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A recombinant Pseudorabies virus expressing TgSAG1 protects against challenge with the virulent Toxoplasma gondii RH strain and pseudorabies in BALB/c mice
Abstract
The major immunodominant surface antigen 1 (TgSAG1) of invasive tachyzoites is a vaccine candidate antigen for Toxoplasma gondii. In this study, we developed a recombinant pseudorabies virus (PRV) expressing TgSAG1 (rPRV/SAG1) based on the PRV vaccine strain Bartha K-61 by homologous recombination, in which partial PK and gG genes were deleted. The growth assay of rPRV/SAG1 showed that the recombinant virus can replicate in vitro as efficiently as PRV Bartha K-61, demonstrating that insertion of the TgSAG1 gene in the PK and gG locus of PRV does not affect the replication of PRV. All mice vaccinated with rPRV/SAG1 developed a high level of specific antibody responses against T. gondii lysate antigen (TLA), a strong increase of the splenocyte proliferative response, and significant levels of IFN-gamma and IL-2 production. And the immunization of mice with rPRV/SAG1 elicited strong cytotoxic T lymphocyte (CTL) responses in vitro. These results demonstrate that rPRV/SAG1 could induce significant humoral and cellular Th1 immune responses. Moreover, rPVR/SAG1 immunization induced partial protection (60%) against a lethal challenge with the highly virulent T. gondii RH strain, and neutralizing antibodies against PRV in a BALB/c mouse model. These results suggest that expression of protective antigens of T. gondii in PRV Bartha K-61 is a novel approach towards the development of a vaccine against both animal toxoplasmosis and pseudorabies.
... Tachyzoites were harvested from the peritoneal cavity of the infected BALB/c mice days after infection and used to give a lethal challenge to immunize the mice. A total lysate antigen was prepared from T. gondii tachyzoites (RH) as described previously (Liu et al., 2008). ...
... Blood samples collected on day 0, day 21 and day 42 were analyzed to determine the T. gondii-specific humoral immune response. An IgG titer against the T. gondii TLA was determined by ELISA as previously described (Liu et al., 2008). ...
... Splenocyte suspensions were prepared from the BALB/c mice immunized with the above-mentioned antigens or PBS on day 42 after vaccination as described elsewhere (Liu et al., 2008), and a lymphocyte proliferation assay was performed as previously described (Liu et al., 2008). In brief, 2 × 10 6 cells/well were cultured in triplicate in 96well plates containing RPMI-1640 medium. ...
... Therefore, T. gondii is of veterinary and medical importance. A vaccine against human infection with T. gondii is not available, but an effective vaccine preventing infection in animals used for human consumption would block the main transmission route to humans (Girish, 2003; Liu et al., 2008). Thus, development of an effective and safe vaccine for controlling T. gondii infection in those animals is an important goal for scientists worldwide. ...
... Thus, development of an effective and safe vaccine for controlling T. gondii infection in those animals is an important goal for scientists worldwide. So far, only a live attenuated vaccine has been available for veterinary use for several years in some countries, but it is expensive, causes side effects, and has the possibility to revert to a pathogenic strain (Mateus-Pinilla et al., 1999; Liu et al., 2008). In order to overcome these problems, subunit, recombinant and DNA vaccines were proposed recently (Bout et al., 2002). ...
... Thereafter, the contents of all wells were discarded, and 100 ml dimethy sulfoxide (DMSO) was added to every well until the deposit was diluted, and the plates were read at 570 nm by a BioRad ELISA reader. Lymphocyte proliferative responses were quantitated by stimulation index (SI), calculated as the ratio of OD 570 nm of stimulated cells to OD 570 nm of unstimulated cells (Liu et al., 2008). ...
To evaluate the protective efficiency of a suicidal DNA vaccine against protozoal parasite Toxoplasma gondii, the microneme protein 3 (MIC3) gene was cloned into suicidal vector pSCA1 and conventional DNA vaccine vector pcDNA3.1+ respectively, their protection against T. gondii challenge were assessed in this study. The recombinant plasmids named pSCA/MIC3 and pcDNA/MIC3 were transfected into BHK-21 cells. The expression of MIC3 in BHK-21 cells was confirmed by RT-PCR and indirect immunofluorescence test. Then BALB/c mice were immunized with pSCA/MIC3 or pcDNA/MIC3. Anti-Tg-MIC3 antibodies were detected by indirect ELISA and the cell immune response were examined by lymphocyte proliferation assay and real time RT-PCR. The results showed that the titre of anti-Tg-MIC3 antibodies, stimulation index (SI) of lymphocyte proliferation response and IFN-gamma expression level induced by pSCA/MIC3 and pcDNA/MIC3 were significantly higher than controls (P<0.05), whereas IL-4 expression level in BALB/c mice immunized with either pSCA/MIC3 or pcDNA/MIC3 was lower than that in control group. After a lethal challenge against T. gondii, survival time of the mice immunized with this suicidal DNA vaccine pSCA/MIC3 and conventional DNA vaccine pcDNA/MIC3 were significantly prolonged in comparison with the control groups (P<0.05), but the difference of protective immune response in BALB/c mice between pSCA/MIC3 and pcDNA/MIC3 was not statistically significant (P>0.05). The findings demonstrated that like conventional DNA vaccine pcDNA/MIC3, suicidal DNA vaccine pSCA/MIC3 also provided favourable efficacy, but it could improve the biosafety of conventional vaccines. This result suggested that suicidal DNA vaccine pSCA/MIC3 is a potential candidate vaccine against toxoplasmosis.
