43 kDa and 66 kDa, two blood stage antigens induce immune response in Plasmodium berghei malaria.

The hunt for an effective vaccine against malaria still continues. Several new target antigens as candidates for vaccine design are being explored and tested for their efficacy. In the present study the sera from mice immunized with 24,000 × g fraction of Plasmodium berghei has been used to identify highly immunogenic blood stage antigens. The protective antibodies present in immune sera were covalently immobilized on CNBr activated sepharose 4B and used for affinity chromatography purification of antigens present in blood stages of P. berghei. Two polypeptides of 66 and 43 kDa molecular weights proved to be highly immunogenic. They exhibited a strong humoral immune response in mice as evident by high titres in ELISA and IFA. Protective immunity by these two antigens was apparent by in vivo and in vitro studies. These two proteins could further be analysed and used as antigens in malaria vaccine design.

Experimental splenectomies and malaria in mice / Esplenectomias e malária experimental em camundongos

PURPOSE: To evaluate the importance of spleen in malaric infection in murino model, comparing the parasitemia and the titles of imunoglobulins in the different groups. METHODS: It was used female mice non-isogenic, in inoculated with Plasmodium berghei, cepa ANKA, intraperitoneally. The parasitemia was analyzed in 23rd, 25th, 27th and 32nd day of the experiment, being the stained blood' exam colored by Giemsa. The titles of the total serum immunoglobulins IgM and IgG were analyzed by Dot-ELISA technique, at 6th, 22nd and 32nd day, when the animals were sacrificed. RESULTS: The parasitemia was gradual in all the inoculated groups. In the end of the experiment, the animals with partial parasitemia present superior parasitemia, but next to the non-splenectomized, while the asplenics present difference bigger than the double. The levels of total serum IgM and IgG didin´t have significant changes with the removal partial or total splenic. CONCLUSION: The techniques conservatives in splenic trauma are possible and necessary. The importance of remaining spleen in the clearance of red blood cells parasitized by Plasmodium berghei showed being efficient, in order to avoid serious complications resulting of the malaria in mice.

OBJETIVO: Avaliar a importância do baço na infecção malárica em modelo murino, comparando a parasitemia e os títulos das imunoglobulinas nos diferentes grupos. MÉTODOS: Utilizaram-se camundongos fêmeos não isogênicos, inoculados com Plasmodium berghei, cepa ANKA, intraperitoneal. A parasitemia foi analisada no 23°, 25°, 27º e 32° dia do experimento, sendo o exame do esfregaço sangüíneo, corado pelo Giemsa. As titulações das imunoglobulinas totais séricas IgM e IgG foram realizadas pela técnica Dot-ELISA, no 6°, 22° e no 32° dia, quando os animais foram sacrificados. RESULTADOS: A parasitemia foi progressiva em todos os grupos inoculados. Ao final do experimento, os animais com esplenectomia parcial apresentaram parasitemia superior, porém próximos as dos não esplenectomizados, enquanto que os asplênicos apresentaram diferença superior a 100 por cento. Os níveis de IgM e IgG totais séricos não foram alterados significativamente com a remoção parcial ou total esplênica. CONCLUSÃO: As técnicas conservadoras no trauma esplênico são possíveis e necessárias. A importância do remanescente esplênico no clearence das hemácias infectadas pelo Plasmodium berghei demonstrou ser eficiente, de modo a evitar sérias complicações decorrentes da malária em camundongos.

Reduced protective effect of Plasmodium berghei immunization by concurrent Schistosoma mansoni infection

Studies on concomitant schistosomiasis and human and experimental malaria have shown a variation in the immunospecific response, as well as an increase in the severity of both parasitoses. In the present study, a murine co-infection model was used to determine the effects of a co-infection with Schistosoma mansoni and Plasmodium berghei on the protective immunity acquired by repeated malarial infections and subsequent curative treatment with chloroquine. Our results have demonstrated that, compared to an infection with P. berghei only, the co-infection increases the malarial parasitaemia and decreases the survival rate. Indeed, mice that were immunized by infection and treatment with drug displayed no mortality whereas co-infected mice showed a reduced protective efficacy of immunization against P. berghei (mortality > 60 percent). Interestingly, this high mortality rate was not associated with high levels of parasitaemia. Our findings support the idea of a suppressive effect of a Schistosoma co-infection on the anti-malarial protection by immunization. This result reveals a possible drawback of the development of anti-malarial vaccines, especially considering the wide endemic areas for both parasitoses.

