Evaluation of two sexual-stage antigens as bivalent transmission-blocking vaccines in rodent malaria.

BACKGROUND: Transmission-blocking vaccine (TBV) is a promising strategy for malaria elimination. It is hypothesized that mixing or fusing two antigens targeting different stages of sexual development may provide higher transmission-blocking activity than these antigens used individually. METHODS: A chimeric protein composed of fragments of Pbg37 and PSOP25 was designed and expressed the recombinant protein in Escherichia coli Rosetta-gami B (DE3). After immunizing mice with individual recombinant proteins Pbg37 and PSOP25, mixed proteins (Pbg37+PSOP25), or the fusion protein (Pbg37-PSOP25), the antibody titers of individual sera were analyzed by ELISA. IFA and Western blot were performed to test the reactivity of the antisera with the native proteins in the parasite. The transmission-blocking activity of the different immunization schemes was assessed using in vitro and in vivo assays. RESULTS: When Pbg37 and PSOP25 were co-administered in a mixture or as a fusion protein, they elicited similar antibody responses in mice as single antigens without causing immunological interference with each other. Antibodies against the mixed or fused antigens recognized the target proteins in the gametocyte, gamete, zygote, and ookinete stages. The mixed proteins or the fusion protein induced antibodies with significantly stronger transmission-reducing activities in vitro and in vivo than individual antigens. CONCLUSIONS: There was no immunological interference between Pbg37 and PSOP25. The bivalent vaccines, which expand the portion of the sexual development during which the transmission-blocking antibodies act, produced significantly stronger transmission-reducing activities than single antigens. Altogether, these data provide the theoretical basis for the development of combination TBVs targeting different sexual stages.

Pre-clinical evaluation of a whole-parasite vaccine to control human babesiosis.

Babesia spp. are tick-transmitted intra-erythrocytic protozoan parasites that infect humans and animals, causing a flu-like illness and hemolytic anemia. There is currently no human vaccine available. People most at risk of severe disease are the elderly, immunosuppressed, and asplenic individuals. B. microti and B. divergens are the predominant species affecting humans. Here, we present a whole-parasite Babesia vaccine. To establish proof-of-principle, we employed chemically attenuated B. microti parasitized red blood cells from infected mice. To aid clinical translation, we produced liposomes containing killed parasite material. Vaccination significantly reduces peak parasitemia following challenge. B cells and anti-parasite antibodies do not significantly contribute to vaccine efficacy. Protection is abrogated by the removal of CD4+ T cells or macrophages prior to challenge. Importantly, splenectomized mice are protected by vaccination. To further facilitate translation, we prepared a culture-based liposomal vaccine and demonstrate that this performs as a universal vaccine inducing immunity against different human Babesia species.

In vitro analyses of Artemisia extracts on Plasmodium falciparum suggest a complex antimalarial effect.

Dried-leaf Artemisia annua L. (DLA) antimalarial therapy was shown effective in prior animal and human studies, but little is known about its mechanism of action. Here IC50s and ring-stage assays (RSAs) were used to compare extracts of A. annua (DLAe) to artemisinin (ART) and its derivatives in their ability to inhibit and kill Plasmodium falciparum strains 3D7, MRA1252, MRA1240, Cam3.11 and Cam3.11rev in vitro. Strains were sorbitol and Percoll synchronized to enrich for ring-stage parasites that were treated with hot water, methanol and dichloromethane extracts of DLA, artemisinin, CoArtem™, and dihydroartemisinin. Extracts of A. afra SEN were also tested. There was a correlation between ART concentration and inhibition of parasite growth. Although at 6 hr drug incubation, the RSAs for Cam3.11rev showed DLA and ART were less effective than high dose CoArtem™, 8 and 24 hr incubations yielded equivalent antiparasitic results. For Cam3.11, drug incubation time had no effect. DLAe was more effective on resistant MRA-1240 than on the sensitive MRA-1252 strain. Because results were not as robust as observed in animal and human studies, a host interaction was suspected, so sera collected from adult and pediatric Kenyan malaria patients was used in RSA inhibition experiments and compared to sera from adults naïve to the disease. The sera from both age groups of malaria patients inhibited parasite growth ≥ 70% after treatment with DLAe and compared to malaria naïve subjects suggesting some host interaction with DLA. The discrepancy between these data and in-vivo reports suggested that DLA's effects require an interaction with the host to unlock their potential as an antimalarial therapy. Although we showed there are serum-based host effects that can kill up to 95% of parasites in vitro, it remains unclear how or if they play a role in vivo. These results further our understanding of how DLAe works against the malaria parasite in vitro.