... The eukaryotic expression plasmids pcDNA/TgSAG1 and pVAX/ mIL-18 have been constructed previously in our laboratory ( Liu et al., 2008;Wei et al., 2008). Plasmids pcDNA/TgSAG1 and pVAX/mIL-18 were sequenced to ensure correct insertions. ...
... Lymphocyte proliferation assay was performed as described ( Liu et al., 2008). Briefly, splenocyte suspensions were prepared from 3 mice per group at day 21 post-vaccination, and 2 Â 10 5 cells/well were cultured in 96-well plates in triplicate at in RPMI-1640, and stimulated with 10 lg/ml of TLA, or unstimulated, respectively, for 72 h at 37 °C in 5% CO 2. Results were expressed as the stimulation index (SI), which is the ratio of OD 570 of stimulated cells to OD 570 of unstimulated ones. ...
More effective vaccines against Toxoplasma gondii may contribute to the control of this pathogen that has major veterinary and public health significance. In this study, two recombinant plasmids pcDNA/TgSAG1 and pVAX/mIL-18 containing T. gondii SAG1 (TgSAG1) and murine cytokine interleukin-18 (IL-18) were evaluated for their ability to protect mice against T. gondii challenge. Mice were given two intramuscular immunizations 3 weeks apart, and challenged with T. gondii 3 weeks later. All animals vaccinated with pcDNA/TgSAG1 alone or with pVAX/mIL-18 developed specific anti-TLA (T. gondii lysate antigen) antibodies and specific lymphocyte proliferative responses. Co-injection of pVAX/mIL-18 significantly increased the production of IFN-gamma and IL-2. Further, challenge experiments showed that co-immunization with pVAX/mIL-18 significantly (P<0.05) increased the survival rate (60%), compared with pcDNA/TgSAG1 alone (40%). Therefore, codelivery of the IL-18-secreting plasmid potentiates the induction and maintenance of the type 1 helper T-cell immune response and may be a potent strategy for enhancing the protective efficacy of vaccines against T. gondii.
... Strikingly, T. gondii (type I-RH strain) modifies gene expression in mouse spleen cells. Gene expression was positively correlated with immune responses in the mice models [101]. ...
Toxoplasma gondii (T. gondii) is an obligate intracellular parasite. During the parasitic invasion, T. gondii creates a parasitophorous vacuole, which enables the modulation of cell functions, allowing its replication and host infection. It has effective strategies to escape the immune response and reach privileged immune sites and remain inactive in a controlled environment in tissue cysts. This current review presents the factors that affect host cells and the parasite, as well as changes in the immune system during host cell infection. The secretory organelles of T. gondii (dense granules, micronemes, and rhoptries) are responsible for these processes. They are involved with proteins secreted by micronemes and rhoptries (MIC, AMA, and RONs) that mediate the recognition and entry into host cells. Effector proteins (ROP and GRA) that modify the STAT signal or GTPases in immune cells determine their toxicity. Interference byhost autonomous cells during parasitic infection, gene expression, and production of microbicidal molecules such as reactive oxygen species (ROS) and nitric oxide (NO), result in the regulation of cell death. The high level of complexity in host cell mechanisms prevents cell death in its various pathways. Many of these abilities play an important role in escaping host immune responses, particularly by manipulating the expression of genes involved in apoptosis, necrosis, autophagy, and inflammation. Here we present recent works that define the mechanisms by which T. gondii interacts with these processes in infected host cells.
... An antigen SAGI, expressed by tachyzoite helps in diagnostic and vaccine preparation purpose. It regulate both humoral and cellular immune response (15) . Goats are mostly raised for milk and meat purpose and some peoples used unpasteurized milk which leads to toxoplasma infection (16). ...
