Vaccination with a Plasmodium chabaudi adami multivalent DNA vaccine cross-protects A/J mice against challenge with P. c. adami DK and virulent Plasmodium chabaudi chabaudi AS parasites.

A current goal of malaria vaccine research is the development of vaccines that will cross-protect against multiple strains of malaria. In the present study, the breadth of cross-reactivity induced by a 30K multivalent DNA vaccine has been evaluated in susceptible A/J mice (H-2a) against infection with the Plasmodium chabaudi adami DK strain and a virulent parasite subspecies, Plasmodium chabaudi chabaudi AS. Immunized A/J mice were significantly protected against infection with both P. c. adami DK (31-40% reduction in cumulative parasitemia) and P. c. chabaudi AS parasites, where a 30-39% reduction in cumulative parasitemia as well as enhanced survival was observed. The 30K vaccine-induced specific IFN-gamma production by splenocytes in response to native antigens from both P. c. chabaudi AS and P. c. adami DK. Specific antibodies reacting with surface antigens expressed on P. c. adami DS and P. c. chabaudi AS infected red blood cells, and with opsonizing properties, were detected. These results suggest that multivalent vaccines encoding conserved antigens can feasibly induce immune cross-reactivity that span Plasmodium strains and subspecies and can protect hosts of distinct major histocompatibility complex haplotypes.

Natural regulatory (CD4+CD25+FOXP+) T cells control the production of pro-inflammatory cytokines during Plasmodium chabaudi adami infection and do not contribute to immune evasion.

Different functions have been attributed to natural regulatory CD4+CD25+FOXP+ (Treg) cells during malaria infection. Herein, we assessed the role for Treg cells during infections with lethal (DS) and non-lethal (DK) Plasmodium chabaudi adami parasites, comparing the levels of parasitemia, inflammation and anaemia. Independent of parasite virulence, the population of splenic Treg cells expanded during infection, and the absolute numbers of activated CD69+ Treg cells were higher in DS-infected mice. In vivo depletion of CD25+ T cells, which eliminated 80% of CD4+FOXP3+CD25+ T cells and 60-70% of CD4+FOXP3+ T cells, significantly decreased the number of CD69+ Treg cells in mice with lethal malaria. As a result, higher parasite burden and morbidity were measured in the latter, whereas the kinetics of infection with non-lethal parasites remained unaffected. In the absence of Treg cells, parasite-specific IFN-gamma responses by CD4+ T cells increased significantly, both in mice with lethal and non-lethal infections, whereas IL-2 production was only stimulated in mice with non-lethal malaria. Following the depletion of CD25+ T cells, the production of IL-10 by CD90(-) cells was also enhanced in infected mice. Interestingly, a potent induction of TNF-alpha and IFN-gamma production by CD4+ and CD90(-) lymphocytes was measured in DS-infected mice, which also suffered severe anaemia earlier than non-depleted infected controls. Taken together, our data suggest that the expansion and activation of natural Treg cells represent a counter-regulatory response to the overwhelming inflammation associated with lethal P.c. adami. This response to infection involves TH1 lymphocytes as well as cells from the innate immune system.

Induction of strain-transcending immunity against Plasmodium chabaudi adami malaria with a multiepitope DNA vaccine.

A major goal of current malaria vaccine programs is to develop multivalent vaccines that will protect humans against the many heterologous malaria strains that circulate in endemic areas. We describe a multiepitope DNA vaccine, derived from a genomic Plasmodium chabaudi adami DS DNA expression library of 30,000 plasmids, which induces strain-transcending immunity in mice against challenge with P. c. adami DK. Segregation of this library and DNA sequence analysis identified vaccine subpools encoding open reading frames (ORFs)/peptides of >9 amino acids [aa] (the V9+ pool, 303 plasmids) and >50 aa (V50+ pool, 56 plasmids), respectively. The V9+ and V50+ plasmid vaccine subpools significantly cross-protected mice against heterologous P. c. adami DK challenge, and protection correlated with the induction of both specific gamma interferon production by splenic cells and opsonizing antibodies. Bioinformatic analysis showed that 22 of the V50+ ORFs were polypeptides conserved among three or more Plasmodium spp., 13 of which are predicted hypothetical proteins. Twenty-nine of these ORFs are orthologues of predicted Plasmodium falciparum sequences known to be expressed in the blood stage, suggesting that this vaccine pool encodes multiple blood-stage antigens. The results have implications for malaria vaccine design by providing proof-of-principle that significant strain-transcending immunity can be induced using multiepitope blood-stage DNA vaccines and suggest that both cellular responses and opsonizing antibodies are necessary for optimal protection against P. c. adami.

