REVIEW OF DNA VACCINE APPROACHES AGAINST THE PARASITE TOXOPLASMA GONDII.

Toxoplasma gondii is an apicomplexan parasite that affects both humans and livestock. Transmitted to humans through ingestion, it is the second-leading cause of foodborne illness-related death. Currently, there exists no approved vaccine for humans or most livestock against the parasite. DNA vaccines, a type of subunit vaccine which uses segments of the pathogen's DNA to generate immunity, have shown varying degrees of experimental efficacy against infection caused by the parasite. This review compiles DNA vaccine efforts against Toxoplasma gondii, segmenting the analysis by parasite antigen, as well as a review of concomitant adjuvant usage. No single antigenic group was consistently more effective within in vivo trials relative to others.

Network meta-analysis comparing the effectiveness of anticoccidial drugs and anticoccidial vaccination in broiler chickens.

With the trend to organic production and concerns about using antibiotic feed additives, the control of infections with Eimeria spp. in broiler flocks has become more difficult. Vaccination against coccidia is an alternative, but there are concerns that the live vaccines used might have negative effects on production parameters and intestinal health. Reports of experiments directly comparing anticoccidial drugs and anticoccidial vaccines are rare. This network meta-analysis (NMA) identified and analyzed 61 articles reporting 63 experiments testing anticoccidial drugs and anticoccidial vaccines under conditions resembling commercial broiler production. The effect sizes were mean differences in body weight/body weight gain (BW/BWG) and feed conversion rate (FCR) between the 175 included groups. The results show that groups vaccinated against coccidia have a similar BW/BWG and FCR at processing age compared to groups given anticoccidial drugs. However, the results tended to be more favorable for anticoccidial drugs than for vaccines. The analysis of eight subsets, containing only groups (1) groups that had not received an AGP in addition to an anticoccidial drug, (2) groups that had not received ionophores, (3) groups that had not received chemicals, (4) groups that had not received an attenuated vaccine, (5) groups that had not received a fully virulent vaccine, (6) groups that were not additionally challenged with bacteria or not challenged, (7) groups that had received a severe challenge as defined by a total infection dose of more than 100,000 oocysts or were not challenged, (8) groups that were challenged on day 15 or earlier or not challenged brought similar results and confirmed the robustness of the NMA. In addition, the analysis exposes unnecessary, as well as inherent, problems with data quality, which every researcher working with coccidia should carefully consider, and identifies under-researched areas that should be addressed in future research.

Antibodies elicited by the CaniLeish® vaccine: long-term clinical follow-up study of dogs in Spain.

The prevention of canine leishmaniosis in healthy dogs requires a multimodal approach combining repellents with an effective vaccine. A vaccine that modulates the cell-mediated immune response against the protozoan has been available in Europe since 2012 (CaniLeish®, Virbac, France). The aim of the present study was to monitor dogs vaccinated with CaniLeish® to examine the kinetics of the antibody response and the safety and tolerance of CaniLeish®. Dogs vaccinated with CaniLeish® were monitored for 12 months. In follow-up visits at baseline (primovaccination or annual booster) (Visit 1, V1), and 1 (V2), 4 (V3), 8 (V4) and 12 (V5) months later, we examined antibody response kinetics using two serology techniques (IFAT and Speed Leish K™). Tolerance to CaniLeish® and its safety were also monitored. Anti-L. infantum IgG antibodies were determined in 242 dogs (125 dogs after primovaccination (Group P) and 117 dogs after booster vaccination (Group B). In addition, 46, 22 and 19 dogs were followed for 2, 3 and 4 years, respectively. At baseline, 100% of dogs in Group P returned negative IFAT and Speed Leish K™ test results while 9.4% (11/117) in Group B tested IFAT positive though Speed Leish K™ negative. In subsequent visits, seropositivity was detected by IFAT in 31.2% (Group P) and 41% (Group B) of the dogs in V2; 16.8% (Group P) and 10.2% (Group B) in V3; 6.4% (Group P) and 8.5% (Group B) in V4; and 3.2% (Group P) and 5.9% (Group B) in V5. All dogs tested Speed Leish K™ negative except two, in which it was later confirmed by molecular testing that they were not infected. Adverse events that could be associated with the vaccine were detected in 20 out of 314 dogs (6.4%). The good clinical status of all dogs was confirmed in an exhaustive clinical exam and haemato-biochemical profile. The Canileish® vaccine was well-tolerated with exceptions that did not appear to be related to age, sex, race or size of vaccinated dogs. Anti-L. infantum antibodies were detected by IFAT in 31.9-40.3% of the dogs 1 month after vaccination, and these antibodies could still be detected in 3.2% of the dogs 1 year later. This means that veterinarians need to use other tools (eg. PCR) to correctly diagnose seropositive dogs.