Background: Toxoplasma has worldwide distribution and every 3 rd animal in the world is affected with toxoplasma gondii. Toxoplasma is mainly transmitted by cat, and also can be transmitted by some warm-blooded intermediate hosts. Objective: To determine the relationship of Sero-positivity against Toxoplasma gondii antibodies with age, body weight, sex, abortion rate and breeds of goat (Caprine) Methodology: Study samples were collected from all three tehsils of district D.G.Khan, Punjab for a period of six months. A total of 410 goats were included in our study. A dichotomous and multiple-choice questionnaire was designed to quantify and identify different determinants of variation among prevalence. 5ml of blood was collected from jugular vein of all the selected goats and transported to University of Agriculture, Faisalabad (UAF). latex agglutination test was used to confirm the presence of antibodies to T.Gondii. Results: Overall prevalence of toxoplasma gondii in goat is 9.75%. Our results showed that prevalence increased with age, goat 3 year or above were found 38.7% positive and 2-2.5-year-old were 8.79% and 0 to 1.5-year-old were found 0% positive. By breed, prevalence in teddy breed was 11.55% as compared to Nachi breed 6.92%. Prevalence in female was 9.94% (higher) as compared to 8.62% in male. Higher body weight goats 47kg or above and 37-47 body weight were found positive as compared to 26-36 kg and 15-25 kg body weight. The goat samples with a toxoplasmosis abortion in history had also higher prevalence Conclusion: Our study concludes that prevalence of toxoplasma gondii is high (9.75%) in goats of Dera Ghazi Khan and it increases with increased age, teddy breed, female gender, high body weight and abortion.
... GRA7 was also used in combination with other antigens, leading to increased survival rates (Jongert et al., 2007(Jongert et al., , 2008aRosenberg et al., 2009). Further studies investigated SAG1, MIC3, and GRA4 as antigens (Chen et al., 2002(Chen et al., , 2009Couper et al., 2003;Ismael et al., 2003Ismael et al., , 2009Martin et al., 2004;Yang et al., 2005;Zhang et al., 2007a;Liu et al., 2008;Fang et al., 2009Fang et al., , 2010Shang et al., 2009). Many of these studies showed promising results at high doses or against non-fetal acquired parasites (Couper et al., 2003;Fachado et al., 2003). ...
Leptodactylus fuscus is a small-sized species widely distributed across South America. However, so far, no works have been addressed to analyze if biotic and abiotic factors may influence the parasite community in this species. Thus, the present work aims to describe the composition and structure of the parasite community and evaluate if biotic and environmental factors correlated to the distribution of the parasite community for this host. We collected 36 L. fuscus specimens from February 2018 to January 2019. The hosts were necropsied, and parasites were collected and identified. To test the effect of environmental variables (temperature, humidity, and precipitation) and morphological characters of the host (snout-vent length and body weight) on helminths of L. fuscus we used a multivariate distance matrix regression (MDMR). We found 10 helminth taxa: Aplectana sp., Aplectana membranosa, Cosmocercidae larvae, Mesocoelium sp., Mesocoelium aff. monas, Mesocoelium aff. sociale, Oswaldocruzia sp., Oxyascaris sp., Ortleppascaris sp. larvae, and Schrankiana formosula. In our study, the helminth community showed an aggregate pattern, and we did not observe a statistically significant correlation of body size and mass of the hosts regarding parasite abundance and richness. Thus, this study represents the first report of M. sociale, A. membranosa, and Ortleppascaris sp. larvae for L. fuscus and new locality reports. We conclude that there is no correlation between variables analyzed and the structure and composition of the parasite community of L. fuscus.
... GRA7 was also used in combination with other antigens, leading to increased survival rates (Jongert et al., 2007(Jongert et al., , 2008aRosenberg et al., 2009). Further studies investigated SAG1, MIC3, and GRA4 as antigens (Chen et al., 2002(Chen et al., , 2009Couper et al., 2003;Ismael et al., 2003Ismael et al., , 2009Martin et al., 2004;Yang et al., 2005;Zhang et al., 2007a;Liu et al., 2008;Fang et al., 2009Fang et al., , 2010Shang et al., 2009). Many of these studies showed promising results at high doses or against non-fetal acquired parasites (Couper et al., 2003;Fachado et al., 2003). ...
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.
... This vaccine was very efficacious as it could raise the protective immunity in mice due to the effect of the multifunctional cytokine that increases both innate and acquired immunity, and potentiates Th1 and Th2 immune responses ( Gracie et al., 2003;Zheng et al., 2015). PRV protects against various infectious agents causing diseases in swine and also in mice as foot and mouth disease virus ( Kamel et al., 2019;Li et al., 2008), porcine circovirus type 2 ( Song et al., 2007;Zheng et al., 2015), hog cholera (van Zijl et al., 1991), japanese encephalitis ( Qian et al., 2015;Xu et al., 2004), transmissible gastroenteritis virus ( Yin et al., 2007), Brucella melitensis in mice ( Yao et al., 2015), rabies virus ( Yuan et al., 2008), T. gondii ( Liu et al., 2008), porcine parvovirus (Chen et al., 2011), H1N1 influenza A virus of swine source (Klingbeil et al., 2015(Klingbeil et al., , 2014), H3N2 subtype swine influenza virus (SIV) ( Tian et al., 2006), Schistosoma japonicum ( Wei et al., 2010), classical swine fever virus ( Wang et al., 2015) and porcine reproductive and respiratory syndrome virus (PRRS) ( Jiang et al., 2007). Their immunization efficacy and responses are listed in Table 1. ...