A bicistronic DNA vaccine containing apical membrane antigen 1 and merozoite surface protein 4/5 can prime humoral and cellular immune responses and partially protect mice against virulent Plasmodium chabaudi adami DS malaria.

The ultimate malaria vaccine will require the delivery of multiple antigens from different stages of the complex malaria life cycle. In order to efficiently deliver multiple antigens with use of DNA vaccine technology, new antigen delivery systems must be assessed. This study utilized a bicistronic vector construct, containing an internal ribosome entry site, expressing a combination of malarial candidate antigens: merozoite surface protein 4/5 (MSP4/5) (fused to a monocyte chemotactic protein 3 chemoattractant sequence) and apical membrane antigen 1 (AMA-1) (fused to a tissue plasminogen activator secretion signal). Transfection of COS 7 cells with bicistronic plasmids resulted in production and secretion of both AMA-1 and MSP4/5 in vitro. Vaccination of BALB/c mice via intraepidermal gene gun and intramuscular routes against AMA-1 and MSP4/5 resulted in antibody production and significant in vitro proliferation of splenocytes stimulated by both AMA-1 and MSP4/5. Survival of BALB/c mice vaccinated with bicistronic constructs after lethal Plasmodium chabaudi adami DS erythrocytic-stage challenge was variable, although significant increases in survival and reductions in peak parasitemia were observed in several challenge trials when the vaccine was delivered by the intramuscular route. This study using a murine model demonstrates that the delivery of malarial antigens via bicistronic vectors is feasible. Further experimentation with bicistronic delivery systems is required for the optimization and refinement of DNA vaccines to effectively prime protective immune responses against malaria.

Induction of specific T-cell responses, opsonizing antibodies, and protection against Plasmodium chabaudi adami infection in mice vaccinated with genomic expression libraries expressed in targeted and secretory DNA vectors.

It has been proposed that a multivalent malaria vaccine is necessary to mimic the naturally acquired resistance to this disease observed in humans. A major experimental challenge is to identify the optimal components to be used in such a multivalent vaccine. Expression library immunization (ELI) is a method for screening genomes of a pathogen to identify novel combinations of vaccine sequences. Here we describe immune responses associated with, and the protective efficacy of, genomic Plasmodium chabaudi adami DS expression libraries constructed in VR1020 (secretory), monocyte chemotactic protein-3 (chemoattractant), and cytotoxic T lymphocyte antigen 4 (lymph node-targeting) DNA vaccine vectors. With splenocytes from vaccinated mice, specific T-cell responses, as well as gamma interferon and interleukin-4 production, were observed after stimulation with P. chabaudi adami-infected erythrocytes, demonstrating the specificity of genomic library vaccination for two of the three libraries constructed. Sera obtained from mice vaccinated with genomic libraries promoted the opsonization of P. chabaudi adami-infected erythrocytes by murine macrophages in vitro, further demonstrating the induction of malaria-specific immune responses following ELI. Over three vaccine trials using biolistic delivery of the three libraries, protection after lethal challenge with P. chabaudi adami DS ranged from 33 to 50%. These results show that protective epitopes or antigens are expressed within the libraries and that ELI induces responses specific to P. chabaudi adami malaria. This study further demonstrates that ELI is a suitable approach for screening the malaria genome to identify the components of multivalent vaccines.

A GRA1 DNA vaccine primes cytolytic CD8(+) T cells to control acute Toxoplasma gondii infection.