Malaria vaccine development: Various immunization regimens for efficient induction of protective anti-malaria immunity

A malária permanece como a maior causa de morbidade e mortalidade humana em todo o mundo, devido à inexist6encia de medidas de controle eficientes para esta infecçäo. Considera-se que a vacinaçäo pode ser um meio eficaz que complementará outras estratégias de prevençäo e controle desta doença no futuro. Embora a possibilidade de uma vacina contra a malária tenha sido demonstrada nos anos 70, o desenvolvimento de uma facina universalmente eficaz contra esta parasitose tem sido uma difícil tarefa devido a diversos problemas complexos. Um dos aspectos é a complexidade do ciclo de vida do parasita, o qual envolve diferentes estágios que possuem antígenos específicos. Muitos antígenos parasitários têm sido investigados como candidatos potenciais à vacinaçäo, e a busca continua, com antígenos adicionais, sendo recentemente indentificados e caracterizados. Alguns desses antígenos estágio-específicos säo capazes de induzir respostas imunoprotetoras celular e humoral no hospedeiro. Todavia, essas respostas imunoprotetoras säo geralmente restritas geneticamente, adicionando outra dificuldade ao desenvolvimento de uma vacina universalmente eficaz. Por fim, o antígeno estágio-específico deve ser introduzido no hospedeiro utilizando-se um sistema de liberaçäo que possa induzir eficientemente respostas protetoras contra os respectivos estágios. No presente trabalho, revemos as diversas tentativas visando a induçäo de imunidade protetora contra todos os estágios do parasita, levando em consideraçäo os aspectos mencionados acima, que säo os antígenos protetores estágio-específicos, as respostas imunoprotetoras do hospedeiro, e os sistemas de liberaçäo antigênica.

Role of macrophages in experimental malaria: VII--studies on adoptive transfer of macrophages.

Adoptive transfer of purified macrophages harvested from normal, Plasmodium berghei infected and latent/cured mice and also macrophages exposed to parasites in vitro were carried out to see the role of macrophages in transferring immunity against P. berghei infection. Macrophages obtained from mice having high parasitaemia at a dose of one million cells/animal showed significant increase in survival period (SP) and K values, compared to controls. Macrophages exposed to low parasite density conferred significant K values only. There was a decrease in prepatent period (PP) in the animal which received macrophages from animals cured 7-11 months compared to controls. The adoptive transfer studies with macrophages conditioned in vitro to parasite contributed towards increased protection of host against P. berghei as expressed by K values only. These studies showed that the macrophages harvested from infected mice were capable of acting as immunogen against P. berghei infection.

Role of macrophages in experimental malaria: VI--Effect of Freund's complete adjuvant in Plasmodium berghei infected mice.

Freund's complete adjuvant (FCA) treated group of mice when challenged with lethal Plasmodium berghei showed increased survival value; survival period (SP) and median survival day (MSD) compared to their respective control groups. K values were affected and mean parasitaemia during infection period was lower than that of control. In general survival rate after 35 days of infection was 10.5% in FCA recipients. The survival rate in a particular group of animals which received 0.2 ml FCA 3 days before challenge was 22.7%. FCA was found to contribute to increased survival of the host against P. berghei infection. The study indicates that adjuvants, like FCA induce protective immunity and future studies should include non-specific immunization against human malaria.

Immune responses to chloroquine--sensitive and resistant populations of Plasmodium berghei in mice.

In order to elucidate the role of the host as a factor in the spread of chloroquine resistance, a study of the host's immune responses in chloroquine resistant (cqr) and chloroquine sensitive (cqs) Plasmodial infections is essential. Course of the infection and the nature of immune responses in mice infected with chloroquine resistant (R) and chloroquine sensitive (S) strains of Plasmodium berghei were compared. Crude parasite antigen activated T cells from both the groups of mice (R and S) and the parasite specific antibodies were detected in the sera of both the groups. The differences in immune responses between the control (uninfected) and infected mice were found to be significant. However, humoral and in vitro cellular responses obtained with T cells from chloroquine resistant P. berghei primed mice was lower in comparison with the responses obtained with T cells from the sensitive infections. Our studies therefore suggest that immunosuppression to parasite antigen is seen in mice primed with chloroquine resistant P. berghei, which may play a role in the development of resistance.

Granulocyte-macrophage colony-stimulating factor production by T lymphocytes in Plasmodium berghei-infected mice.