Throughout the past few decades, numerous viral species have been generated as vaccine vectors. Every viral vector has its own distinct characteristics. For example, the family herpesviridae encompasses several viruses that have medical and veterinary importance. Attenuated herpesviruses are developed as vectors to convey heterologous immunogens targeting several serious and crucial pathogens. Some of these vectors have already been licensed for use in the veterinary field. One of their prominent features is their capability to accommodate large amount of foreign DNA, and to stimulate both cell-mediated and humoral immune responses. A better understanding of vector-host interaction builds up a robust foundation for the future development of herpesviruses-based vectors. At the time, many molecular tools are applied to enable the generation of herpesvirus-based recombinant vaccine vectors such as BAC technology, homologous and two-step en passant mutagenesis, codon optimization, and the CRISPR/Cas9 system. This review article highlights the most important techniques applied in constructing recombinant herpesviruses vectors, advantages and disadvantages of each recombinant herpesvirus vector, and the most recent research regarding their use to control major animal diseases.
... Many types of research have focused on the selection of immunoreactive and protective antigens, which could be candidate for vaccine against toxoplasmosis. Some of these proteins used in the combination of experimental vaccines are dense granule proteins GRA1 (29,30), GRA2 (31), GRA4 (32), GRA6 (33), rhoptry proteins ROP2 (34), ROP16 (35), ROP18 (36,37), micronemal proteins MIC3 (38,39), MIC6 (40), and surface antigen of tachyzoites SAG1 (41,42), SAG2 (43), SAG3 (44). ...
Background:
Toxoplasmosis is a parasitic disease caused by the intracellular protozoan parasite, Toxoplasma gondii, which can infect humans and warm-blooded animals. This infection can lead to still birth and abortion among some susceptible hosts especially sheep and human in pregnancy. Development of a vaccine against T. gondii infection is very important-especially for use in immunocompromised patients, pregnant women, and sheep. Different antigens of T. gondii can be potential candidates for immunization. The aims of this study were to identify the immunodominant and antigenic proteins of T. gondii in sheep and man.
Methods:
Tachyzoites' proteins were separated by two-dimensional polyacrylamide gel electrophoresis (2-DE), and subjected to western blot analysis probed with T. gondii positive sera of sheep and human (Biotechnology Department of Pasteur Institute of Tehran, Iran, from April 2016 to March 2017). Finally, the immunoreactive proteins were identified by mass spectrometry (MALDI-TOF/MS and MS/MS) technique.
Results:
Five immunoreactive and antigenic proteins were recognized by Toxoplasma positive sera of human and sheep. These identified proteins were Enolase 2, rhoptry protein 4 (ROP4), dense granular protein 14 (GRA14), rhoptry protein 15 (ROP15) and rhoptry protein 9 (ROP9).
Conclusion:
The identified immunodominant proteins have potential to be used as diagnostic antigens and as diagnostic markers of Toxoplasma infection in sheep and human.
... Vector vaccines based on Salmonella typhimurium (Cong et al. 2005), adenovirus (Caetano et al. 2006), and pseudorabies (Liu et al. 2008) expressing the SAG1 have also been used. All of them have shown a high protective response against Toxoplasma infection. ...
Toxoplasmosis is a disease caused by the protozoan parasite Toxoplasma gondii. This parasite is transmitted by ingestion of cysts found in the tissues of infected animals or by ingestion of food or water contaminated with oocysts released in the feces of infected cats. T. gondii is able to infect all warm-blooded animals, including humans. Vaccination against T. gondii is the most efficient and safe method to prevent this infectious disease. Since the portal of entry of T. gondii is the mucosa, an efficient stimulation of the mucosa and an adequate systemic response constitute a priority which could be accomplished by the administration of an oral or nasal vaccine. In this context, plant-based expression systems represent an interesting production platform for oral vaccine development due to their reduced manufacturing costs and high scalability. However, the heterologous expression of parasitic antigens in plants has been poorly explored. Despite the lack of interest aroused by the proteins derived from parasites to be expressed in plants, the results shown in the present chapter are auspicious. The antigens of T. gondii in plants orally administered in low doses and without the use of adjuvants have been shown to be able to induce a protective immune response. In fact, the degree of protection observed is similar to that reported by other authors who assayed these proteins as recombinant proteins or DNA vaccines in combination with adjuvants. Both GRA4 and SAG1 expressed in plants are good candidates for the development of a multi-antigenic vaccine against Toxoplasma. In summary, the expression of T. gondii antigens in plants is a realistic strategy to develop an anti-T. gondii vaccine. The joint expression of SAG1 and GRA4 in plants may provide an excellent opportunity to explore the application of an oral vaccine against toxoplasmosis based on edible plant tissues. © 2014 Springer Science+Business Media New York. All rights reserved.
