavirus, administered by the oral route and the nasal vaccine against influenza. Most of these vaccines have in common the use of live attenuated microorganisms, which represent a risk for immunosuppressed individuals. Up to now, there are no human vaccines for mucosal administration based on subunits antigens (the instability and degradation of such candidate vaccines remaining the main obstacles for their development). Therefore, the current project attempts to explore a new strategy in the development of mucosal vaccines which would confer stability, resistance to degradation and potential stimulation of mucosal immunity. The production of an effective mucosal vaccine candidate against TB with the possibility to be administered as an edible or inhalable vaccine among other routes could represent a game changer in the field of TB vaccine development. This project will fall in nicely within the End TB Strategy.

What sort of innovation are you bringing in your project?

A new strategy in the development of mucosal vaccines which would confer stability, resistance to degradation and potential stimulation of mucosal immunity. 

Role and Responsibility of Each Partner

Each Partner will implement the following activities:

1. UNIVERSITI SAINS MALAYSIA, MALAYSIA (USM) - DESIGNATED GRANTEE.

・Coordinator and administrator of the project.

・Design and obtain genetic constructs.

・Evaluation of the specific mucosal and systemic cellular and antibody responses & biodistribution of the vaccine candidate.

・Data Analysis & Report preparation.

2. THE UNIVERSITY OF TOKYO, JAPAN (UT)

・Production of Ag85B from E. coli.

・Evaluation of the specific mucosal and systemic cellular and antibody responses & biodistribution of the vaccine candidate.

・Data Analysis & Report preparation.

3. INSTITUTO DE NUTRICION “SALVADOR ZUBIRAN”, MEXICO (IN)

・Evaluation of the specific mucosal and systemic cellular and antibody responses & biodistribution of the vaccine candidate.

・Data Analysis & Report preparation.

4. UNIVERSIDAD DE CONCEPCION, CHILE (UC)

・Design and obtain genetic constructs.

・Expression and purification of the vaccine candidate.

・Data Analysis & Report preparation.

Final Report

1. Project objective 

The main objective of this project was to design and produce a new tuberculosis vaccine candidate for mucosal administration. Mucosal administration is appropriate because the tuberculosis bacterium enters the body through the lungs and strengthening the local immune system at the site of entry may stop infection more effectively. The candidate vaccine was constructed as a chimeric protein comprising the Mycobacterium tuberculosis 85B antigen fused to a host protein to improve its effectiveness. This chimeric protein was then administered intranasally to mice to evaluate the specific mucosal and systemic humoral and cellular immune response against the tuberculosis antigen.

2. Project design

The strategy of the project was based on the design of a chimeric molecule containing the M. tuberculosis 85B antigen fused to a host protein which could facilitate its effectiveness. The chimeric protein was then produced and purified. This protein which is the vaccine candidate was administered by the intranasal route to mice and different immune parameters were determined in the nasal-associated lymphoid tissue (NALT) and lungs together with the evaluation of signs of local toxicity. The specific mucosal and systemic immune response were also determined after the immunization.

3. Results, lessons learned

Despite some technical challenges, we were successful in producing and purifying the chimeric protein as our candidate vaccine. Tests revealed that the vaccine candidate contains all the necessary components of the host protein and tuberculosis antigen as intended. After the immunization of mice by the intranasal route, immune activation in the NALT and lungs was detected without any signs of toxicity. Specific mucosal immune responses, based on the production of specific IgA anitbodies were detected after the immunization. In addition, specific serum total IgG and IgG subclasses antibodies were also detected. These results indicated that the humoral immune response was triggered by the candidate vaccine both at the site of immunization and systemically. In addition, tests also revealed that immune cells in immunized mice responded vigorously against exposure to the tuberculosis antigen. There was strong proliferation of immune cells against the tuberculosis antigen as well as the production of various relevant cytokines, molecules that trigger the immune system, by the immune cells of vaccine–immunized animals but not of control animals. The results obtained suggest that our strategy in constructing a chimeric protein containing a tuberculosis antigen could be used as a mucosal vaccine and was effective in inducing the appropriate immune responses at the site of administration as well as systemically in the animals. Our findings support the future evaluation of this vaccine candidate in Mycobacterium tuberculosis challenge experiments which, if successful, suggests the potential of our chimeric protein to induce protection at the port of entry and throughout the body against tuberculosis.