Synergistic Effect of Two Nanotechnologies Enhances the Protective Capacity of the Theileria parva Sporozoite p67C Antigen in Cattle.

East Coast fever (ECF), caused by Theileria parva, is the most important tick-borne disease of cattle in sub-Saharan Africa. Practical disadvantages associated with the currently used live-parasite vaccine could be overcome by subunit vaccines. An 80-aa polypeptide derived from the C-terminal portion of p67, a sporozoite surface Ag and target of neutralizing Abs, was the focus of the efforts on subunit vaccines against ECF and subjected to several vaccine trials with very promising results. However, the vaccination regimen was far from optimized, involving three inoculations of 450 µg of soluble p67C (s-p67C) Ag formulated in the Seppic adjuvant Montanide ISA 206 VG. Hence, an improved formulation of this polypeptide Ag is needed. In this study, we report on two nanotechnologies that enhance the bovine immune responses to p67C. Individually, HBcAg-p67C (chimeric hepatitis B core Ag virus-like particles displaying p67C) and silica vesicle (SV)-p67C (s-p67C adsorbed to SV-140-C18, octadecyl-modified SVs) adjuvanted with ISA 206 VG primed strong Ab and T cell responses to p67C in cattle, respectively. Coimmunization of cattle (Bos taurus) with HBcAg-p67C and SV-p67C resulted in stimulation of both high Ab titers and CD4 T cell response to p67C, leading to the highest subunit vaccine efficacy we have achieved to date with the p67C immunogen. These results offer the much-needed research depth on the innovative platforms for developing effective novel protein-based bovine vaccines to further the advancement.

Evaluation of Leishmanization Using Iranian Lizard Leishmania Mixed With CpG-ODN as a Candidate Vaccine Against Experimental Murine Leishmaniasis.

Background and Objectives: The live non-pathogenic Leishmania tarantolae has recently provided a promising approach as an effective vaccine candidate against experimental leishmaniasis (ILL). Here, we evaluated the immunoprotective potential of the live Iranian Lizard Leishmania mixed with CpG adjuvant against L. major infection in BALB/c mice. Methods: Four groups of female BALB/c mice were included in the study. The first and second groups received PBS and CpG, respectively. The immunized groups received 2 × 105 ILL promastigotes and the CpG-mixed ILL (ILL+CpG). Injections were performed subcutaneously in the right footpad. Three weeks later, all mice were challenged with 2 × 105 metacyclic promastigotes of Leishmania majorEGFP ; inoculation was done in the left footpad. The measurement of footpad swelling and in vivo fluorescent imaging were used to evaluate disease progress during infection course. Eight weeks after challenge, all mice were sacrificed and the cytokines levels (IFN-γ, IL-4, and IL-10) and sera antibodies concentrations (IgG2a and IgG1) using ELISA assay, nitric oxide production using Griess assay, and arginase activity in cultured splenocytes, were measured. In addition, direct fluorescent microscopy analysis and qPCR assay were used to quantify the splenic parasite burden. Result: The results showed that mice immunized with ILL+CpG were protected against the development of the dermal lesion. Moreover, they showed a significant reduction in the parasite load, in comparison to the control groups. The observed protection was associated with higher production of IFN-γ, as well as a reduction in IL-4 level. Additionally, the results demonstrated that arginase activity was decreased in ILL+CpG group compared to other groups. Conclusion: Immunization using ILL+CpG induces a protective immunity; indicating that ILL with an appropriate adjuvant would be a suitable choice for vaccination against leishmaniasis.

Anti-Sarcocystis Antibodies in Lambs Deprived of Colostrum.