Evaluation of 4 merozoite antigens as candidate vaccines against Eimeria tenella infection.

Coccidiosis, caused by parasites of the genus Eimeria, is one of the most widespread and economically detrimental diseases in the global poultry industry. Because the merozoite stage of Eimeria tenella is immunologically vulnerable, motile, and functionally important for the parasites, the proteins expressed in these stages are considered to be potentially immunoprotective antigens, especially the secreted antigens and surface antigens. Here, we detected a previously unidentified MIC2-associated protein (Et-M2AP) from E. tenella and determined its localization. An immunofluorescence assay revealed that Et-M2AP was distributed in the apical part of second generation merozoites and sporozoites. In addition, an expression profile analysis revealed that the transcriptional level of Et-M2AP is significantly higher in the merozoite stage. To assess the potential of Et-M2AP protein as a coccidiosis vaccine, we expressed recombinant Et-M2AP (rEt-M2AP) and compared the immune protective efficacy of rEt-M2AP with 3 surface antigens that are highly expressed by merozoites (rEt-SAG23, rEt-SAG16, and rEt-SAG2 proteins). The immune protective efficacy of these vaccine candidates was assessed based on survival rate, lesion score, BW gain, relative BW gain, and oocyst output. The results show that the survival rate was 90%, which are significantly higher than those in the challenge control group. The BW gain rate was 42% (P < 0.001) in rEt-M2AP-immunized chickens, which are significantly higher than those in the challenge control group and rEt-SAG23, rEt-SAG16, and rEt-SAG2 proteins-immunized chickens. In addition, chickens immunized with rEt-M2AP (88% oocyst output decrease rate, P < 0.001) had the least oocyst output, compared with those immunized with rEt-SAG16 (59.2% oocyst output decrease rate, P < 0.001), rEt-SAG23 (22% oocyst output decrease rate), and rEt-SAG2 (1.36% oocyst output decrease rate). These results demonstrate that rEt-M2AP provided effective protection against challenge with E. tenella, suggesting that rEt-M2AP is a promising candidate antigen gene for development as a coccidiosis vaccine.

Epitope Analysis and Efficacy Evaluation of Phosphatase 2C (PP2C) DNA Vaccine Against Toxoplasma gondii Infection.

Toxoplasma gondii infects almost all warm-blooded animals and negatively affects the health of a wide range of these animals, including humans. Protein phosphatase 2C (PP2C) is a T. gondii protein secreted by rhoptry organelles during host cell invasion. However, very little is known about whether this protein can induce protective immunity against T. gondii. In this study, bioinformatics analysis of PP2C revealed some useful information in the context of anti-toxoplasmosis treatments and vaccine research. In addition, the PP2C gene was amplified, and a eukaryotic expression vector (pEGFP-PP2C) was successfully constructed to express PP2C. Finally, the constructed pEGFP-PP2C was injected into mice to evaluate whether it could induce immunoprotection. Compared with the control groups, we found that immunizations with the pEGFP-PP2C plasmid could elicit specific IgG antibodies and cytokines against T. gondii infection. The survival of mice immunized with the pEGFP-PP2C plasmid was significantly prolonged compared with that of the control group mice. Based on the ability of pEGFP-PP2C to induce specific immune responses against T. gondii, we propose that PP2C merits consideration as a potential vaccine candidate against toxoplasmosis.

Immune protection provided by a precocious line trivalent vaccine against rabbit Eimeria.

