Recombinant BCG expressing the LTAK63 adjuvant increased memory T cells and induced long-lasting protection against Mycobacterium tuberculosis challenge in mice

Vaccine-induced protection against Mycobacterium tuberculosis (Mtb) is usually ascribed to the induction of Th1, Th17, and CD8+ T cells. However, protective immune responses should also involve other immune cell subsets, such as memory T cells. We have previously shown improved protection against Mtb challenge using the rBCG-LTAK63 vaccine (a recombinant BCG strain expressing the LTAK63 adjuvant, a genetically detoxified derivative of the A subunit from E. coli heat-labile toxin). Here we show that mice immunized with rBCG-LTAK63 exhibit a long-term (at least until 6 months) polyfunctional Th1/Th17 response in the draining lymph nodes and in the lungs. This response was accompanied by the increased presence of a diverse set of memory T cells, including central memory, effector memory and tissue-resident memory T cells. After the challenge, the T cell phenotype in the lymph nodes and lungs were characterized by a decrease in central memory T cells, and an increase in effector memory T cells and effector T cells. More importantly, when challenged 6 months after the immunization, this group demonstrated increased protection in comparison to BCG. In conclusion, this work provides experimental evidence in mice that the rBCG-LTAK63 vaccine induces a persistent increase in memory and effector T cell numbers until at least 6 months after immunization, which correlates with increased protection against Mtb. This improved immune response may contribute to enhance the long-term protection

Expression of Schistosoma mansoni antigens in recombinant BCG.

O BCG é um bacilo atenuado de Mycobacterium bovis atualmente investigado na expressão de antígenos heterólogos. Genes oriundos de vírus, bactérias e parasitas são clonados em vetores de expressão micobacterianos e a partir deles, são geradas cepas de BCG recombinante, rBCG. Duas cepas micobacterianas, BCG e M. smegmatis, demonstraram a expressão do antígeno SmStoLP-2 e animais imunizados com este BCG não demonstra diferença na proteção contra a infecção por cercárias, apesar da resposta imune diferenciada. A otimização do códon de SmTSP-2 e SmVAL-5 permitiu sua expressão em M. smegmatis recombinante, mas não em BCG. M. smegmatis transformado com uma biblioteca de plasmídeos contendo o promotor L5p mutagenizado à montante de egfp demonstra acentuada diferença na fluorescência. 3 destes plasmídeos definidos como \'\'forte\'\' e \'\'fraco\'\', com base em sua capacidade de produzir GFP foram utilizados para expressar em BCG o antígeno Sm29 em maior e menor nível.BCG is an attenuated bacillus derived from Mycobacterium bovis currently being investigated to express foreign antigens. Genes from viruses, bacteria and parasites are cloned to mycobacterial vectors and with them, recombinant BCG strains are created. Recombinant BCG and M. smegmatis strains where capable to express SmStoLP-2 and mice immunized with these rBCGs do not show better protection against infection with cercariae, although the distinct immune response observed. The use of codon optimization made possible the expression of SmTSP-2 and SmVAL-5 in M. smegmatis but not BCG. M. smegmatis transformed with a library of plasmids containing the L5p upstream egfp gene show a wide range in fluorescence. 3 of these plasmids strong and weak defined as their ability to produce GFP were used to express in BCG the antigen Sm29 at different levels by each promoter

Robust Immune Response and Protection against Lethal Pneumococcal Challenge with a Recombinant BCG-PspA-PdT Prime/Boost Scheme Administered to Neonatal Mice

Pneumococcal diseases are an important public health problem, with high mortality rates in young children. Although conjugated pneumococcal vaccines offer high protection against invasive pneumococcal diseases, this is restricted to vaccine serotypes, leading to serotype replacement. Furthermore, the current vaccines do not protect neonates. Therefore, several protein-based pneumococcal vaccines have been studied over the last few decades. Our group established a recombinant BCG expressing rPspA-PdT as a prime/rPspA-PdT boost strategy, which protected adult mice against lethal intranasal pneumococcal challenge. Here, we immunized groups of neonate C57/Bl6 mice (6–10) (at 5 days) with rBCG PspA-PdT and a boost with rPspA-PdT (at 12 days). Controls were saline or each antigen alone. The prime/boost strategy promoted an IgG1 to IgG2c isotype shift compared to protein alone. Furthermore, there was an increase in specific memory cells (T and B lymphocytes) and higher cytokine production (IFN-γ, IL-17, TNF-α, IL-10, and IL-6). Immunization with rBCG PspA-PdT/rPspA-PdT showed 100% protection against pulmonary challenge with the WU2 pneumococcal strain; two doses of rPspA-PdT showed non-significant protection in the neonates. These results demonstrate that a prime/boost strategy using rBCG PspA-PdT/rPspA-PdT is effective in protecting neonates against lethal pneumococcal infection via the induction of strong antibody and cytokine responses

Recombinant BCG expressing the LTAK63 adjuvant induces increased early and long-term immune responses against Mycobacteria

The development of more effective vaccines against Mycobacterium tuberculosis has become a world priority. Previously, we have shown that a recombinant BCG expressing the LTAK63 adjuvant (rBCG-LTAK63) displayed higher protection than BCG against tuberculosis challenge in mice. In order to elucidate the immune effector mechanisms induced by rBCG-LTAK63, we evaluated the immune response before and after challenge. The potential to induce an innate immune response was investigated by intraperitoneal immunization with BCG or rBCG-LTAK63: both displayed increased cellular infiltration in the peritoneum with high numbers of neutrophils at 24 h and macrophages at 7 d. The rBCG-LTAK63-immunized mice displayed increased production of Nitric Oxide at 24 h and Hydrogen Peroxide at 7 d. The number of lymphocytes was higher in the rBCG-LTAK63 group when compared to BCG. Immunophenotyping of lymphocytes showed that rBCG-LTAK63 immunization increased CD4+ and CD8+ T cells. An increased long-term Th1/Th17 cytokine profile was observed 90 d after subcutaneous immunization with rBCG-LTAK63. The evaluation of immune responses at 15 d after challenge showed that rBCG-LTAK63-immunized mice displayed increased TNF-α-secreting CD4+ T cells and multifunctional IL-2+ TNF-α+ CD4+ T cells as compared to BCG-immunized mice. Our results suggest that immunization with rBCG-LTAK63 induces enhanced innate and long-term immune responses as compared to BCG. These results can be correlated with the superior protection induced against TB