INTRODUCTION: The objective of this study was to evaluate the presence of anti-Sarcocystis spp. specific IgG antibodies in serum samples from precolostral lambs to determine the occurrence of transplacental transmission of Sarcocystis spp. in sheep. METHODS: Blood samples were collected from 80 ewes and their respective lambs, immediately after lambing and before colostrum ingestion, respectively. The presence of anti-Sarcocystis spp. IgG was evaluated in serum samples using the indirect fluorescent antibody test (IFAT). Positive samples of the lambs were submitted to titration and IFAT to detect anti-T. gondii and anti-N. caninum specific IgG. RESULTS: Anti-Sarcocystis spp. IgG was detected in 62.5% of the ewes (50/80) and in 4% of the lambs of the seropositive ewes (2/50). None of the lambs from seronegative ewes were positive. The final titers of the positive lambs were 80. No cross reaction was detected among the positive samples to anti-Sarcocystis spp., anti-N. caninum, and anti-T. gondii IgG. The detection of anti-Sarcocystis spp. antibodies in serum samples of lambs deprived of colostrum suggests transplacental transmission of infection. Thus, the vertical transmission may be an alternative route of infection of Sarcocystis spp. also in sheep. Further studies are warranted to confirm transplacental transmission in sheep and to explain the importance of this infection pathway.

Protective efficacy induced by DNA prime and recombinant protein boost vaccination with Toxoplasma gondii GRA14 in mice.

Toxoplasma gondii, the etiological agent of toxoplasmosis, can cause severe or lethal damages in both animals and man. So, tends to develop a more effective vaccine to prevent this disease is extremely needed and would be so prominent. The novel dense granule antigen 14 (GRA14) has been identified as a potential vaccine candidate against T. gondii infection. The aim of this study was evaluation of protective immunity induced by prime/boost vaccination strategy of GRA14 antigen with calcium phosphate (CaPNs) or Aluminum hydroxide (Alum) nano-adjuvants in BALB/c mice. The finding showed that immunization with the prime-boost strategy using plasmid DNA (pcGRA14) and recombinant protein (rGRA14) with nano-adjuvants significantly elicited levels of specific IgG antibodies and cytokines against T. gondii infection. Given that, there were the high levels of total IgG, IgG2a, IFN-γ in mice of rGRA14-CaPNs and pcGRA14 + rGRA14-CaPNs groups, which indicating a Th-1 type response. While immunization of mice with Alum based rGRA14 and pcGRA14 + rGRA14 elicited specific IgG1 and IL-4 levels, which was confirmed a Th-2 type response. Mice immunized with DNA prime-protein boost vaccine with nano-adjuvants produce more vigorous specific lymphoproliferative responses than mice immunized with other antigen formulations. In addition, the CaPNs-based prime-boost vaccine of pcGRA14 + rGRA14 showed the longest survival time in mice and the lowest parasitic load in their brain tissue compared to the other groups. The results obtained in this study show that the use of GRA14 based DNA prime-protein boost vaccination regime with CaPNs can dramatically enhanced both humoral and cellular immune responses. Therefore, this strategy can provide a promising approach to the development of an effective vaccine against T. gondii infection in the future.

The impact of early life exposure to Plasmodium falciparum on the development of naturally acquired immunity to malaria in young Malawian children.

BACKGROUND: Antibodies targeting malaria blood-stage antigens are important targets of naturally acquired immunity, and may act as valuable biomarkers of malaria exposure. METHODS: Six-hundred and one young Malawian children from a randomized trial of prenatal nutrient supplementation with iron and folic acid or pre- and postnatal multiple micronutrients or lipid-based nutrient supplements were followed up weekly at home and febrile episodes were investigated for malaria from birth to 18 months of age. Antibodies were measured for 601 children against merozoite surface proteins (MSP1 19kD, MSP2), erythrocyte binding antigen 175 (EBA175), reticulocyte binding protein homologue 2 (Rh2A9), schizont extract and variant surface antigens expressed by Plasmodium falciparum-infected erythrocytes (IE) at 18 months of age. The antibody measurement data was related to concurrent malaria infection and to documented episodes of clinical malaria. RESULTS: At 18 months of age, antibodies were significantly higher among parasitaemic than aparasitaemic children. Antibody levels against MSP1 19kD, MSP2, schizont extract, and IE variant surface antigens were significantly higher in children who had documented episodes of malaria than in children who did not. Antibody levels did not differ between children with single or multiple malaria episodes before 18 months, nor between children who had malaria before 6 months of age or between 6 and 18 months. CONCLUSIONS: Antibodies to merozoite and IE surface antigens increased following infection in early childhood, but neither age at first infection nor number of malaria episodes substantially affected antibody acquisition. These findings have implications for malaria surveillance during early childhood in the context of elimination. Trials registration Clinical Trials Registration: NCT01239693 (Date of registration: 11-10-2010). URL: http://www.ilins.org.