... A rPRV was constructed expressing SAG1 from the protozoan parasite, T. gondii [57]. The SAG1 protein domain belongs to a group of glycosylphosphatidylinositol (GPI)-linked proteins with SAG1 related sequences that can be found on the surface of the parasite. ...
Pseudorabies virus (PRV) is a double-stranded, DNA-based swine virus with a genome approximating 150 kb in size. PRV has many nonessential genes which can be replaced with genes encoding heterologous antigens but without deleterious effects on virus propagation. Recombinant PRVs expressing both native and foreign antigens are able to stimulate immune responses. In this paper, we review the current status of live attenuated recombinant PRVs and live PRV-based vector vaccines with potential for controlling viral infections in animals.
... Liu et al. developed a recombinant pseudorabies virus (rPRV/SAG1) that expressed TgSAG1. The recombinant pseudorabies virus-based anti-Toxoplasma vaccine induced a strong Th1-type response and provided higher levels of protection against a virulent challenge with T. gondii RH strain in a mouse model [40]. Qu et al. constructed a multiantigenic DNA vaccine that expressed SAG1 and MIC3 antigens carried by attenuated Salmonella typhimurium [41]. ...
... A modified vaccinia ankara vector expressing ROP2 could increase the life span after lethal challenge with T. gondii RH (Roque- Resendiz et al. 2004). Recently, 60% survival was obtained after vaccination with a SAG1 expressing Pseudorabies virus, in mice challenged with T. gondii RH (Liu et al. 2008). Table III shows studies using live, attenuated vectors. ...
Development of vaccines against Toxoplasma gondii infection in humans is of high priority, given the high burden of disease in some areas of the world like South America, and the lack of effective drugs with few adverse effects. Rodent models have been used in research on vaccines against T. gondii over the past decades. However, regardless of the vaccine construct, the vaccines have not been able to induce protective immunity when the organism is challenged with T. gondii, either directly or via a vector. Only a few live, attenuated T. gondii strains used for immunization have been able to confer protective immunity, which is measured by a lack of tissue cysts after challenge. Furthermore, challenge with low virulence strains, especially strains with genotype II, will probably be insufficient to provide protection against the more virulent T. gondii strains, such as those with genotypes I or II, or those genotypes from South America not belonging to genotype I, II or III. Future studies should use animal models besides rodents, and challenges should be performed with at least one genotype II T. gondii and one of the more virulent genotypes. Endpoints like maternal-foetal transmission and prevention of eye disease are important in addition to the traditional endpoint of survival or reduction in numbers of brain cysts after challenge.
Many recent studies have been conducted to find new DNA vaccines based on Toxoplasma gondii antigens. DNA vaccines encoding complex of different antigens showed better immune responses compared to single antigen vaccine. In this study, we constructed a DNA vaccine encoding SAG1, SAG3, MIC4, GRA5, GRA7, AMA1 and BAG1 against T. gondii, and evaluated the immune response it induced in BALB/c mice. For this purposes, thirty BALB/c mice were randomly divided into three groups containing tenmice each. There were two negative control groups (PBSand pVAX1 vector) and one vaccination group (pVAX1-MAF, Multantigenic Fragment). On days 0, 14 and 28, the mice were immunized intramuscularly, and 5 weeks later they were challenged with T. gondii RH strain. The immune responses were evaluated using lymphocyte proliferation assay, T-cell subsets detection, and measurement of antibody and cytokine levels. The results showed that mice immunized with pVAX1-MAF developed high levels of IL-2, IL-12, IgG and IFN- γ as well as CD3⁺CD4⁺ T cells. In addition, the survival time of mice immunized by pVAX1-MAF was longer than that control mice. In conclusion, our results show that the multiple DNA vaccine encodingSAG1, SAG3, mic4, GRA5, GRA7, AMA and BAG1effectively enhanced humoral and cellular immune responses, and prolonged the survival time. Together this would suggest that further investigation may result in a promising candidate vaccine to treat toxoplasmosis.
Pseudorabies virus (PRV) is a double-stranded DNA virus with a genome approximating 150 kb in size. PRV contains many non-essential genes that can be replaced with genes encoding heterogenous antigens without affecting viral propagation. With the ability to induce cellular, humoral and mucosal immune responses in the host, PRV is considered to be an ideal and potential live vector for generation of animal vaccines. In this review, we summarize the advances in attenuated recombinant PRVs and design of PRV-based live vaccines as well as the challenge of vaccine application.
