Marcelo Mendonça

Human toxocariasis: current advances in diagnostics, treatment, and interventions

Toxocariasis is a neglected zoonosis caused by the nematodes Toxocara canis and Toxocara cati. This disease is widespread in many countries, reaching high prevalence independently of the economic conditions. However, the true number of cases of toxocariasis is likely to be underestimated owing to the lack of adequate surveillance programs. Although some diagnostic tests are available, their sensitivity and specificity need to be improved. In addition, treatment options for toxocariasis are limited and are non-specific. Toxocariasis is listed as one of the five most important neglected diseases by the CDC. This review presents recent advances related to the control of toxocariasis, including new immunodiagnostics, therapies, and drug formulations, as well as novel interventions using DNA vaccines, immunomodulators, and probiotics.

Production and evaluation of a recombinant chimeric vaccine against clostridium botulinum neurotoxin types C and D.

Bovine botulism is a fatal disease that is caused by botulinum neurotoxins (BoNTs) produced by Clostridium botulinum serotypes C and D and that causes great economic losses, with nearly 100% lethality during outbreaks. It has also been considered a potential source of human food-borne illness in many countries. Vaccination has been reported to be the most effective way to control bovine botulism. However, the commercially available toxoid-based vaccines are difficult and hazardous to produce. Neutralizing antibodies targeted against the C-terminal fragment of the BoNT heavy chain (HC) are known to confer efficient protection against lethal doses of BoNTs. In this study, a novel recombinant chimera, consisting of Escherichia coli heat-labile enterotoxin B subunit (LTB), a strong adjuvant of the humoral immune response, fused to the HC of BoNT serotypes C and D, was produced in E. coli. Mice vaccinated with the chimera containing LTB and an equivalent molar ratio of the chimera without LTB plus aluminum hydroxide (Al(OH)3) developed 2 IU/mL of antitoxins for both serotypes. Guinea pigs immunized with the recombinant chimera with LTB plus Al(OH)3 developed a protective immune response against both BoNT/C (5 IU/mL) and BoNT/D (10 IU/mL), as determined by a mouse neutralization bioassay with pooled sera. The results achieved with guinea pig sera fulfilled the requirements of commercial vaccines for prevention of botulism, as determined by the Brazilian Ministry of Agriculture, Livestock and Food, Supply. The presence of LTB was essential for the development of a strong humoral immune response, as it acted in synergism with Al(OH)3. Thus, the vaccine described in this study is a strong candidate for the control of botulism in cattle.

Listeria monocytogenes isolates from food and food environment harbouring tetM and ermB resistance genes

Listeria monocytogenes is a foodborne pathogen that has become an important cause of human and animal diseases worldwide. The purpose of this study was to evaluate the serotypes, virulence potential, antimicrobial resistance profile, and genetic relationships of 50 L. monocytogenes isolates from food and food environment in southern Brazil. In this study, the majority of L. monocytogenes isolates belonged to the serotypes 1/2b (42%) and 4b (26%), which are the main serotypes associated with human listeriosis. In addition, all isolates harboured internalin genes (inlA, inlC, inlJ), indicating a virulence potential. The isolates were sensitive to most of the antimicrobial compounds analysed, and five isolates (10%) were multi‐resistant. Two isolates harboured antimicrobial resistance genes (tetM and ermB) and in one of them, the gene was present in the plasmid. Moreover, according to the pulsed field gel electrophoresis assay, two multi‐resistant isolates were a single clone isolated from food and the processing plant. The isolates were susceptible to the most frequently used antibiotics for listeriosis treatment. However, the presence of multidrug‐resistant isolates and antimicrobial resistance genes including in the plasmid could even be transferred between bacterial species, suggesting a potential health risk to consumers and a potential risk of spreading multi‐resistance genes to other bacteria.

Evaluation of the Leptospira interrogans Outer Membrane Protein OmpL37 as a Vaccine Candidate

The identification of potential vaccine candidates against leptospirosis remains a challenge. However, one such candidate is OmpL37, a potentially surface-exposed antigen that has the highest elastin-binding ability described to date, suggesting that it plays an important role in host colonization. In order to evaluate OmpL37’s ability to induce a protective immune response, prime-boost, DNA and subunit vaccine strategies were tested in the hamster model of lethal leptospirosis. The humoral immune response was evaluated using an indirect ELISA test, and the cytokine profile in whole blood was determined by quantitative real-time PCR. Unlike the DNA vaccine, the administration of recombinant OmpL37 induced a strong IgG antibody response. When individually administrated, both formulations stimulated a TNF-α mediated inflammatory response. However, none of the OmpL37 formulations or vaccination strategies induced protective immunity. Further studies are required towards the identification of new vaccine targets against leptospirosis.

Recombinant Botulinum Toxoids: A Practical Guide for Production

Clostridium botulinum is a Gram-positive, spore-forming, anaerobic bacillus that produces a potent neurotoxin. Botulinum neurotoxins (BoNTs) are classified from serotypes A to H, and even though they have similar mechanisms of action, they show preferential hosts. In veterinary medicine, BoNT serotypes C and D are the most important, once several animal species are susceptible to them. Since BoNTs are the most potent toxins known in nature, the best way to control botulism in animals is through vaccination. However, current commercial vaccines are based on inactivated toxins (toxoids) and cells (bacterins) and present many drawbacks, such as a time-consuming production with variable antigen yield and biosafety risks. Recombinant vaccines, especially those produced by Escherichia coli expression system, have proved to be an interesting alternative to overcome these problems. E. coli is a very well-known microorganism that allows the production of large amounts of nontoxic recombinant antigens in a short period using simple culture medium reducing the production complexity and decreasing most of the biosafety risks involved in the process. We describe herein a method for the production of recombinant vaccines for veterinary medicine application, involving initial steps of gene design up to vaccine formulation and evaluation itself.