A pilot study was conducted to evaluate the pathogenicity and immunogenicity of vaccinated rabbits with different doses of oocysts (5 × 102, 1 × 103, 1 × 104, and 5 × 104) of a precocious line, including Eimeria magna, E. intestinalis or E. media following the challenge with their corresponding parent strains. Our results showed that each precocious line had weak pathogenicity but good immunogenicity in terms of clinical symptoms, average daily weight gains (ADGs), and oocyst outputs. Therefore, a precocious line trivalent vaccine, including E. magna, E. intestinalis, and E. media was formulated. A total of sixty 40-day-old coccidia-free rabbits were allocated to ten treatments with a 2 × 3 factorial arrangement that included 2 vaccination doses (5 × 102 or 1 × 103 oocysts of the precocious line). Groups I to Ⅷ and Unimmunized Challenged Control group were challenged with mixed oocysts of their corresponding parent strains (1 × 104 oocysts of each parent strain) 14 days after vaccination. No clinical symptoms were observed in the immunized groups after vaccination. Average daily weight gains (ADGs) were similar to those of unimmunized unchallenged controls (P > 0.05) after vaccination or after challenge. Oocyst outputs in the vaccinated challenged groups were significantly different from those of unimmunized challenged controls (P < 0.01) after challenge. These results indicated that the trivalent vaccine could provide immune protection against coccidiosis and therefore, it could be used as a candidate vaccine.

Low Levels of Polymorphisms and Negative Selection in Plasmodum knowlesi Merozoite Surface Protein 8 in Malaysian Isolates.

Human infections due to the monkey malaria parasite Plasmodium knowlesi is increasingly being reported from most Southeast Asian countries specifically Malaysia. The parasite causes severe and fatal malaria thus there is a need for urgent measures for its control. In this study, the level of polymorphisms, haplotypes and natural selection of full-length pkmsp8 in 37 clinical samples from Malaysian Borneo along with 6 lab-adapted strains were investigated. Low levels of polymorphism were observed across the full-length gene, the double epidermal growth factor (EGF) domains were mostly conserved, and non-synonymous substitutions were absent. Evidence of strong negative selection pressure in the non-EGF regions were found indicating functional constrains acting at different domains. Phylogenetic haplotype network analysis identified shared haplotypes and indicated geographical clustering of samples originating from Peninsular Malaysia and Malaysian Borneo. This is the first study to genetically characterize the full-length msp8 gene from clinical isolates of P. knowlesi from Malaysia; however, further functional characterization would be useful for future rational vaccine design.

Babesiosis Vaccines: Lessons Learned, Challenges Ahead, and Future Glimpses.

The incidence and prevalence of babesiosis in animals and humans is increasing, yet prevention, control, or treatment measures remain limited and ineffective. Despite a growing body of new knowledge of the biology, pathogenicity, and virulence of Babesia parasites, there is still no well-defined, adequately effective and easily deployable vaccine. While numerous published studies suggest that the development of such anti-Babesia vaccines should be feasible, many others identify significant challenges that need to be overcome in order to succeed. Here, we review historic and recent attempts in babesiosis vaccine discovery to avoid past pitfalls, learn new lessons, and provide a roadmap to guide the development of next-generation babesiosis vaccines.

Immuno-efficacy of DNA vaccines encoding PLP1 and ROP18 against experimental Toxoplasma gondii infection in mice.

We constructed a new plasmid pIRESneo/ROP18/PLP1 that was injected intramuscularly into Kunming mice to evaluate its immune efficacy. The immunized mice exhibited significantly increased serum IgG2a levels, lymphocyte counts and Th1-type cytokine (IL-2, IL-12 and IFN-γ) levels. Moreover, the immunized mice exhibited longer survival times (44.7 ± 2.1 days for ROP18/PLP1 and 47.2 ± 2.9 days for ROP18/PLP1 + IL-18) and lower brain cyst burden (68.9% for ROP18/PLP1 and 72.4% for ROP18/PLP1 + IL-18) than control mice after T. gondii challenge. Our results demonstrate that the multiple-gene DNA vaccine including both ROP18 and PLP1 elicits greater protection against T. gondii challenge and stronger immunogenicity than single-gene vaccines.

Vaccine potential of antigen cocktails composed of recombinant Toxoplasma gondii TgPI-1, ROP2 and GRA4 proteins against chronic toxoplasmosis in C3H mice.

