Protection against Toxoplasma gondii cysts in pigs immunized with rROP2 plus Iscomatrix / Proteção contra cistos de Toxoplasma gondii em suínos imunizados com rROP2 associado à Iscomatrix

Abstract This study aimed to evaluate the humoral immune response in pigs immunized intranasally and intramuscularly with recombinant Toxoplasma gondii rROP2 protein in combination with the adjuvant Iscomatrix. Twelve mixed breed pigs divided into three groups (n=4) were used, G1 received recombinant ROP2 proteins (200 µg/dose) plus Iscomatrix, G2 received PBS plus Iscomatrix, and G3 as the control group. The intranasal (IN) and intramuscular (IM) routes were used. Animals were challenged orally with VEG strain oocysts and treated on day three after challenge. Fever, anorexia, and prostration were the clinical signs observed in all animals. All the G1 animals produced antibodies above the cut-off on the day of the challenge, while the G2 and G3 remained below the cut-off. Better partial protection against parasitemia and cyst tissue formation was observed in G1 than G3. The protection factors against tissue cyst formation were 40.0% and 6.1% for G1 and G2, respectively, compared to G3. In conclusion, there were not systemic antibody responses in pigs with IN immunization with rROP2+Iscomatrix; however, after IM immunization, those animals produced higher titers than animal controls. We associated these results with partial protection obtained against parasitemia and tissue cysts formation.

Resumo O objetivo deste estudo foi avaliar a resposta imune humoral em suínos imunizados pelas vias intranasal e intramuscular com proteínas recombinantes rROP2 do Toxoplasma gondii associadas ao adjuvante Iscomatrix. Doze suínos cruzados divididos em 3 grupos (n=4) foram utilizados. O G1 recebeu proteína recombinante ROP2 (200mg/dose) associada ao adjuvante Iscomatrix; o G2 recebeu PBS associado ao Iscomatrix; e o G3 foi o grupo controle. As vias intranasal (IN) e intramuscular (IM) foram utilizadas. Os animais foram desafiados por via oral com a cepa VEG e tratados no dia três após o desafio. Febre, anorexia e prostração foram os sinais clínicos observados em todos os animais. Todos os animais do G1 produziram anticorpos acima do ponto de corte no dia do desafio, enquanto os animais do G2 e G3 permaneceram abaixo do ponto de corte no desafio. Proteção parcial contra parasitemia e formação de cistos teciduais foram observadas nos suínos do G1 comparados ao G3. Os fatores de proteção contra a formação de cistos teciduais foram 40,0% e 6,1% no G1 e G2, respectivamente, comparados com o G3. Como conclusão, não houve estimulação da resposta imune humoral sistêmica nos suínos após as imunizações IN com rROP2+Iscomatrix. Estes animais, porém, após a imunização IM, produziram títulos de anticorpos mais altos que os animais controles. Esses resultados foram associados a uma proteção parcial contra a parasitemia e formação de cistos teciduais.

rROP2 from Toxoplasma gondii as a potential vaccine against oocyst shedding in domestic cats / rROP2 de Toxoplasma gondii como potencial vacina contra a eliminação de oocistos em gatos domésticos

Abstract The aim of the present study was to evaluate oocyst shedding in cats immunized by nasal route with T. gondii proteins ROP2. Twelve short hair cats (Felis catus) were divided in three groups G1, G2 and G3 (n=4). Animals from G1 received 100 μg of rROP2 proteins plus 20 μg of Quil-A, G2 received 100 μg of BSA plus 20 μg of Quil-A, and the G3 only saline solution (control group). All treatments were done by intranasal route at days 0, 21, 42, and 63. The challenge was performed in all groups on day 70 with ≅ 800 tissue cysts of ME-49 strain by oral route. Animals from G1 shed less oocysts (86.7%) than control groups. ELISA was used to detect anti-rROP2 IgG and IgA, however, there were no correlation between number of oocyst shedding by either IgG or IgA antibody levels. In the present work, in spite of lesser oocysts production in immunized group than control groups, it was not possible to associate the use of rROP2 via nostrils with protection against oocyst shedding. For the future, the use of either other recombinant proteins or DNA vaccine, in combination with rROP2 could be tested to try improving the efficacy of this kind of vaccine.