Protective immunity against Toxoplasma gondii is known to be mediated mainly by T lymphocytes and gamma interferon (IFN-gamma). The contribution of CD4(+) and CD8(+) T-lymphocyte subsets to protective immune responses against T. gondii infection, triggered by a GRA1 (p24) DNA vaccine, was assessed in this study. In vitro T-cell depletion experiments indicated that both CD4(+) and CD8(+) T-cell subsets produced IFN-gamma upon restimulation with a T. gondii lysate. In addition, the GRA1 DNA vaccine elicited CD8(+) T cells that were shown to have cytolytic activity against parasite-infected target cells and a GRA1-transfected cell line. C3H mice immunized with the GRA1 DNA vaccine showed 75 to 100% protection, while 0 to 25% of the mice immunized with the empty control vector survived challenge with T. gondii cysts. In vivo T-cell depletion experiments indicated that CD8(+) T cells were essential for the survival of GRA1-vaccinated C3H mice during the acute phase of T. gondii infection, while depletion of CD4(+) T cells led to an increase in brain cyst burden during the chronic phase of infection.

CD4+ T cells contain Mycobacterium tuberculosis infection in the absence of CD8+ T cells in mice vaccinated with DNA encoding Ag85A.

The contribution of CD8+ and CD4+ T cell-mediated effector functions against Mycobacterium tuberculosis infection elicited by i.m. vaccination with plasmid DNA encoding the immunodominant Ag85A antigen of M. tuberculosis was studied. Ag85A DNA-vaccinated beta2-microglobulin gene-deficient (beta2m-/-) mice, which lack CD8+ T cells, produced Ag85-specific antibodies and Th1 type cytokines similar to wild-type mice. Although beta2m-/- mice were more susceptible to M. tuberculosis infection, following vaccination they efficiently controlled bacterial replication in spleen and lungs 4 weeks post-infection. In contrast, mice lacking CD4+ T cells were neither sensitized by the Ag85A DNA vaccine to produce Ag85-specific antibodies or Th1 type cytokines nor did they contain a M. tuberculosis challenge infection. In addition, Ag85A DNA-vaccinated IFN-gamma gene knockout mice produced Ag85-specific antibodies and IL-2 but died rapidly following a M. tuberculosis challenge infection. Collectively, these data support the view that IFN-gamma-producing CD4+ T cells, independently of CD8+ T cells, may mediate the protective effect of the Ag85A DNA vaccine.

DNA vaccination with genes encoding Toxoplasma gondii antigens GRA1, GRA7, and ROP2 induces partially protective immunity against lethal challenge in mice.

C57BL/6, C3H, and BALB/c mice were vaccinated with plasmids encoding Toxoplasma gondii antigens GRA1, GRA7, and ROP2, previously described as strong inducers of immunity. Seroconversion for the relevant antigen was obtained in the majority of the animals. T. gondii lysate stimulated specific T-cell proliferation and secretion of gamma interferon (IFN-gamma) in spleen cell cultures from vaccinated BALB/c and C3H mice but not in those from control mice. Although not proliferating, stimulated splenocytes from DNA-vaccinated C57BL/6 mice also produced IFN-gamma. No interleukin-4 was detected in the supernatants of lysate-stimulated splenocytes from DNA-vaccinated mice in any of the mouse strains evaluated. As in infected animals, a high ratio of specific immunoglobulin G2a (IgG2a) to IgG1 antibodies was found in DNA-vaccinated C3H mice, suggesting that a Th1-type response had been induced. For BALB/c mice, the isotype ratio of the antibody response to DNA vaccination was less polarized. The protective potential of DNA vaccination was demonstrated in C3H mice. C3H mice vaccinated with plasmid encoding GRA1, GRA7, or ROP2 were partially protected against a lethal oral challenge with cysts of two different T. gondii strains: survival rates increased from 10% in controls to at least 70% after vaccination in one case and from 50% to at least 90% in the other. In vaccinated C3H mice challenged with a nonlethal T. gondii dose, the number of brain cysts was significantly lower than in controls. DNA vaccination did not protect BALB/c or C57BL/6 mice. Our results demonstrate for the first time in an animal model a partially protective effect of DNA vaccination against T. gondii.

Hemozoin is a key factor in the induction of malaria-associated immunosuppression.