Pseudorabies virus (PRV) is considered as an emerging zoonotic pathogen since its isolation from a human case. Meanwhile, the disease caused by PRV infection has led huge economic losses to Chinese pig industry since 2011. In this study, we constructed a recombinant PRV stably expressing the enhanced green fluorescent protein (EGFP) by homologous recombination technology for evaluating its susceptibility to different human cell lines and screening antiviral compounds. Stably expressed EGFP by this designed rPRVHuN-EGFP virus was confirmed in the infected cells, moreover, the growth kinetics of which was similar to that of wild type strain. Importantly, the application of this rPRV allowed us to easily verify its infectivity in all tested human cell lines, although the infection efficiencies were lower than that in PK15 cells. Meanwhile, the antiviral activities of Harmine and PHA767491 were also conveniently validated in vitro, as directly reflected by the reduced EGFP signals. These results demonstrate that this recombinant PRV virus should be a useful tool for basic virology researches and antiviral agent screening.
Current study deals with a novel multi-epitope vaccine designed in silico and its confirmation experiments for potential efficacy in BALB/c mice. Major histocompatibility complex (MHC)-binding and B-cell binding epitopes of five Toxoplasma antigens (SAG1, ROP16, GRA12, MIC4 and M2AP) were predicted. Selected epitopes were fused together using SAPGTP linker, and antigenicity, allergenicity, physico-chemical features, secondary and tertiary structures and validations were all performed via bioinformatics servers. Then, vaccine construct was cloned into pLEXSY-neo 2.1 vector. After Leishmania tarentolae transfection, live recombinant and wild parasites were subcutaneously injected into 6-8 week female BALB/c mice and immune responses were measured. Results showed that the peptide possessed 282 amino acid residues with average molecular weight of 28.06 kDa. About 90% of the peptide residues were incorporated in favored and allowed regions of the Ramachandran plot. Vaccinated mice showed remarkably elevated levels of specific antibodies (P < 0.05) with predominance of IgG2a production. Also, a Th1 immune response with production of IFN-γ and relatively increased survival rate against intraperitoneal challenge with RH strain was demonstrated in immunized mice than control groups (P < 0.05). Also, very low levels of IL-4 were demonstrated, which showed statistically significant association with controls (P < 0.05). The findings clarified that multi-epitope vaccine expressed in Leishmania tarentolae induced significant immune responses against acute toxoplasmosis.
Toxoplasma gondii is an intracellular parasite that infects a broad range of animal species and humans. As the main surface antigen of the tachyzoite, SAG1 is involved in the process of recognition, adhesion and invasion of host cells. The aim of the current systematic review study is to clarify the latest status of studies in the literature regarding SAG1-associated recombinant proteins or SAG1-associated recombinant DNAs as potential vaccines against toxoplasmosis. Data were systematically collected from six databases including PubMed, Science Direct, Web of Science, Google Scholar, EBSCO and Scopus, up to 1st of January 2019. A total of 87 articles were eligible for inclusion criteria in the current systematic review. The most common antigens used for experimental cocktail vaccines together with SAG1 were ROP2 and SAG2. In addition, the most parasite strains used were RH and ME49. Freund's adjuvant and cholera toxin have been predominantly utilized. Furthermore, regarding the animal models, route and dose of vaccination, challenge methods, measurement of immune responses and cyst burden have been discussed in the text. Most of these experimental vaccines induce immune responses and have a high degree of protection against parasite infections, increase survival rates and duration and reduce cyst burdens. The data demonstrated that SAG1 antigen has a high potential for use as a vaccine and provided a promising approach for protecting humans and animals against toxoplasmosis.
The surface antigen 1 of Toxoplasma gondii (TgSAG1) is a major immunodominant antigen and is widely considered an ideal candidate for the development of an effective recombinant vaccine against toxoplasmosis. Eimeria tenella, an affinis apicomplexan parasite with T. gondii, is a potential vaccine vector carrying exogenous antigens that stimulates specific immune responses. Here, we engineered TgSAG1 into E. tenella and obtained a stably transfected E. tenella line (Et-TgSAG1). We found TgSAG1 localized on the cell surface of Et-TgSAG1, which is similar to its native distribution in T. gondii tachyzoites. We immunized the chickens with Et-TgSAG1 orally and detected TgSAG1-specific immune responses, which partly reduced T. gondii infection. In the mouse model, we immunized the mice with Et-TgSAG1 sporozoites intraperitoneally and challenged them with T. gondii tachyzoites RH strain. We found that the mice immunized with Et-TgSAG1 showed a TgSAG1 specific Th 1-dominant immune response and a prolonged survival time compared with wild-type E. tenella and non-immunized mice. Collectively, our results demonstrated that Et-TgSAG1, utilized as a recombinant vaccine against toxoplasmosis, could be applied in both chickens and mice. Our findings also provide a promising persuasion for the development of transgenic Eimeria as vaccine vectors for use in birds and mammals.