The production of granulocyte-macrophage colony-stimulating factor (GM-CSF) by lymphocytes was examined in murine malaria. When spleen cells or lymph node cells from P. berghei-infected mice were cultured in vitro with malaria antigen, the GM-CSF production correlated with the incubation time up to 72 hours. When lymphocytes obtained at various days after infection were cultured with the antigen, GM-CSF became detectable as early as 2 days after infection, reached a peak at day 9 and then rapidly decreased. Production of GM-CSF was antigen-specific, and related to the dose of antigen. Treatment of lymphocytes with anti-Thy-1.2 antibody and complement resulted in almost complete loss of GM-CSF-producing activity, while treatment with either anti-CD4 or anti-CD8 antibody and complement resulted in partial loss of GM-CSF-producing activity, indicating that both CD4+ and CD8+ T cells are involved in GM-CSF production in malaria. GM-CSF exhibits glycoprotein nature, and has an apparent molecular weight of 36,000. The molecular properties of this T-cell derived GM-CSF were compared with those of known lymphokine GM-CSF.

Dextran sulfate induced suppression of Plasmodium berghei parasitaemia.

Effect of dextran sulfate (DS, Mr 500,000) on parasitaemia in mice (Balb/c) infected with erythrocytic stage of P. berghei was investigated. Intraperitoneal injection of DS caused marked suppression of patent parasitaemia and also enhanced the survival time of the infected animals.

Stimulation by adjuvants of cell-mediated immune response in Plasmodium berghei infected mice.

Immunological adjuvants (alum, liposomes and saponin) were utilized to stimulate cell-mediated immune response in Plasmodium berghei infected Balb/c mice. It was shown that malaria antigen mixed with adjuvant induced appreciably delayed type hypersensitivity and production of migration inhibition factor compared to antigen alone.

Use of adjuvants in modulating the behaviour of Plasmodium berghei.

Different adjuvants were assessed for their role in conferring protection against the rodent malarial parasite P. berghei and compared with the classical Freund's complete adjuvant (FCA). Pretreatment of mice with trehalose dimycolate (TDM) mixed with antigen (Ag), sulpholipids (SL) mixed with Ag, muramyl dipeptide (MDP) alone, liposomes containing Ag and phosphomannoinositides (PIM) mixed with Ag were ineffective in conferring protection. However, MDP given with squalane (Sq) and Ag, MDP with incomplete Freund's adjuvant (IFA) and Ag, palmitoyl-MDP with Sq and Ag, aluminium hydroxide adsorbed Ag, and FCA with Ag were effective in conferring varying degrees of protection to mice. Complete protection in rats was obtained with MDP mixed with Sq and Ag, and FCA mixed with Ag, and a partial protection with liposomes containing Ag.

Potentiation of immune response against malaria in immunocompromised mice through glucan as an immunoadjuvant.

Untreated mice were fully immunocompetent but their treatment with various immunosuppressors rendered them immunocompromised with respect to one or the other or both limbs of immunity. Both, humoral immune response or cell mediated immune response suppressed mice were only partially protected against the challenge with Plasmodium berghei following their immunization. Hydrocortisone treated mice, in which both types of immune responses were suppressed, were not protected against the challenge with P. berghei following their immunization. In contrast, untreated immunized mice, were fully protected against the challenge with P. berghei. The results suggest that glucan potentiated both limbs of immunity and both were involved in the host defence against malaria.

Uso de sonda de DNA para detectar inmunidad celular contra un parasitismo intracelular / Use of DNA probe to detect cellular immunity against intracellular parasitism

By using a specific, repetitive DNA probe, we have been able to detect picograms of P. berghei DNA. With this probe we have determined that: a) P. berghei, innoculated into Norway Brown rats, reaches its peak of proliferation in the liver 44 after infection; b) gamma interferon inhibits in a dose-dependent fashion the development of liver exoerythrocytic forms (EEF) in vivo and in vitro, and; c) endogenous gamma interferon inhibits the development of EEF in hostys immunized with irradiated sporozoites. Related with and derived from these findings, was have found that, in order to obtain an effective immunity against malaria in experimental animal models, effector mechanisms mediated by cells are required. This is substantiated by the following facts: a) immune hosts innoculated with monoclonal antibodies aginst gamma interferon reversed their immunity against a sporozoite challenge; b) This immunity was also reversed when the animals were depleted from their CD8 positive cytotoxic T cells. Therfore, sterile immunity against this parasite requires not only the presence of antibodies but also the inhibition of the EEF by gamma interferon with participation of CD8 positive T cells

Prevention of malaria-induced foetal abnormalities following immunization of mice with Plasmodium berghei merozoite antigen.