Effectiveness of the sesquiterpene (-)-α-bisabolol in dogs with naturally acquired canine leishmaniosis: an exploratory clinical trial.

The use of natural products is a promising approach for treating visceral leishmaniosis. (-)-α-Bisabolol is a sesquiterpene that have been proved active in vivo on Leishmania infantum-infected mice without showing toxicity. A single-centre, parallel-group, randomized, exploratory study was designed to assess its efficacy in a canine leishmaniosis model involving naturally infected dogs. In this clinical trial, 12 dogs were allocated into two groups and were treated with either meglumine antimoniate (100 mg/kg) through subcutaneous route or (-)-α-bisabolol (30 mg/kg) through oral route for two treatment series of 30 days, separated by a 30-day interval. A 4-month follow-up period was established as well. Parasite loads in bone marrow, lymph node and blood were estimated through quantitative PCR. Antibody titres were determined through immunofluorescence antibody test and cytokine expression values were estimated through real-time reverse transcription-PCR. Treatment safety was assessed through the evaluation of weight, gastrointestinal alterations and hematological and biochemical parameters in blood. Analyses were performed before and after treatment, and after a 4-months follow-up period. Treatment with the sesquiterpene was effective at decreasing parasite loads and increasing gamma-interferon expression level. Dogs treated with (-)-α-bisabolol did not show any toxicity sign. These results were better than those obtained using the reference drug, meglumine antimoniate. The natural compound seemed to induce a Th1 immune response that led to parasitological and clinical improvement without showing any safety issue, suggesting a high potential for the treatment of canine and human visceral leishmaniosis.

Evaluation of Saccharum officinarum L. bagasse-derived polysaccharides as native immunomodulatory and anticoccidial agents in broilers.

Coccidiosis is one of the most important protozoal diseases of the poultry industry, inflicting heavy economic losses in the form of high mortality and morbidity in affected birds. Under these circumstances, the development of nonchemical consumer-friendly strategies for its effective control is of paramount importance. Therefore, this study was conducted to evaluate the sugarcane (Saccharum officinarum L.) bagasse-derived polysaccharides as native immunomodulatory and anticoccidial agents in commercial broilers. Polysaccharides were recovered from sugarcane bagasse (PSCB) and analyzed by high-performance liquid chromatography (HPLC). Five different sugars including melezitose, maltose, glucose, mannose, and fructose were detected in hydrolyzed solution of PSCB. The isolated PSCB were orally administered to the broilers in three graded doses ranging from 10 to 50mg/kg of body weight/day for 3 consecutive days, i.e., fifth through seventh days of life. Results showed significantly enhanced (p<0.05) lymphoproliferative and humoral responses to T-cell mitogen (PHA-P) and sheep red blood cells (SRBCs) in PSCB-administered chickens. In a challenge experiment, percent protection and daily weight gains were significantly higher (p<0.05), whereas mean oocyst counts and lesion scores were lower (p<0.05) in PSCB-administered chickens as compared to control. ELISA showed that PSCB significantly enhanced (p<0.05) antibody titers against the Eimeria species used for the induction of infection in chickens of PSCB-administered and control groups. In conclusion, PSCB showed the potential to modulate the immune responses in industrial broiler chickens with subsequent protection against coccidial infection.

Cisplatin along with herbal drug treatment reduces the percentage of regulatory T cells and decreased the severity of experimental visceral leishmaniasis.