The rabies virus glycoprotein is an efficient vaccine antigen particularly for the oral vaccine of wildlife. Pseudorabies virus contains many non-essential genes and was usually used to construct recombinant vaccines as a viral vector to deliver foreign genes into the target animals. In this study, a recombinant pseudorabies virus rPRV/SRV9G/EGFP, expressing EGFP and rabies virus (SRV9 strain) glycoprotein was constructed by homologous recombination in the UL21 gene. The immunogenicity was evaluated as a vaccine in mice. In this trial, thirty mice were administered with the recombinant vaccine orally. This recombinant virus was demonstrated to be safe for mice by oral administration and could induce neutralizing antibody level above 0.5 IU mL -1 at 3 weeks after the vaccination. The results suggest that rPRV/SRV9G/EGFP was an effective oral vaccine candidate against rabies and UL21 gene could be a target insertion area for foreign genes.
A vaccine capable of protecting against Toxoplasma gondii would have both beneficial medical and veterinary impacts. Successful vaccination of humans would not only reduce mortality and morbidity, but also reduce the financial burden of lifelong care required by those worst affected. A veterinary vaccine would have the dual advantages of increasing livestock productivity while reducing the public health risk associated with eating contaminated meat. Herein we review progress towards these goals using both large animal studies and the murine models of disease. Early approaches were largely empirical and used attenuated organisms, parasite extracts, or defined sub-units based upon the limited genomic data previously available. The recent elucidation of the T. gondii genome, understanding of T. gondii population structures, predicative algorithms for MHC binding peptides, facile manipulation of T. gondii taken together with a wealth of immunological knowledge should significantly promote new vaccine development.
Toxoplasmosis is an opportunistic infection caused by the protozoan parasite Toxoplasma gondii. T. gondii is widespread globally and causes severe diseases in individuals with impaired immune defences as well as congenitally infected infants. The high prevalence rate in some parts of the world such as South America and Africa, coupled with the current drug treatments that trigger hypersensitivity reactions, makes the development of immunotherapeutics intervention a highly important research priority. Immunotherapeutics strategies could either be a vaccine which would confer a pre-emptive immunity to infection, or passive immunization in cases of disease recrudescence or recurrent clinical diseases. As the severity of clinical manifestations is often greater in developing nations, the development of well-tolerated and safe immunotherapeutics becomes not only a scientific pursuit, but a humanitarian enterprise. In the last few years, much progress has been made in vaccine research with new antigens, novel adjuvants, and innovative vaccine delivery such as nanoparticles and antigen encapsulations. A literature search over the past 5 years showed that most experimental studies were focused on DNA vaccination at 52%, followed by protein vaccination which formed 36% of the studies, live attenuated vaccinations at 9%, and heterologous vaccination at 3%; while there were few on passive immunization. Recent progress in studies on vaccination, passive immunization, as well as insights gained from these immunotherapeutics is highlighted in this review.
Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus associated with the development of both lymphoid and epithelial tumors. EBNA1 is the only viral protein expressed in all EBV-associated malignancies and plays important roles in EBV latency. Thus, EBNA1 is thought to be a promising antigen for immunotherapy of all EBV-associated malignancies. This study was undertaken to produce recombinant EBNA1 protein in Pichia pastoris and evaluate its immunogenicity. The truncated EBNA1 (E1ΔGA, codons 390-641) was expressed as a secretory protein with an N-terminal histidine tag in the methylotrophic yeast P. pastoris and purified by Ni-NTA affinity chromatography. The purified proteins were then used as antigens to immunize BALB/c mice for production of polyclonal antibodies. Western blot analysis showed that the polyclonal antibodies specifically recognized the EBNA1 protein in B95-8 cell lysates. The recombinant E1ΔGA also induced strong lymphoproliferative and Th1 cytokine responses in mice. Furthermore, mice immunized with E1ΔGA developed CD4(+) and CD8(+) T cell responses. These findings showed that the yeast-expressed E1ΔGA retained good immunogenicity and might be a promising vaccine candidate against EBV-associated malignancies.
