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.

Peptide linker increased the stability of pneumococcal fusion protein vaccine candidate.

Streptococcus pneumoniae is a bacterial pathogen exclusive to humans, responsible for respiratory and systemic diseases. Pneumococcal protein vaccines have been proposed as serotype-independent alternatives to currently used conjugated polysaccharide vaccines, which have presented limitations regarding their coverage. Previously in our group, pneumococcal surface protein A (PspA) and detoxified pneumolysin (PdT) were genetically fused and the hybrid protein protected mice against pneumococcal challenge, offered higher cross-protection against different strains and showed greater opsonophagocytosis rate than co-administered proteins. As juxtaposed fusion was unstable to upscale production of the protein, flexible (PspA-FL-PdT) and rigid (PspA-RL-PdT) molecular linkers were inserted between the antigens to increase stability. This work aimed to produce recombinant fusion proteins, evaluate their stability after linker insertion, both in silico and experimentally, and enable the production of two antigens in a single process. The two constructs with linkers were cloned into Escherichia coli and hybrid proteins were purified using chromatography; purity was evaluated by SDS-PAGE and stability by Western blot and high performance size exclusion chromatography. PspA-FL-PdT showed higher stability at -20°C and 4°C, without additional preservatives. In silico analyses also showed differences regarding stability of the fusion proteins, with molecule without linker presenting disallowed amino acid positions in Ramachandran plot and PspA-FL-PdT showing the best scores, in agreement with experimental results. Mice were immunized with three doses and different amounts of each protein. Both fusion proteins protected all groups of mice against intranasal lethal challenge. The results show the importance of hybrid protein structure on the stability of the products, which is essential for a successful bioprocess development.

Recombinant BCG expressing the LTAK63 adjuvant improves a short-term chemotherapy schedule in the control of tuberculosis in mice.

Tuberculosis (TB) is one of the deadliest infectious diseases around the world. Prevention is based on the prophylactic use of BCG vaccine, effective in infants but as protection wanes with time, adults are less protected. Additionally, chemotherapy requires the use of many antibiotics for several months to be effective. Immunotherapeutic approaches can activate the immune system, intending to assist chemotherapy of TB patients, improving its effectiveness, and reducing treatment time. In this work, the recombinant BCG expressing LTAK63 (rBCG-LTAK63) was evaluated for its immunotherapeutic potential against TB. Bacillary load, immune response, and lung inflammation were evaluated in mice infected with Mycobacterium tuberculosis (Mtb) and treated either with BCG or rBCG-LTAK63 using different routes of administration. Mice infected with Mtb and treated intranasally or intravenously with rBCG-LTAK63 showed a reduced bacillary load and lung inflammatory area when compared to the group treated with BCG. In the spleen, rBCG-LTAK63 administered intravenously induced a higher inflammatory response of CD4+ T cells. On the other hand, in the lungs there was an increased presence of CD4+IL-10+ and regulatory T cells. When combined with a short-term chemotherapy regimen, rBCG-LTAK63 administered subcutaneously or intravenously decreases the Mtb bacillary load, increases the anti-inflammatory response, and reduces tissue inflammation. These findings highlight the potential of rBCG-LTAK63 in assisting chemotherapy against Mtb.

CRISPR/Cas9 Approach to Generate an Auxotrophic BCG Strain for Unmarked Expression of LTAK63 Adjuvant: A Tuberculosis Vaccine Candidate.

Tuberculosis is one of the deadliest infectious diseases and a huge healthcare burden in many countries. New vaccines, including recombinant BCG-based candidates, are currently under evaluation in clinical trials. Our group previously showed that a recombinant BCG expressing LTAK63 (rBCG-LTAK63), a genetically detoxified subunit A of heat-labile toxin (LT) from Escherichia coli, induces improved protection against Mycobacterium tuberculosis (Mtb) in mouse models. This construct uses a traditional antibiotic resistance marker to enable heterologous expression. In order to avoid the use of these markers, not appropriate for human vaccines, we used CRISPR/Cas9 to generate unmarked mutations in the lysA gene, thus obtaining a lysine auxotrophic BCG strain. A mycobacterial vector carrying lysA and ltak63 gene was used to complement the auxotrophic BCG which co-expressed the LTAK63 antigen (rBCGΔ-LTAK63) at comparable levels to the original construct. The intranasal challenge with Mtb confirmed the superior protection induced by rBCGΔ-LTAK63 compared to wild-type BCG. Furthermore, mice immunized with rBCGΔ-LTAK63 showed improved lung function. In this work we showed the practical application of CRISPR/Cas9 in the tuberculosis vaccine development field.