BACKGROUND: Visceral leishmaniasis is the most alarming and devastating amongst the various forms of leishmaniases. It is caused by Leishmania donovani, an obligate intracellular parasite of macrophages that survives through immunosuppression. Absence of T regulatory cells provides complete clearance of the parasite. A few immunoprophylactics have been sought to battle instinctive leishmaniasis, with fluctuating achievement. Our previous studies have shown that treatment of L. donovani infected mice with cisplatin along with herbal drugs resulted in decreased parasite load with heightened delayed type hypersensitivity responses (DTH), increased levels of IgG2a, IFN-γ, IL-2, CD4+ cells, NK 1.1 cells over that of IgG1, IL-4, 1L-10, CD8+ and CD19 in infected mice. METHODS: Along the above lines, the present study further evaluated the percentage of CD4+ CD25+ FoxP3+ T regulatory cells and ultra structural changes in kidney, liver and spleen. Cisplatin (5 mg/kg b.wt. daily for 5 days, i.p.) along with Tinosporacordifolia (100 mg/kg b.wt. daily for 15 days, p.o.) or Withaniasomnifera (350 mg/kg b.wt. daily for 15 days, p.o.) or Asparagusracemosus (650 mg/kg b.wt. daily for 15 days, p.o.) was administered to L. donovani infected BALB/c and after 30 days post treatment mice were sacrificed. RESULTS: The findings uncover a significant reduction in parasite load coupled with decreased percentage of Treg cells and no pathological changes at ultra structural level. CONCLUSION: In this manner, results acquired recommend that the decrease in percentage of T reg cells may further help the antileishmanial remedial impact of cisplatin alongside natural medications.

Giardia and Cryptosporidium infections in neonatal reindeer calves: Relation to the acute phase response.

This longitudinal observational study was conducted to investigate the spontaneous effect of Giardia and Cryptosporidium infections on acute phase response (APR) in reindeer calves (Rangifer tarandus tarandus) in Finnish Lapland. Serum (n=609) and faecal samples (n=366) were collected from 54 reindeer calves aged zero to 33days. The samples were analysed for Giardia, Cryptosporidium, acute phase proteins (APP) and γ-globulins. Linear regression models were used to investigate associations of early Giardia infection (before 12days of life) with the response of APPs and acquiring of passive immunity. Giardia was detected in 100% and Cryptosporidium in 23% of calves. There was a negative association between early Giardia infection and γ-globulin concentrations (p=0.032) and a positive association with serum amyloid A (SAA) concentrations (p=0.042). The results suggest a protective effect of colostrum against Giardia infection and that early infection may induce activation of APR.

In vitro screening of the open source Pathogen Box identifies novel compounds with profound activities against Neospora caninum.

Neospora caninum is a major cause of abortion in cattle and represents an important veterinary health problem of great economic significance. The Medicines for Malaria Venture (MMV) Pathogen Box, an open-source collection of 400 compounds with proven anti-infective properties against a wide range of pathogens, was screened against a N. caninum beta-galactosidase reporter strain grown in human foreskin fibroblasts. A primary screening carried out at 1µM yielded 40 compounds that were effective against N. caninum tachyzoites. However, 30 of these compounds also affected the viability of the host cells. The 10 remaining compounds exhibited IC50 values between 4 and 43nM. Three compounds with IC50 values below 10nM, namely MMV676602, MMV688762 and MMV671636, were further characterized in vitro in more detail with respect to inhibition of invasion versus intracellular proliferation, and only MMV671636 had an impact on intracellular proliferation of tachyzoites. This was confirmed by transmission electron microscopy, showing that the primary target of MMV671636 was the mitochondrion. MMV671636 treatment of experimentally infected mice significantly reduced the number of animals with lung and brain infection, and these mice also exhibited a significantly reduced titer of antibodies directed against N. caninum antigens. Thus, MMV671636 is a promising starting point for the development of a future neosporosis therapy.

Spatial and simultaneous representative seroprevalence of anti-Toxoplasma gondii antibodies in owners and their domiciled dogs in a major city of southern Brazil.

