Intranasal vaccination with messenger RNA as a new approach in gene therapy: use against tuberculosis

Abstract

Background: mRNAs are highly versatile, non-toxic molecules that are easy to produce and store, which can allow transient protein expression in all cell types. The safety aspects of mRNA-based treatments in gene therapy make this molecule one of the most promising active components of therapeutic or prophylactic methods. The use of mRNA as strategy for the stimulation of the immune system has been used mainly in current strategies for the cancer treatment but until now no one tested this molecule as vaccine for infectious disease.

Results: We produce messenger RNA of Hsp65 protein from Mycobacterium leprae and show that vaccination of mice with a single dose of 10 μg of naked mRNA-Hsp65 through intranasal route was able to induce protection against subsequent challenge with virulent strain of Mycobacterium tuberculosis. Moreover it was shown that this immunization was associated with specific production of IL-10 and TNF-alpha in spleen. In order to determine if antigen presenting cells (APCs) present in the lung are capable of capture the mRNA, labeled mRNA-Hsp65 was administered by intranasal route and lung APCs were analyzed by flow cytometry. These experiments showed that after 30 minutes until 8 hours the populations of CD11c+, CD11b+ and CD19+ cells were able to capture the mRNA. We also demonstrated in vitro that mRNA-Hsp65 leads nitric oxide (NO) production through Toll-like receptor 7 (TLR7).

Conclusions: Taken together, our results showed a novel and efficient strategy to control experimental tuberculosis, besides opening novel perspectives for the use of mRNA in vaccines against infectious diseases and clarifying the mechanisms involved in the disease protection we noticed as well.

Figure 1

(A) - Electrophoretic profile of in vitro synthesized messenger RNAs. Lane M: 0.5-10 kb RNA Ladder (Invitrogen), lane 1: 1 μg of mRNA-Hsp65, 2: 1 μg of EF-1α mRNA. The electrophoresis was performed in 1.5% denaturing agarose gel stained with ethidium bromide. (BP-Base pair, M-Marker). (B) - Detection of the integrity of the mRNA-Hsp65 by RT-PCR. After transfection of HEK 293 cells with the mRNA-Hsp65 cells were maintained in culture for different periods of time, as showed in the figure, in order to analyze the presence of mRNA-Hsp65. Electrophoresis was run in 1.5% agarose gel and stained with ethidium bromide (NC-Negative PCR control, NT-Not transfected, PC-Positive PCR control). (C) - After contact mRNA-Hsp65 for different periods of time, the total cell lysate was subjected to polyacrylamide gel electrophoresis (12.5%) and the bands transferred to nitrocellulose membrane and incubated with anti-Hsp65 for 2 hours. The reaction was revealed with secondary antibody anti-mouse IgG in the presence of DAB. (RC-Recombinant protein Hsp65, NT-Not transfected)

Figure 2

Assessment of the amount of professional antigen-presenting cells capable of capturing mRNA-Hsp65 labeled with Alexa488. Five BALB/c mice per group were immunized by intranasal route with one dose of 10 μg of Alexa488 labeled mRNA-Hsp65. The control group received Ringer's solution. The kinetics of the capture was made in different times (indicated in the legend) and the cellular phenotype detected by flow cytometry. Data represent the mean positive cell counts ± SD of five mice per group of one of three independent experiments

Figure 3

Intranasal vaccination with mRNA-Hsp65 protects mice against tuberculosis. Five BALB/c mice per group were immunized by intranasal route with a dose of different formulations containing mRNA-Hsp65 or EF-1α mRNA. BCG group received subcutaneously 1 dose of BCG Moreau. Non vaccinated group received only the Ringer's solution. Thirty days after the last immunization, animals were challenged with 10⁵ bacilli of the virulent H37Rv strain of M. tuberculosis by intranasal route. Thirty days after challenge, animals were euthanized and their lungs extracted for the evaluation of the bacterial load. Data represent the mean log10 CFU counts ± SD of five mice per group of one of three independent experiments. *p < 0.05 were considered significant when compared to non vaccinated group.

Figure 4

Histological analysis of lungs of BALB/c mice vaccinated and challenged with virulent strain of Mycobacterium tuberculosis. Five BALB/c mice per group were immunized by intranasal route with a dose of different formulations containing mRNA-Hsp65 or EF-1α mRNA. The BCG group received 1 dose of BCG Moreau subcutaneously. Thirty days after the last immunization animals were challenged with 10⁵ bacilli of the virulent strain of M. tuberculosis by H37RV intranasal route. Thirty days after challenge, animals were sacrificed and their lungs extracted for the recovery histology. 1-Non vaccinated group, 2-BCG group, 3-EF-1α mRNA 5 ug group, 4-EF-1α mRNA 10 μg group, 5-mRNA-Hsp65 5 ug group, 6-mRNA-Hsp65 10 μg group. Staining was performed with hematoxylin and eosin (HE), increased original image of 100×.

Figure 5

Immunostimulatory activity of mRNA-Hsp65. Five BALB/c mice per group mice were immunized with one dose of mRNA-Hsp65 (10 μg per mouse in 100 μL volume). Not immunized group received only the Ringer's solution. Two weeks after the immunization splenic cells are isolated and stimulated with 20 μg/ml of Hsp65 protein, 48 hours after stimulation the production of (A) IFN-gamma and (B) TNF-alpha by were determined by ELISA. *p < 0.05 were considered significant when compared to not immunized group.

Figure 6

TLR7 activation by hsp65 mRNA. HEK293T cells (3 × 10⁵ cells/well) were co-transfected in triplicate with the TLR expression plasmids. The next day, cells were treated with hsp65 mRNA or following TLRs agonists: poly-IC 100 μg/ml (TLR3), LPS 1 μg/ml (O55:B55) (TLR4) and R-848 1 μg/ml (TLR7 and 8) for 7 hours. The amount of NO release was measured form culture supernatant using the method of Griess. Data are presented as the means ± SD of triplicate cultures. *p < 0.05 were considered significant when compared to empty vector.