Systems Biology Analysis of the Radiation-Attenuated Schistosome Vaccine Reveals a Role for Growth Factors in Protection and Hemostasis Inhibition in Parasite Survival.

In spite of several decades of research, an effective vaccine against schistosomiasis remains elusive. The radiation-attenuated (RA) cercarial vaccine is still the best model eliciting high protection levels, although the immune mechanisms have not yet been fully characterized. In order to identify genes and pathways underlying protection we investigated patterns of gene expression in PBMC and skin draining Lymph Nodes (LN) from mice using two exposure comparisons: vaccination with 500 attenuated cercariae versus infection with 500 normal cercariae; one versus three doses. Vaccinated mice were challenged with 120 normal parasites. Integration of PBMC and LN data from the infected group revealed early up-regulation of pathways associated with Th2 skewing and polarization of IgG antibody profiles. Additionally, hemostasis pathways were downregulated in infected mice, correlating with platelet reduction, potentially a mechanism to assist parasite migration through capillary beds. Conversely, up regulation of such mechanisms after vaccination may explain parasite blockade in the lungs. In contrast, a single exposure to attenuated parasites revealed early establishment of a Th1 bias (signaling of IL-1, IFN-γ; and Leishmania infection). Genes encoding chemokines and their receptors were more prominent in vaccinated mice, indicating an enhanced capacity for inflammation, potentially augmenting the inhibition of intravascular migration. Increasing the vaccinations from one to three did not dramatically elevate protection, but there was a clear shift towards antibody-mediated effectors. However, elements of the Th1 bias were still evident. Notable features after three vaccinations were markers of cytotoxicity (including IL-6 and NK cells) together with growth factors and their receptors (FGFR/VEGF/EGF) and the apoptosis pathway. Indeed, there is evidence for the development of anergy after three vaccinations, borne out by the limited responses detected in samples after challenge. We infer that persistence of a Th1 response puts a limit on expression of antibody-mediated mechanisms. This feature may explain the failure of multiple doses to drive protection towards sterile immunity. We suggest that the secretions of lung stage parasites would make a novel cohort of antigens for testing in protection experiments.

Optimization and scale-up production of Zika virus ΔNS1 in Escherichia coli: application of Response Surface Methodology.

Diagnosing Zika virus (ZIKV) infections has been challenging due to the cross-reactivity of induced antibodies with other flavivirus. The concomitant occurrence of ZIKV and Dengue virus (DENV) in endemic regions requires diagnostic tools with the ability to distinguish these two viral infections. Recent studies demonstrated that immunoassays using the C-terminal fragment of ZIKV NS1 antigen (ΔNS1) can be used to discriminate ZIKV from DENV infections. In order to be used in serological tests, the expression/solubility of ΔNS1 and growth of recombinant E. coli strain were optimized by Response Surface Methodology. Temperature, time and IPTG concentration were evaluated. According to the model, the best condition determined in small scale cultures was 21 °C for 20 h with 0.7 mM of IPTG, which predicted 7.5 g/L of biomass and 962 mg/L of ΔNS1. These conditions were validated and used in a 6-L batch in the bioreactor, which produced 6.4 g/L of biomass and 500 mg/L of ΔNS1 in 12 h of induction. The serological ELISA test performed with purified ΔNS1 showed low cross-reactivity with antibodies from DENV-infected human subjects. Denaturation of ΔNS1 decreased the detection of anti-ZIKV antibodies, thus indicating the contribution of conformational epitopes and confirming the importance of properly folded ΔNS1 for the specificity of the serological analyses. Obtaining high yields of soluble ΔNS1 supports the viability of an effective serologic diagnostic test capable of differentiating ZIKV from other flavivirus infections.

The Challenges Imposed by Dengue, Zika, and Chikungunya to Brazil.