The development of an effective and safe vaccine to prevent Toxoplasma gondii infection is an important aim due to the great clinical and economic impact of this parasitosis. We have previously demonstrated that immunization with the serine protease inhibitor-1 (TgPI-1) confers partial protection to C3H/HeN and C57BL/6 mice. In order to improve the level of protection, in this work, we combined this novel antigen with ROP2 and/or GRA4 recombinant proteins (rTgPI-1+rROP2, rTgPI-1+rGRA4, rTgPI-1+rROP2+rGRA4) to explore the best combination against chronic toxoplasmosis in C3H/HeN mice. All tested vaccine formulations, administered following a homologous prime-boost protocol that combines intradermal and intranasal routes, conferred partial protection as measured by the reduction of brain cyst burden following oral challenge with tissue cysts of Me49 T. gondii strain. The highest level of protection was achieved by the mixture of rTgPI-1 and rROP2 proteins with an average parasite burden reduction of 50% compared to the unvaccinated control group. The vaccine-induced protective effect was related to the elicitation of systemic cellular and humoral immune responses that included antigen-specific spleen cell proliferation, the release of Th1/Th2 cytokines, and the generation of antigen-specific antibodies in serum. Additionally, mucosal immune responses were also induced, characterized by secretion of antigen-specific IgA antibodies in intestinal lavages and specific mesenteric lymph node cell proliferation. Our results demonstrate that rTgPI-1+rROP2 antigens seem a promising mixture to be combined with other immunogenic proteins in a multiantigenic vaccine formulation against toxoplasmosis.

Are Eimeria Genetically Diverse, and Does It Matter?

Eimeria pose a risk to all livestock species as a cause of coccidiosis, reducing productivity and compromising animal welfare. Pressure to reduce drug use in the food chain makes the development of cost-effective vaccines against Eimeria essential. For novel vaccines to be successful, understanding genetic and antigenic diversity in field populations is key. Eimeria species that infect chickens are most significant, with Eimeria tenella among the best studied and most economically important. Genome-wide single nucleotide polymorphism (SNP)-based haplotyping has been used to determine population structure, genotype distribution, and potential for cross-fertilization between E. tenella strains. Here, we discuss recent developments in our understanding of diversity for Eimeria in relation to its specialized life cycle, distribution across the globe, and the challenges posed to vaccine development.

Protective efficacy of pVAX-RON5p against acute and chronic infections of Toxoplasma gondii in BALB/c mice.

Toxoplasma gondii can infect all the warm-blooded animals and humans and causes serious diseases especially in immuno-compromised patients and pregnant women. Rhoptry neck proteins (RONs) play an important role in the formation of moving junction, which mediates the invasion of this parasite. A recombinant plasmid pVAX-RON5p, which can express part of RON5 protein in the eukaryocyte, was generated and used to immune BALB/c mice for evaluating the protective efficacy against the acute and chronic infections of T. gondii. Both humoral and cellular immune responses were evoked in mice by pVAX-RON5p immunization, and a slightly prolonged survival period was detected in the immunized group (7.6 ± 3.31 days) compared to the blank control (4.9 ± 0.32 days) after acute T. gondii infection (P < 0.05). For chronic infection of T. gondii, the number of cysts in the brain of pVAX-RON5p-immunized mice decreased 25.8% compared to blank control (P < 0.05). Our data suggested that RON5p DNA vaccine can induce partial protective immunity against acute and chronic T. gondii infections.

Identification and immunogenicity of microneme protein 2 (EbMIC2) of Eimeria brunetti.

There have been only a few antigen genes of Eimeria brunetti reported up to now. In this study, the gene encoding the microneme protein 2 (EbMIC2) was isolated from oocysts of E. brunetti by RT-PCR and the immunogenicity of recombinant EbMIC2 was observed. The EbMIC2 was cloned into vector pMD19-T for sequencing. The sequence was compared with the published EbMIC2 gene from GenBank revealed homology of the nucleotide sequence and amino acids sequence were 99.43 and 98.63%, respectively. The correct recombinant pMD-EbMIC2 plasmid was inserted into the pET-28a (+) expressing vector and transformed into competent Escherichia coli BL21 cells for expression. The expressed product was analyzed using SDS-PAGE and Western-blot. The results indicated that the recombinant EbMIC2 protein was recognized strongly by serum from naturally infected chicken with E. brunetti. Rat rcEbMIC2 antisera bound to bands of about 36 kDa in the somatic extract of E. brunetti sporozoites. The recombinant plasmid pVAX1-EbMIC2 was constructed and then the efficacies of recombinant plasmid and recombinant protein were evaluated. The results of IgG antibody level and cytokines concentration suggested that recombinant EbMIC2 could increase the IgG antibody level and induce the expressions of cytokines. Animal challenge experiments demonstrated that the recombinant EbMIC2 protein and recombinant plasmid pVAX1-EbMIC2 could significantly increase the average body weight gains, decrease the mean lesion scores and the oocyst outputs of the immunized chickens and presented high anti-coccidial index. All results suggested that EbMIC2 could become an effective candidate for the development of new vaccine against E. brunetti infection.