Resumo O objetivo do presente estudo foi avaliar a eliminação de oocistos de Toxoplasma gondii em gatos imunizados pela via nasal com proteínas ROP2 de T. gondii. Doze gatos sem raça definida (Felis catus) foram divididos em três grupos experimentais G1, G2 e G3 (n = 4). Os animais do G1 receberam 100 μg de proteínas de rROP2 mais 20 μg de Quil-A, G2 recebeu 100 μg de albumina de soro bovino (BSA) junto com 20 μg de Quil-A, e o G3 recebeu apenas solução salina (grupo de controle). Todos os tratamentos foram realizados pela via intranasal nos dias 0, 21, 42 e 63. O desafio foi realizado em todos os grupos no dia 70 com aproximadamente 800 cistos de tecido da cepa ME-49 por via oral. Os animais de todos os grupos tiveram as suas fezes examinadas e o número de oocistos foi determinado durante 20 dias após o desafio. Os animais de G1 eliminaram menos oocistos (86,7%) do que os grupos controles. O ELISA foi utilizado para detectar IgG e IgA anti-rROP2, no entanto, não houve correlação entre o número de eliminhação de oocistos com os níveis de anticorpos IgG ou IgA. No presente trabalho, apesar da menor produção de oocistos no grupo imunizado (G1) em relação aos grupos controles (G2 e G3), não foi possível associar o uso de rROP2 pela via nasal com proteção contra eliminação de oocistos de T. gondii. Para o futuro, a utilização de outras proteínas recombinantes, ou mesmo vacina de DNA, em combinação com rROP2 poderia ser utilizada para tentar melhorar a eficácia deste tipo de vacina.

A Novel Recombinant BCG Vaccine Encoding Eimeria tenella Rhomboid and Chicken IL-2 Induces Protective Immunity Against Coccidiosis

A novel recombinant Bacille Calmette-Guerin (rBCG) vaccine co-expressed Eimeria tenella rhomboid and cytokine chicken IL-2 (chIL-2) was constructed, and its efficacy against E. tenella challenge was observed. The rhomboid gene of E. tenella and chIL-2 gene were subcloned into integrative expression vector pMV361, producing vaccines rBCG pMV361-rho and pMV361-rho-IL2. Animal experiment via intranasal and subcutaneous route in chickens was carried out to evaluate the immune efficacy of the vaccines. The results indicated that these rBCG vaccines could obviously alleviate cacal lesions and oocyst output. Intranasal immunization with pMV361-rho and pMV361-rho-IL2 elicited better protective immunity against E. tenella than subcutaneous immunization. Splenocytes from chickens immunized with either rBCG pMV361-rho and pMV361-rho-IL2 had increased CD4+ and CD8+ cell production. Our data indicate recombinant BCG is able to impart partial protection against E. tenella challenge and co-expression of cytokine with antigen was an effective strategy to improve vaccine immunity.

Efficacy of a DNA Vaccine Carrying Eimeria maxima Gam56 Antigen Gene against Coccidiosis in Chickens

To control coccidiosis without using prophylactic medications, a DNA vaccine targeting the gametophyte antigen Gam56 from Eimeria maxima in chickens was constructed, and the immunogenicity and protective effects were evaluated. The ORF of Gam56 gene was cloned into an eukaryotic expression vector pcDNA3.1(zeo)+. Expression of Gam56 protein in COS-7 cells transfected with recombinant plasmid pcDNA-Gam56 was confirmed by indirect immunofluorescence assay. The DNA vaccine was injected intramuscularly to yellow feathered broilers of 1-week old at 3 dosages (25, 50, and 100 microg/chick). Injection was repeated once 1 week later. One week after the second injection, birds were challenged orally with 5x10(4) sporulated oocysts of E. maxima, then weighed and killed at day 8 post challenge. Blood samples were collected and examined for specific peripheral blood lymphocyte proliferation activity and serum antibody levels. Compared with control groups, the administration of pcDNA-Gam56 vaccine markedly increased the lymphocyte proliferation activity (P<0.05) at day 7 and 14 after the first immunization. The level of lymphocyte proliferation started to decrease on day 21 after the first immunization. A similar trend was seen in specific antibody levels. Among the 3 pcDNA-Gam56 immunized groups, the median dosage group displayed the highest lymphocyte proliferation and antibody levels (P<0.05). The median dosage group had the greatest relative body weight gain (89.7%), and the greatest oocyst shedding reduction (53.7%). These results indicate that median dosage of DNA vaccine had good immunogenicity and immune protection effects, and may be used in field applications for coccidiosis control.

Immunological correlates of cure in the first American Cutaneous Leishmaniasis patient treated by immunotherapy in Argentina: A case report. / Correlatos inmunológicos de curación en el primer paciente con Leishmaniasis Cutánea Americana tratado con inmunoterapia en Argentina: Reporte de un caso

A patient with localized cutaneous leishmaniasis due to Leishmania (Leishmania) amazonensis infection was treated with an antigen containing heat-killed L. (L.) amazonensis promastigotes plus BCG. Expression of T-cell differentiation, memory and senescence receptors markers were analyzed on T cell subpopulations, in order to establish the correlation between the percentages of expression of these receptors and his clinical status, at different stages of his follow up. The following case reports on the achievement of a successful clinical outcome with complete resolution after receiving immunotherapy. A thorough clinical and immunological follow up supporting the healing process of this patient’s lesion is presented in detail.

