EVALUATION OF MYCOBACTERIUM TUBERCULOSIS RECOMBINANT ANTIGENS ESAT-6 AND CFP10 FOR DELAYED TYPE HYPERSENSITIVITY RESPONSES IN GUINEA PIG (CAVIA PORCELLUS)
Name: MIRLEIDE DE ARAÚJO CÁO
Publication date: 25/10/2024
Examining board:
Name |
Role |
|---|---|
| GUSTAVO MODESTO | Examinador Externo |
| LENIR CARDOSO PORFÍRIO | Examinador Externo |
| LUCIANA POLACO COVRE | Examinador Interno |
| MOISES PALACI | Presidente |
| NOEME SOUSA ROCHA | Examinador Externo |
Summary: Tuberculosis is an infectious disease that still presents challenges for effective control and
remains a global health threat, requiring faster and more affordable diagnostic methods. In this
study, we examined the response of recombinant proteins in the diagnostic use of latent
tuberculosis in the literature. Next, we selected the proteins that had the highest response
compared to PPD and based on the synthesis of these proteins from the RD1 region of M.
Tuberculosis, the experiments were carried out. We used different groups, sensitized with M.
bovis, which were tested after 30 days to standardize the technique, and with M. tuberculosis,
which were tested after 30, 60, and 180 days with PPD RT-23, in comparison with the synthetic
proteins ESAT-6 and CFP-10 separately at two concentrations (0.3mg/ml and 3.0mg/ml) and
at the combined concentrations. The minimum time for induration formation after sensitization
is 30 days and the average time for induration formation was 24 hours. The induration was
measured after 24 hours and biopsy of the application site was performed, followed by
immunohistochemistry to verify the infiltration of cells such as T lymphocytes and
macrophages or B lymphocytes, by means of the cytokines CD3, CD68, Tbet, and verification
of the dispersion of proteins at the site by ESAT-6 and CFP-10 labeling. The data were
statistically analyzed by the Fisher and Dunnet tests, respectively. In parallel, micro-needles of
different constructions were tested, to later be used in the delivery of these substances to the
skin. The mean induration between the antigens and the PPD-RT23 did not show significant
variability, and the indurations are compatible between them, presenting a positive response. In
our studies, we found that the CFP10 and ESAT-6 antigens provoked almost identical DTH
reactions in TB-infected guinea pigs, and these were comparable to the responses induced by
PPD. The results showed that the animals with 180 days of sensitization showed a prominent
cutaneous hypersensitivity reaction when challenged with the recombinant proteins associated
with the recombinant proteins CFP-10 and ESAT-6 of M. tuberculosis, inducing a good delayed
cutaneous hypersensitivity response in guinea pigs at the two concentrations alone and
combined. When comparing the positive and low positive categories between cytokines and
PPD, considering a significance level of 10%, there is no evidence that the performance of PPD
is different from the concentrations for the expression of CD3, TBET and CFP-10, i.e., they
have the same or greater capacity than PPD to induce the expression of CD3 and Tbet in the
tissue, which does not occur when we evaluate CD68 expression, showing that there is no
presence of cells that induce the response by type B lymphocytes. The ESAT-6 and CFP10
proteins, at the concentrations of ESAT-6+CFP-10 (100%) [3mg/ml] and ESAT-
6(100%)+CFP-10(10%) [3mg/ml+0.3mg/ml], induced delayed hypersensitivity more
efficiently in the animals, resulting in the longest observed indurations. In addition, the
recombinant proteins provided by the Pace Diagnostics laboratory demonstrated a superior
ability to induce DTH compared to the proteins used in similar studies conducted by other
researchers.