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.

Approaches for the vaccination and treatment of Neospora caninum infections in mice and ruminant models.

Neospora caninum is a leading cause of abortion in cattle, and is thus an important veterinary health problem of high economic significance. Vaccination has been considered a viable strategy to prevent bovine neosporosis. Different approaches have been investigated, and to date the most promising results have been achieved with live-attenuated vaccines. Subunit vaccines have also been studied, and most of them represented components that are functionally involved in (i) the physical interaction between the parasite and its host cell during invasion or (ii) tachyzoite-to-bradyzoite stage conversion. Drugs have been considered as an option to limit the effects of vertical transmission of N. caninum. Promising results with a small panel of compounds in small laboratory animal models indicate the potential value of a chemotherapeutical approach for the prevention of neosporosis in ruminants. For both, vaccines and drugs, the key for success in preventing vertical transmission lies in the application of bioactive compounds that limit parasite proliferation and dissemination, without endangering the developing fetus not only during an exogenous acute infection but also during recrudescence of a chronic infection. In this review, the current status of vaccine and drug development is presented and novel strategies against neosporosis are discussed.

Immunization with Neospora caninum profilin induces limited protection and a regulatory T-cell response in mice.

Profilins are actin-binding proteins that regulate the polymerization of actin filaments. In apicomplexan parasites, they are essential for invasion. Profilins also trigger the immune response of the host by activating TLRs on dendritic cells (DCs), inducing the production of pro-inflammatory cytokines. In this study we characterized for the first time the immune response and protection elicited by a vaccine based on Neospora caninum profilin in mice. Groups of eight BALB/c mice received either two doses of a recombinant N. caninum profilin expressed in Escherichia coli. (rNcPRO) or PBS, both formulated with an aqueous soy-based adjuvant enriched in TLR-agonists. Specific anti-profilin antibodies were detected in rNcPRO-vaccinated animals, mainly IgM and IgG3, which were consumed after infection. Splenocytes from rNcPRO-immunized animals proliferated after an in vitro stimulation with rNcPRO before and after challenge. An impairment of the cellular response was observed in NcPRO vaccinated and infected mice following an in vitro stimulation with native antigens of N. caninum, related to an increase in the percentage of CD4+CD25+FoxP3+. Two out of five rNcPRO-vaccinated challenged mice were protected; they were negative for parasite DNA in the brain and showed no histopathological lesions, which were found in all PBS-vaccinated animals. As a whole, our results provide evidence of a regulatory response elicited by immunization with rNcPRO, and suggest a role of profilin in the modulation and/or evasion of immune responses against N. caninum.

Cell mediated immune responses in the placenta following challenge of vaccinated pregnant heifers with Neospora caninum.

The aim of the present study was to investigate and correlate the cell-mediated immune response and pathological changes at the maternal-fetal interface of Neospora-challenged pregnant cattle previously immunized with live and inactivated experimental vaccines. Pregnant heifers naïve to Neospora caninum were divided in 5 groups of 4 animals, each one immunized before mating: Group A heifers were intravenously (iv) immunized with 6.25 × 10(7) live tachyzoites of the NC-6 strain; group B heifers were immunized twice subcutaneously (sc) 3 weeks apart with native antigen extract of the NC-6 strain formulated with ISCOMs; group C heifers were sc immunized twice 3 weeks apart with three recombinant proteins (rNcSAG1, rNcHSP20, rNcGRA7) of the NC-1 strain formulated with ISCOMs; group D heifers were sc injected with sterile phosphate-buffered saline (PBS) and group E heifers received sc ISCOM-matrix (ISCOMs without antigen). All groups were iv-challenged with 4.7 × 10(7) NC-1 tachyzoites at 70 days of gestation. Heifers were culled at day 104 of gestation and placentomes were examined to evaluate lesions and local cellular immune responses using histopathology, immunohistochemistry and real time-PCR. Immunohistochemistry was performed using bovine leucocyte specific antibodies. Cytokine expression and levels (IFN-γ, IL-4, IL-10, IL-12 and TNF-α) were measured using real-time reverse transcription-PCR and ELISA, respectively. Minimal inflammation was observed in group A placentomes; while placentomes from group B, C, D and E had moderate to severe infiltration with CD3(+), CD4(+), γδ-T cells, CD8(+) cells and macrophages being more numerous in groups B and E placentomes, when compared with groups C and D (P<0.001). Cytokine levels were significantly increased in the caruncles of animals of groups B and C in comparison with the other animal groups (P < 0.001). The results from this study showed that the strongest cellular immune responses were observed in the placentomes of animals that were immunized with inactivated vaccines (groups B and C) and in the placentomes of animals that were sc-sham-inoculated (groups D and E). On the other hand, animals that were immunized with live tachyzoites showed a milder immune cell infiltration to the placenta possibly due to the existence of a protective systemic maternal immune response that helped to minimize N. caninum infection at the maternal-fetal interface.