Un paciente con leishmaniasis cutánea localizada producida por Leishmania (Leishmania) amazonensis fue tratado con un antígeno compuesto por promastigotes de L. (L.) amazonensis muertos por calor combinado con BCG. Se analizó la expresión de distintos receptores de diferenciación, de memoria y de senescencia en las subpoblaciones de células T, con el fin de establecer una relación entre los porcentajes de expresión de dichos receptores y la clínica del paciente en diferentes momentos del seguimiento. Se reporta en este caso un resultado exitoso, con resolución completa de la lesión después de recibir la inmunoterapia, y se presenta en detalle un seguimiento clínico e inmunológico completo durante el proceso de curación.

CD8+ T-cell Activation in Mice Injected with a Plasmid DNA Vaccine Encoding AMA-1 of the Reemerging Korean Plasmodium vivax

Relatively little has been studied on the AMA-1 vaccine against Plasmodium vivax and on the plasmid DNA vaccine encoding P. vivax AMA-1 (PvAMA-1). In the present study, a plasmid DNA vaccine encoding AMA-1 of the reemerging Korean P. vivax has been constructed and a preliminary study was done on its cellular immunogenicity to recipient BALB/c mice. The PvAMA-1 gene was cloned and expressed in the plasmid vector UBpcAMA-1, and a protein band of approximately 56.8 kDa was obtained from the transfected COS7 cells. BALB/c mice were immunized intramuscularly or using a gene gun 4 times with the vaccine, and the proportions of splenic T-cell subsets were examined by fluorocytometry at week 2 after the last injection. The spleen cells from intramuscularly injected mice revealed no significant changes in the proportions of CD8+ T-cells and CD4+ T-cells. However, in mice immunized using a gene gun, significantly higher (P<0.05) proportions of CD8+ cells were observed compared to UB vector-injected control mice. The results indicated that cellular immunogenicity of the plasmid DNA vaccine encoding AMA-1 of the reemerging Korean P. vivax was weak when it was injected intramuscularly; however, a promising effect was observed using the gene gun injection technique.

Immunotherapy, immunochemotherapy and chemotherapy for American cutaneous leishmaniasis treatment

O tratamento de primeira escolha para leishmaniose tegumentar americana é o antimonial pentavalente. Embora este tratamento seja na maioria das vezes efetivo e indicado, devem ser consideradas as desvantagens tais como efeitos colaterais, longa duracão do tratamento e contra-indicacão para cardiopatas, nefropatas, idosos, grávidas e outras condicões. Com o advento da vacina antileishmaniose tegumentar americana para fins profiláticos e terapêuticos, associando-a ou não a outros fármacos, muitas pesquisas têm sido desenvolvidas, sendo a vacina a principal entre os atuais recursos no tratamento e prevencão da leishmaniose tegumentar americana. Em conclusão, a associacão do antimônio com a vacina (imunoquimioterapia) apresentou o mesmo índice de cura em relacão ao tratamento padrão (100%), e ainda reduziu o volume do sal em 17,9% e o tempo de cura significativamente, de 87 para 62 dias; conseqüentemente, reduzindo os efeitos colaterais.

The FML vaccine (fucose-mannose ligand) protects hamsters from experimental kala-azar

Visceral leishmaniais or kala-azar, is a chronic and frequently lethal disease, caused by Leishmania donovani. Clinical signs include malaise, hepatosplenomegaly, hypergammaglobulinemia, fever, cachexia and progressive suppresion of the cellular immune response. Only few studies on prophylactic immunization against this disease have been understaken, mostly with crude antigens, and no vaccine against kala-azar is yet available. In previous studies, we have isolated the Fucose-Mannose Ligand (FML) of L. donovani that strongly and specifically inhibits the in vitro infection of macrophages by promastigotes and amastigotes. The FML behaves as a pontent immunogen for rabbits and mice, and is specifically recognized by kala-azar patient sera. The protective pontential of FML on kala-azar was now analyzed in the CB-hamster model. We studied the efect of three intraperitoneal weekly doses of FML (100 mg) in saponin (100 mg), folowed by an intracardiac injection of 107 amastigotes. Saponin- and saline-treated controls were also included. Protection was highly significant regarding the enhancement of anti-FML antibodies titers, the splenocyte proliferative response, and the intradermal delayed hypersensitivity reaction to antigen, as well as the decrease of the parasite burden in spleen and of splenomegaly. Protection to kala-azar was due to specific FML antigenic properties, since the results obtained by saponin alone were significantly different. We conclude that the use of FML and saponin as a vaccine reduced the disease impact and retarded its onset.