Toxoplasmosis, caused by Toxoplasma gondii, has traditionally been considered an important water and foodborne protozoonosis with important public health considerations. Although felids play a well-established role as definitive hosts, canine epidemiological involvement in the parasite's life cycle remains questionable and controversial. The increasing closeness of the human-dog bond, particularly seen in urban settings, has been recognized as a historically unprecedented worldwide movement. Sharing daily lives in the same households, dogs may be exposed to similar associated risks of T. gondii infection as their owners. Thus, epidemiological assessment of the intra-domiciled environment, especially among socio-economically different human populations, may provide novel information regarding the actual role of dogs in animal and human toxoplasmosis. Despite spatial approaches being recently used for other water and foodborne diseases, no study has been conducted on the simultaneous spatial seroprevalence of both human and animal IgG anti-T. gondii antibodies in urban areas of major cities. Accordingly, the aim of the present study was to assess the seroprevalence and associated variables of Toxoplasma infection in owners and their domiciled dogs in Londrina, southern Brazil. Human and canine seroprevalence rates and variables associated with seroprevalence were investigated through representative random sampling among 564 households, which included 597 owners and 729 dogs. Overall, statistically significant differences between the seroprevalence of human and dog anti-T. gondii antibodies were found by Immunofluorescence Antibody Testing in 248/597 (41.54%) owners and 119/729 (16.32%) dogs. Through multiple analysis, significant concomitant variables for seropositivity of household individuals (people and dogs) were determined, including public sewer service, yard cleaning frequency, and having a dirty yard. Although no statistically significant multiple logistic model was observed among owners, univariate analysis detected associations with monthly income, soil contact, and occupation. Among dogs, the absence of other dogs and the absence of a dirty yard were concomitant significantly protective associated factors. Age differences between seropositive and seronegative individuals was significant only for human beings, with the median age of negative individuals significantly higher than positive individuals. Although no spatial clusters were identified for humans or residences, a significant cluster was identified for dogs. In conclusion, characteristics of urban toxoplasmosis may include significantly higher owner seroprevalence than their owned dogs, with canine seroprevalence directly associated with having more dogs and a dirty backyard, and spatial differences in both human and dog exposures. Although not a good indicator for human foodborne diseases, dogs may be a reliable sentinel for environmental infection. Moreover, such a holistic approach may provide crucial information for more focused prevention and monitoring programs, particularly in households with multiple pets and trash-filled backyards.

Cysteine mutagenesis improves the production without abrogating antigenicity of a recombinant protein vaccine candidate for human chagas disease.

A therapeutic vaccine for human Chagas disease is under development by the Sabin Vaccine Institute Product Development Partnership. The aim of the vaccine is to significantly reduce the parasite burden of Trypanosoma cruzi in humans, either as a standalone product or in combination with conventional chemotherapy. Vaccination of mice with Tc24 formulated with monophosphoryl-lipid A (MPLA) adjuvant results in a Th1 skewed immune response with elevated IgG2a and IFNγ levels and a statistically significant decrease in parasitemia following T. cruzi challenge. Tc24 was therefore selected for scale-up and further evaluation. During scale up and downstream process development, significant protein aggregation was observed due to intermolecular disulfide bond formation. To prevent protein aggregation, cysteine codons were replaced with serine codons which resulted in the production of a non-aggregated and soluble recombinant protein, Tc24-C4. No changes to the secondary structure of the modified molecule were detected by circular dichroism. Immunization of mice with wild-type Tc24 or Tc24-C4, formulated with E6020 (TLR4 agonist analog to MPLA) emulsified in a squalene-oil-in-water emulsion, resulted in IgG2a and antigen specific IFNγ production levels from splenocytes that were not significantly different, indicating that eliminating putative intermolecular disulfide bonds had no significant impact on the immunogenicity of the molecule. In addition, vaccination with either formulated wild type Tc24 or Tc24-C4 antigen also significantly increased survival and reduced cardiac parasite burden in mice. Investigations are now underway to examine the efficacy of Tc24-C4 formulated with other adjuvants to reduce parasite burden and increase survival in pre-clinical studies.

Ginseng Stem-and-Leaf Saponin (GSLS)-Enhanced Protective Immune Responses Induced by Toxoplasma gondii Heat Shocked Protein 70 (HSP70) Against Toxoplasmosis in Mice.