Pre-pregnancy immunization of Swiss albino mice with merozoite antigen of P. berghei entrapped in multilamellar phosphatidyl choline liposomes resulted in (i) increased prepatent period, (ii) either no or low parasitaemic levels, (iii) reduced mortality, and (iv) normal foetal and placental development, upon challenge with P. berghei on 13th gestational day. The unimmunized animals which received either phosphate buffered saline or empty multilamellar phosphatidyl choline liposomes before pregnancy developed high parasitaemic and 30-40 per cent animals died before parturition while 60-70 per cent unimmunized animals revealed foetal abnormalities such as low body weight and larger spleen size. Placentae of unprotected animals had hyperplasia of trophoblastic membrane and plugging of placental sinusoids with parasitized erythrocytes and malarial pigments. The data suggest that prior immunization of animals with merozoite antigen entrapped in multilamellar phosphatidyl choline liposomes could abrogate the ill effects induced by malaria infection under the stress of pregnancy.

Immunoprotection by beta-1,3 glucan antigen combination in Plasmodium berghei infection in mice.

In an attempt to protect mice against experimental infection with P. berghei, mice were immunized against soluble extract of P. berghei in combination with beta-1,3 glucan or FCA and also independently. Mice immunized against P. berghei antigen-glucan developed well defined cell mediated and humoral immune responses, while mice injected with antigen FCA or antigen alone developed only an antibody response. Antigen-glucan immunization afforded a high degree of immune protection to the host against the challenge with live parasites.

Evaluation of CIEP and ELISA in a rodent malaria model.

Circulating antigens could be detected by both ELISA and CIEP in the sera of mice infected with Plasmodium berghei. However, ELISA was positive for circulating antigen even though the Giemsa stained smears did not show significant parasites. Hence, ELISA can have a diagnostic value for detection of antigens as a marker of active infection.

Effects of mycobacterium bovis BCG, bacterial lipopolysaccharide and hydrocortisone on the development of immunity to Plasmodium berghei

Mycobacterium bovis (BCG) aumenta significantemente o desenvolvimento da imunidade nos camundongos CFW, C57BL/6, C57BL/10ScN e BALB/c (Nu/+) para os estágios eritrocitos do Plasmodium berghei. Camundongos tratados com BCG requerem menos ciclos de infecçäo com P. berghei e cura pelo Fansidar (pirimetamina + sulfadoxina) para desenvolverem imunidade sólida a este parasita do que os controles. Contudo, os animais que receberam BCG 30 dias antes do início da imunizaçäo evidenciaram uma perda precoce da imunidade adquirida para o P. berghei, quando comparado com os animais que receberam BCG 14 dias antes ou que näo receberam BCG. Assim, sendo, o BCG aumenta a induçäo na resposta imune do hospedeiro ao P. berghei no curso de infecçöes subseqüentes. O tratamento de camundongos CFW, BALB/c e C57BL/6 com lipopolissacarídeo bacteriano ou hidrocortisona faz com que os animais requeiram um número maior de ciclos de infecçäo e cura para tornarem-se imunes ao P. berghei que os controles. O tratamento dos camundongos C57BL/10ScN com hidrocortisona aboliu completamente a sua habilidade de sobrevida subseqüente a ciclos de infecçäo com P. berghei e cura pelo Fansidar

Protection of athymic (NU/NU) BALB/c mice against Plasmodium berghei by splenocytes from normal (NU/+) BALB/c mice

Camundongos atímicos BALB/c (Nu/Nu) sucumbem entre 7-13 dias após a inoculaçäo (DAI) da cepa NK65 de Plasmodium berghei. Todavia, seus singenêicos heterozigotos (Nu/+) morrem em 7-8 DAI. Camundongos nude (Nu/Nu) reconstituídos com 2x10 esplenócitos de camundongos heterozigotos singenêicos normais näo infectados (Nu/+) 20 dias antes de inoculaçäo a (DBI) do parasita, sucumbem 2 dias antes que os animais controles. Camundongos nude reconstituídos 10 ou 2 DBI, vivem 2-4 dias a mais que os animais controles e alguns deles sobrevivem. Esses achados indicam que a cepa NK65 de P. berghei induz, no mínimo, dois imunofenômenos dependentes de linfócitos T; um supressivo e outro estimulatório. A reconstituiçäo de camundongos nude com células T de camundongos BALB/c (Nu/+) parece reduzir ou "Bypass" a atividade supressora das células T, o qual leva à formaçäo de uma resposta imune protetora por alguns dos camundongos nude