Babesia divergens apical membrane antigen-1 (BdAMA-1): A poorly polymorphic protein that induces a weak and late immune response.

Babesiosis is an important veterinary and zoonotic tick borne disease caused by the hemoprotozoan Babesia spp. which infects red blood cell of its vertebrate host. In order to control the infection, vaccination that targets molecules involved in the invasion process of red blood cells could provide a good alternative to chemotherapy. Among these molecules, Apical Membrane Antigen-1 (AMA-1) has been described as an excellent vaccine candidate in Plasmodium spp. In this paper, we have investigated AMA-1 of Babesia divergens (BdAMA-1) as vaccine candidate by evaluating its polymorphism and by studying the humoral response against BdAMA-1 of sheep experimentally infected with B. divergens. Polymorphism of BdAMA-1 was investigated by sequencing the corresponding gene of 9 B. divergens isolates from different geographical areas in France. Two Bdama-1 haplotypes (A and B) could be defined based on 2 non-synonymous point mutations. In silico prediction of linear epitopes revealed that the antigenicity of the 2 haplotypes is very similar. Antibody production against the extracellular domain of BdAMA-1 is weak and late, between 1 and 5 months after the inoculation of parasites. Both IgG1 and IgG2 are components of the anti-BdAMA-1 response. These results indicate that while BdAMA-1 may not be an immuno-dominant antigen, it could induce a mixed type 1 and type 2 immune response. In light of these results, the potential of BdAMA-1 as vaccine candidate is discussed.

A vaccine formulation combining rhoptry proteins NcROP40 and NcROP2 improves pup survival in a pregnant mouse model of neosporosis.

Currently there are no effective vaccines for the control of bovine neosporosis. During the last years several subunit vaccines based on immunodominant antigens and other proteins involved in adhesion, invasion and intracellular proliferation of Neospora caninum have been evaluated as targets for vaccine development in experimental mouse infection models. Among them, the rhoptry antigen NcROP2 and the immunodominant NcGRA7 protein have been assessed with varying results. Recent studies have shown that another rhoptry component, NcROP40, and NcNTPase, a putative dense granule antigen, exhibit higher expression levels in tachyzoites of virulent N. caninum isolates, suggesting that these could be potential vaccine candidates to limit the effects of infection. In the present work, the safety and efficacy of these recombinant antigens formulated in Quil-A adjuvant as monovalent vaccines or pair-wise combinations (rNcROP40+rNcROP2 and rNcGRA7+rNcNTPase) were evaluated in a pregnant mouse model of neosporosis. All the vaccine formulations elicited a specific immune response against their respective native proteins after immunization. Mice vaccinated with rNcROP40 and rNcROP2 alone or in combination produced the highest levels of IFN-γ and exhibited low parasite burdens and low IgG antibody levels after the challenge. In addition, most of the vaccine formulations were able to increase the median survival time in the offspring. However, pup survival only ensued in the groups vaccinated with rNcROP40+rNcROP2 (16.2%) and rNcROP2 (6.3%). Interestingly, vertical transmission was not observed in those survivor pups immunized with rNcROP40+rNcROP2, as shown by PCR analyses. These results show a partial protection against N. caninum infection after vaccination with rNcROP40+rNcROP2, suggesting a synergistic effect of the two recombinant rhoptry antigens.

Gene-therapy for malaria prevention.

The limited number of tools for malaria prevention and the inability to eradicate the disease have required large investments in vaccine development, as vaccines have been the only foreseeable type of immunoprophylaxis against malaria. An alternative strategy named vectored immunoprophylaxis (VIP) now would allow genetically transduced host cells to assemble and secrete antibodies that neutralize the infectivity of the malaria parasite and prevent disease.