Toxoplasma gondii is an obligate intracellular protozoan parasite and is able to infect birds and mammals including humans. In order to find effective antigen-adjuvant combinations that can boost the immunogenicity and protection of antigen vaccines against toxoplasmosis, we examined the protective efficacy in mice immunized with recombinant protein HSP70 when co-administered with ginseng stem-and-leaf saponins (GSLS) isolated from Panax ginseng . All immunized mice produced significantly high levels of specific antibodies against rTgHSP70, and splenocytes from mice presented strong proliferative immune responses. Vaccinated mice displayed a significantly increased percentage of CD4+ and CD8+ T cells, indicating a strong immune response was triggered. The cellular and humoral immune responses were enhanced, which could be reflected of the increased mRNA levels of IFN-γ and IL-4, respectively. Immunization with rTgHSP70 and GSLS prolonged survival time of the treated mice compared to the controls, which died within 6 days after challenge with the virulent T. gondii RH strain. Our data demonstrate that by addition with GSLS, rTgHSP70 induced a strong immune response and provided partial protection against T. gondii ; therefore GSLS could be used as a promising vaccine adjuvant against acute toxoplasmosis.

Association between malaria immunity and pregnancy outcomes among Malawian pregnant women receiving nutrient supplementation.

BACKGROUND: Malaria antibody responses measured at delivery have been associated with protection from maternal anaemia and low birth weight deliveries. Whether malarial antibodies present in the first half of pregnancy may protect from these or other poor birth outcomes is unclear. To determine whether malaria antibodies in the first half of pregnancy predict pregnancy outcomes, antibodies were measured to a range of merozoite antigens and to antigens expressed on the surface of parasitized red blood cells (pRBCs) in plasma samples collected at 14-20 weeks of gestation from Malawian women. The latter antibodies were measured as total IgG to pRBCs, and antibodies promoting opsonic phagocytosis of pRBCs. Associations between antibodies and maternal haemoglobin in late pregnancy or newborn size were investigated, after adjusting for potential covariates. RESULTS: Antibodies to pRBC surface antigens were associated with higher haemoglobin concentration at 36 weeks. Total IgG to pRBCs was associated with 0.4 g/l [(95% confidence interval (0.04, 0.8)] increase in haemoglobin, and opsonizing antibody with 0.5 (0.05, 0.9) increase in haemoglobin for each 10% increase in antibody. These antibodies were not associated with birthweight, placental malaria, or newborn anthropometrics. Antibodies to merozoite antigens and non-placental-binding IEs were not associated with decreased risk of any of these outcomes. In some instances, they were negatively associated with outcomes of interest. CONCLUSION: Antibodies to placental-binding infected erythrocytes may be associated with higher haemoglobin levels in pregnancy, whereas antibodies to other malaria antigens may instead be markers of malaria exposure. Trial registration clinicaltrials.gov NCT01239693. Registered Nov 10, 2010.

TLR-2 and TLR-4 transcriptions in unstimulated blood from dogs with leishmaniosis due to Leishmania infantum at the time of diagnosis and during follow-up treatment.

Innate immunity, in particular, the role of toll-like receptors (TLRs), has not been extensively studied in canine L. infantum infection. The main aim of this study was to determine the transcription of TLR2 and TLR4 in the blood of dogs with natural clinical leishmaniosis at the time of diagnosis and during treatment follow-up and subsequently correlate these findings with clinical, serological and parasitological data. Forty-six Leishmania-seropositive sick dogs with a high antibody level at the time of diagnosis were studied and compared with 34 healthy seronegative dogs. Twenty-two of these sick dogs were treated with meglumine antimoniate and allopurinol and followed-up at 30, 180 and 365days following the start of treatment. Clinical status was defined by a thorough physical examination, complete blood count, biochemistry profile, electrophoresis of serum proteins, and urinary protein/creatinine ratio (UPC). EDTA blood was stored in RNAlater® solution before RNA extraction and cDNA production were performed. TLR2, TLR4 and three reference genes (HPRT-1, CG14980 and SDHA) were studied in each blood sample by real time PCR. The relative quantification of TLR2 was higher (mean 3.5) in sick dogs when compared with seronegative healthy dogs (mean 1.3; P=0.0001) while the relative quantification of TLR4 was similar in both groups. In addition, the relative quantification of TLR2 significantly decreased during follow-up at all time points compared with day 0 whereas no changes were observed with TLR4 transcription. A significant positive correlation was noted between TLR2 and UPC, total protein, beta and gamma globulins, specific L. infantum antibodies and blood parasite load while a negative correlation was observed with albumin, albumin/globulin ratio, hematocrit and hemoglobin. TLR4 transcript did not correlate with any parameter. These findings indicate an up-regulation of TLR2 transcription in unstimulated blood in naturally infected sick dogs as compared to healthy dogs suggesting active innate immune and proinflammatory responses. In addition, TLR2 transcription is reduced with clinical improvement during treatment. In contrast, TLR4 transcription appears to be similar among groups at the time of diagnosis with no changes during treatment follow-up suggesting a less important role for this TLR in clinical canine leishmaniosis.