Saccharomyces boulardii improves humoral immune response to DNA vaccines against leptospirosis

Purpose. Saccharomyces boulardii may improve the immune response by enhancing the production of anti‐inflammatory cytokines, T‐cell proliferation and dendritic cell activation. The immunomodulator effect of this probiotic has never been tested with DNA vaccines, which frequently induce low antibody titers. This study evaluated the capacity of Saccharomyces boulardii to improve the humoral and cellular immune responses using DNA vaccines coding for the leptospiral protein fragments LigAni and LigBrep. BALB/c mice were fed with rodent‐specific feed containing 108 c.f.u. of Saccharomyces boulardii per gram. Methodology. Animals were immunized three times intramuscularly with 100 &mgr;g of pTARGET plasmids containing the coding sequences for the above mentioned proteins. Antibody titers were measured by indirect ELISA. Expression levels of IL‐4, IL‐10, IL‐12, IL‐17, IFN‐&ggr; and TGF‐&bgr; were determined by quantitative real‐time PCR from RNA extracted from whole blood, after an intraperitoneal boost with 50 &mgr;g of the recombinant proteins. Results/Key findings. Antibody titers increased significantly after the second and third application when pTARGET/ligAni and pTARGET/ligBrep were used to vaccinate the animals in comparison with the control group (P<0.05). In addition, there was a significant increase in the expression of the IL‐10 in mice immunized with pTARGET/ligBrep and fed with Saccharomyces boulardii. Conclusion. The results suggested that Saccharomyces boulardii has an immunomodulator effect in DNA vaccines, mainly by stimulating the humoral response, which is often limited in this kind of vaccine. Therefore, the use of Saccharomyces boulardii as immunomodulator represents a new alternative strategy for more efficient DNA vaccination.

Xanthan gum enhances humoral immune response elicited by a DNA vaccine against leptospirosis in mice

Background Traditional vaccines (killed or inactivated) played vital roles in controlling and eradicating infectious diseases for a long time. Antigen-specific T cell response can be induced when mice are intramuscularly inoculated with naked plasmid DNA. Therefore, DNA vaccines were evaluated in many studies, demonstrating its safety, stability and easy production. Furthermore, it has been reported that the gene expression lasted for one year after intramuscular injection of the plasmid DNA. However, some disadvantages such as the low transfection rate and low immunogenicity make the use of multiple doses necessary [1]. In light of this context, several studies have been performed to improve the immune response induced by DNA vaccines. The xanthan gum is an extracellular polysaccharide produced during fermentation of bacteria of the genus Xanthomonas and has been studied as a new vaccine adjuvant [2]. However, it has not yet been evaluated as an adjuvant for DNA vaccines. The aim of this study was to evaluate the capacity of the xanthan gum to increase the humoral immune response of mice inoculated with a DNA vaccine against a fragment of the leptospiral antigen LigAni cloned in the mammalian expression plasmid pTARGET.

Label-Free Real-Time Monitoring of Reactions Between Internalin A and Its Antibody by an Oblique-Incidence Reflectivity-Difference Method

Surface protein internalin (InlA) is a major virulence factor of the food-borne pathogen L. monocytogenes. It plays an important role in bacteria crossing the hosts barrier by specific interaction with the cell adhesion molecule E-cadherin. Study of this protein will help to find better ways to prevent listeriosis. In this study, a monoclonal antibody against InlA was used to detect InlA. The reaction was label-free and monitored in real time with an oblique-incidence reflectivity-difference (OI-RD) technique. The kinetic constants k on and k off and the equilibrium dissociation constant K d for this reaction were also obtained. These parameters indicate that the antibody is capable of detecting InlA. Additionally, the results also demonstrate the feasibility of using OI-RD for proteomics research and bacteria detection.

Partial characterization of two novel monoclonal antibodies for Listeria spp. Immunodetection

Background The Listeria genus comprises ten different species, however, only two of them L. monocytogenes and L. ivanovii are pathogenic. Although being a well-known pathogen, its detection methodologies are still limited. The goldstandard technique involves enrichment, isolation, and biochemical characterization, which can take about 7 days [1]. In addition, since both pathogenic and non-pathogenic species are present in contaminated food, it is important to detect them both [2]. This way, new methods, such as the immunodetection using monoclonal antibodies (MAbs), have been developed to reduce the detection time of Listeria spp.. Recently, our group described a hybridoma clone secreting a MAb of the IgM isotype, called MAb3F8, that reacts exclusively with an antigen of 30 kDa (P30) present in all Listeria spp. evaluated [3]. In the present work, we produced and initially characterized, by ELISA and Western blot, other two novel antibodies against P30 to be used in Listeria spp. immunodetection assays.

Saccharomyces boulardii ingestion increases the humoral response of a DNA vaccine against leptospirosis in mice

Background DNA vaccines are a good option to generate a desired antigen using the cellular machinery of the vaccinated subject. This kind of vaccine can induce both of the humoral and cellular immune response, and show high stability and easy working, as well as offer a low-cost production. and safety for immune compromised patients [1]. However, some disadvantages, such as the low transfection rate and low immunogenicity, makes necessary the use of multiple doses [2,3]. Therefore, several studies have been conducted trying to increase the immune response generated by the DNA vaccines. Sacharomyces boulardii, a probiotic yeast that is capable to increase the host immune response [4], was not previously evaluated as an adjuvant for DNA vaccines. Thereby, in the present study we evaluated the S. boulardii capacity to increase the humoral response of mice using DNA vaccines against a fragment of the leptospiral antigen LigAcloned in the mammalian expression plasmid pTARGET [4,5].