Infection-associated immunoincompetence during malaria might result from macrophage dysfunction. In the present study, we investigated the role of macrophages as target for immunosuppression during infection, using the murine Plasmodium c. chabaudi model. Special attention has been paid to the analysis of processing/presentation of protein antigens and presentation of peptides, using cocultures of peritoneal exudate cells (PECs) from infected mice and antigen-specific T-cell hybridomas. The results obtained indicate a defective processing of protein antigens that becomes maximal at acute parasitemias. In addition, macrophages from acutely infected mice suppress the interleukin-2 production by the antigen-activated T-cell hybridomas. This effect was independent of prostaglandin and nitric oxide production by the macrophage. The possible role of parasite components in the impaired accessory cell function of PECs was investigated and hemozoin, the end-product of the hemoglobin catabolism by intraerythrocytic malaria parasites, was found to induce similar infection-associated deficiencies in vitro. Moreover, hemozoin, was shown to mimic the immunosuppressive effects induced in PECs during in-vivo infections with P. chabaudi. In conclusion, we propose that hemozoin is a key factor in the malaria-associated immunosuppression, affecting both the antigen processing and immunomodulatory functions of macrophages.

Opsonization of Toxoplasma gondii tachyzoites with nonspecific immunoglobulins promotes their phagocytosis by macrophages and inhibits their proliferation in nonphagocytic cells in tissue culture.

We have recently shown that Toxoplasma gondii tachyzoites grown in in vitro culture can bind unspecific immunoglobulin (Ig) through their Fc moiety. We show now that Fc receptors are also present on T. gondii within the host animal, and that intraperitoneal parasites in immunocompetent mice are saturated with unspecific Ig. We have also investigated the effect of the parasite's Fc receptor on the interaction of tachyzoites with mammalian cells, using the Vero cell line as a model for nonphagocytic host cells and murine peritoneal macrophages in primary culture as a model for phagocytic cells. Coating of tachyzoites with parasite-unrelated Ig did not enhance their invasive capacity in either target cell type, but slightly decreased the parasite proliferation. Moreover, phagocytosis by macrophages was increased by approximately 50% when parasites were coated with unspecific Ig. These results indicate that the Fc receptor on T. gondii affects the balance between invasion and phagocytosis in a way that is detrimental to the parasites.

[Palm oil derivatives with different concentration of palmitic acid and antioxidants. Effects upon plasmatic lipids and platelet aggregation]. / Derivados del aceite de palma con concentraciones diferentes de ácido palmítico y antioxidantes. Efectos sobre lípidos plasmáticos y agregación plaquetaria.

It was evaluated the effect of diet rich with cholesterol (0.1%) and different concentration of palmitic acid (16:0) and antioxidants (vitamin C, alpha tocopherol and retinol) upon plasmatic lipids and platelet aggregability in rabbits. The animals were distributed in three groups: I. Standard chow meal (Rp Conejarina) + cholesterol (chol) 0.1%; II. Standard chow meal + chol 0.1% + semipurified palm oil 10% (16:0 = 39.8%, oleic acid 48.7%, linoleic acid 11.4%, retinol 7.3 ug/dL, alpha tocopherol 157.6 ug/dL; III. Standard chow meal + chol 0.1% + crude palm oil 10% (16:0 = 45.3%, oleic acid 46.3%, linoleic acid 7.9%, retinol 96.4 ug/dL, alpha tocopherol 322.8 ug/dL). Monthly determination of plasmatic lipids were done (Enzymatic methods) and at ten months platelet aggregability with ADP, plasmatic vitamin C, retinol and, alpha tocopherol determination were done. Total plasmatic cholesterol (TC) and LDLc increased significantly in the three groups of animals. Significant differences between groups were not found. Platelet aggregability was lower in the animals fed with palmitic acid rich diet (groups II and III) (P = 0.002 and 0.001). Retinol, alpha tocopherol plasmatic concentrations revealed no significant differences. Vitamin C in the groups I was lower than groups II and III (P < 0.05 < 0.02). In this study hypercholesterolemic rabbits fed with rich diets (crude and semipurified) had lower platelet aggregability without changes in plasmatic lipids concentrations.

MIF-1 can accelerate neuromotor, EEG and behavioral development in mice.

Newborn mice were injected SC daily with 1 mg/kg of MIF-1 or saline during the first 19 days of life. The progress of each pup was monitored for physical (body weight, eye and ear opening), neurobehavioral (reflexes) and neurophysiological (EEG) development until the weaning stage. In early adulthood (40 days of age) mice were tested on a maze learning task. Results indicate that MIF-1 can accelerate neurologic (days 3-9), somatic (days 10-14) and electroencephalographic (days 16-19) parameters, and that the effects of treatment last into the early adult stage with increased learning abilities in an appetitive task.