Evaluation of recombinant P23 protein as a vaccine for passive immunization of newborn calves against Cryptosporidium parvum.

Cryptosporidiosis is a zoonotic protozoan disease that affects the gastrointestinal tract of animals and humans. Diarrhoea as the most important indication of the infection leads to high economic losses in livestock industries and is a life threatening infection in immunocompromised individuals. In the absence of the effective drugs, vaccine has an effective role in the prevention of infection. For this purpose we developed a vaccine utilizing recombinant P23 protein and immunized pregnant cows four times from 70 days to parturition every 2 weeks. After parturition, each calf received his dam colostrum and challenged with 1 × 10(7) Cryptosporidium parvum oocysts at 12 h of age. Results showed that in contrast with the control group, the antibody titre in the sera and first milking colostra of the immunized cows significantly increased and calves fed hyperimmune colostrum did not show cryptosporidiosis signs. Moreover, enriched colostrum not only reduced significantly the amount of oocyst excretion but also delayed its onset. Our study showed that recombinant P23 protein could be used for passive immunization of newborn calves against Cryptosporidium parvum.

Co-administration of interleukins 7 and 15 with DNA vaccine improves protective immunity against Toxoplasma gondii.

Toxoplasma gondii is an obligatory intracellular parasite, which can infect all warm-blooded animals including humans. Cytokines, including IL-15 and IL-7, play a critical role in the regulation of the homeostasis of naive and memory T cells. Co-administration the DNA vaccine with cytokines may improve its efficacy. IL-7 and IL-15 from splenic tissues of Kunming mice were cloned, and eukaryotic plasmid pVAX-IL-7-IL-15 was constructed. Kunming mice were administrated with DNA vaccine expressing T. gondii calcium-dependent protein kinase 1 (TgCDPK1), pVAX-CDPK1, in the presence or absence of IL-7 and IL-15 plasmids (pVAX-IL-7-IL-15), immune responses were analyzed including lymphoproliferative assay, cytokine and serum antibody measurements, flow cytometric surface markers on lymphocytes, and thus protective immunity against acute and chronic T. gondii infection was estimated. Mice injected with pVAX-CDPK1 supplemented with pVAX-IL-7-IL-15 showed higher Toxoplasma-specific IgG2a titers, Th1 responses associated with the production of IFN-γ, IL-2 as well as cell-mediated cytotoxic activity where stronger frequencies of IFN-γ secreting CD8+ and CD4+ T cells (CD8+/CD4+ IFN-γ+ T cells) compared to controls. Co-administration of pVAX-IL-7-IL-15 and pVAX-CDPK1 significantly (P < 0.05) increased survival time (18.07 ± 5.43 days) compared with pVAX-CDPK1 (14.13 ± 3.85 days) or pVAX-IL-7-IL-15 (11.73 ± 1.83 days) alone, and pVAX-IL-7-IL-15 + pVAX-CDPK1 significantly reduced the number of brain cysts (73.5%) in contrast to pVAX-CDPK1 (46.0%) or pVAX-IL-7-IL-15 alone (45.0%). Our results indicate that supplementation of DNA vaccine with IL-7 and IL-15 would facilitate specific humoral and cellular immune responses elicited by DNA vaccine against acute and chronic T. gondii infection in mice.