Brazil has a well-established immunization program in which vaccines are provided through the Public Health System free of charge to the whole population, obtaining high coverage and reducing the incidence of important infectious diseases in children and adults. However, the environmental changes and high mobility rates of the population occurring in the last decades have triggered the sequential introduction of a series of vector-borne emerging infectious diseases, such as Dengue, Zika, and Chikungunya, that have imposed a considerable burden on the population, with yet unmet solutions. The first to be introduced in Brazil was the Dengue virus, reaching epidemic levels in 2010, with over 1 million cases annually, maintaining high infection rates until 2016. Brazil has invested in vaccine development. The Zika virus infection, initially assumed to have appeared during the World Cup in 2014, was later shown to have arrived earlier in 2013. Its emergence mobilized the Brazilian scientific community to define priorities and strategies, that rapidly investigated mechanisms of pathogenesis, differential diagnostics, and determined that Zika virus infection per se causes relatively mild symptoms, however, in pregnant women can cause microcephaly in the newborns. The diagnostics of Zika infection is confusing given its similar symptoms and cross-reactivity with Dengue, which also hindered the appraisal of the extent of the epidemics, which peaked in 2015 and finished in 2016. Another complicating factor was the overlap with Chikungunya virus infection, which arrived in Brazil in 2014, being prevalent in the same regions, with similar symptoms to both Dengue and Zika. Although Dengue infection can be fatal and Zika infection in pregnant woman can lead to newborns with microcephaly or an array of neurodegenerative manifestations, the Chikungunya infection is a debilitating disease leaving chronic sequelae, which unfortunately has received less attention. Precise differential diagnostics of Dengue, Zika, and Chikungunya will be necessary to evaluate the actual extent of each of these diseases during this overlapping period. Here we review the impact of these emerging infections on public health and how the scientific community was mobilized to deal with them in Brazil.

Current status and perspectives on protein-based pneumococcal vaccines.

Despite the efforts to expand the availability of conjugate vaccines, pneumococcal diseases still pose an enormous burden worldwide. Therefore, several proteins have been investigated as alternative vaccines, alone or in combination with other antigens. With an increasing array of techniques, many of which arose from the publication of the bacterial genome, several proteins have been identified as potential vaccine candidates, and some have even progressed to clinical trials. Also, whole cell vaccines are being studied for the induction of broad ranging protective responses. Here, we briefly summarize the current knowledge on pneumococcal proteins that are being investigated as potential vaccine candidates against pneumococcal infections, and provide an insight on the future generation of protein-based vaccines against Streptococcus pneumoniae.

Screening the Schistosoma mansoni transcriptome for genes differentially expressed in the schistosomulum stage in search for vaccine candidates.

Schistosomiasis affects more than 200 million people worldwide; another 600 million are at risk of infection. The schistosomulum stage is believed to be the target of protective immunity in the attenuated cercaria vaccine model. In an attempt to identify genes up-regulated in the schistosomulum stage in relation to cercaria, we explored the Schistosoma mansoni transcriptome by looking at the relative frequency of reads in EST libraries from both stages. The 400 genes potentially up-regulated in schistosomula were analyzed as to their Gene Ontology categorization, and we have focused on those encoding-predicted proteins with no similarity to proteins of other organisms, assuming they could be parasite-specific proteins important for survival in the host. Up-regulation in schistosomulum relative to cercaria was validated with real-time reverse transcription polymerase chain reaction (RT-PCR) for five out of nine selected genes (56%). We tested their protective potential in mice through immunization with DNA vaccines followed by a parasite challenge. Worm burden reductions of 16-17% were observed for one of them, indicating its protective potential. Our results demonstrate the value and caveats of using stage-associated frequency of ESTs as an indication of differential expression coupled to DNA vaccine screening in the identification of novel proteins to be further investigated as potential vaccine candidates.

Schistosoma mansoni Annexin 2: molecular characterization and immunolocalization.

We here describe the cloning and characterization of the Schistosoma mansoni Annexin 2, previously identified in the tegument by proteomic studies, and as an up-regulated gene in schistosomulum stage by microarray data. In silico analysis predicts a conserved core containing four repeat domains of Annexin (ANX) and a variable N-terminal region similar to that described for mammalian isoforms. Real-time RT-PCR and Western blot analysis determined that S. mansoni Annexin 2 is significantly up-regulated in the transition from free-living cercaria to schistosomulum and adult worm parasitic stages. Immunolocalization experiments and tegument membrane preparations confirmed Annexin 2 as a protein mainly localized in the tegument of schistosomula and adult worms. Furthermore, it binds to the tegument surface membranes in a calcium-dependent manner. These results suggest that S. mansoni Annexin 2 is closely associated to the tegument arrangement, being a potential target for immune intervention.