Ascariasis caused by Ascaris is the most common parasite problem in humans and pigs worldwide. No vaccines are available for the prevention of Ascaris infections. In the present study, the gene encoding Ascaris suum enolase (As-enol-1) was amplified, cloned and sequenced. Amino acid sequence alignment indicated that As-enol-1 was highly conserved between different nematodes and shared the highest identity (87%) with enolase from Anisakis simplex s.l. The recombinant pVAX-Enol was successfully expressed in Marc-145 cells. The ability of the pVAX-Enol for inducing immune protective responses against challenge infection with A. suum L3 was evaluated in Kunming mice. The immune response was evaluated by lymphoproliferative assay, cytokine and antibody measurements, and the reduction rate of recovery larvae. The results showed that the mice immunized with pVAX-Enol developed a high level of specific antibody responses against A. suum, a strong lymphoproliferative response, and significant levels of IFN-γ, IL-2, IL-4 and IL-10 production, compared with the other groups immunized with empty plasmid or blank controls, respectively. There was a 61.13% reduction (P<0.05) in larvae recovery compared with that in the blank control group. Our data indicated that A. suum enolase is a potential vaccine candidate against A. suum infection.
Infection with the intracellular protozoan parasite Toxoplasma gondii causes serious public health problems and is of great economic importance worldwide. The rhomboid proteins which are responsible for adhesion and invasion of host cells have been suggested as vaccine candidates against toxoplasmosis. A DNA vaccine (pVAX-ROM1) encoding T. gondii rhomboid protein 1 (TgROM1) gene was constructed and the immune response and protective efficacy of this vaccine against lethal challenge in BALB/c mice were evaluated. The results indicated that specific antibody and lymphocyte proliferative responses were elicited in mice receiving pVAX-ROM1. The production levels of IFN-γ, IL-2, IL-4, and IL-10, as well as the percentage of CD4(+) cells in mice vaccinated with pVAX-ROM1 were significantly increased respectively, compared to controls receiving either pVAX1 alone or PBS. After lethal challenge, the mice immunized with pVAX-ROM1 showed an increased survival time compared with the mice in the controls. Our data suggested that a DNA vaccine pVAX-ROM1 encoding T. gondii rhomboid protein 1 triggered strong humoral and cellular responses, and prolonged survival time against T. gondii infection in BALB/c mice.
Toxoplasma gondii is one of the most common parasitic pathogens in humans and warm-blooded animals, causing toxoplasmosis. One of the efficient ways to control this disease is immunization. In this study, two recombinant pseudorabies virus (PRV) expressing TgSAG1 (rPRV-SAG1) and TgMIC3 (rPRV-MIC3) based on the PRV vaccine strain were developed by homologous recombination and used for immunizing BALB/c mice. Ninety BALB/c mice were randomly divided into five groups including four experimental groups (inoculated twice in 4 weeks interval with PRV TK-/gG-/EGFP+, rPRV-SAG1, rPRV-MIC3, rPRV-SAG1+rPRV-MIC3, respectively) and one control group (inoculated with medium). All mice vaccinated with rPRV developed a high level of specific antibody responses against T. gondii lysate antigen (TLA), a strong increase of the splenocyte proliferative response, and significant levels of IFN-γ and IL-2 production. These results demonstrated that rPRV could induce significant humoral and cellular Th1 immune responses. Moreover, rPRV immunization induced partial protection against a lethal challenge with T. gondii RH strain, and neutralizing antibodies against PRV in a BALB/c mouse model. The mice immunized with the rPRV-SAG1 and rPRV-MIC3 cocktail could develop higher T. gondii-specific IgG antibodies and lymphocyte proliferative responses and conferred more efficient protection against T. gondii challenge. These results suggested that expression of protective antigens of T. gondii in PRV is a novel approach towards the development of a vaccine against both animal pseudorabies and toxoplasmosis.
A DNA vaccine (pVAX1-TgADF) encoding Toxoplasma gondii actin depolymerizing factor (ADF) gene was constructed and the immune response and protective efficacy of this vaccine against homologous challenge in BALB/c mice were evaluated. High titers of specific antibody and increases in the percentage of CD4(+) and CD8(+) T lymphocyte cells were observed from BALB/c mice vaccinated with pVAX1-TgADF (P<0.05), when PBS group was used as control. The survival time of BALB/c mice in pVAX1-TgADF group was longer than those in control groups. The numbers of brain cysts in the experimental BALB/c mice immunized with pVAX1-TgADF reduced significantly compared with those in PBS group (P<0.05), and the rate of reduction could reach to around 42.8%. These results suggested that the DNA vaccine pVAX1-TgADF could generate specific humoral and cellular immune responses, prolong survival times, and reduce brain cysts load against T. gondii infection in BALB/c mice.
Toxoplasma gondii can infect a large variety of domestic and wild animals and human beings, sometimes causing severe pathology. Rhoptries are involved in T. gondii invasion and host cell interaction and have been implicated as important virulence factors. In this study, we constructed a DNA vaccine expressing rhoptry protein 16 (ROP16) of T. gondii and evaluated the immune responses it induced in Kunming mice. The gene sequence encoding ROP16 was inserted into the eukaryotic expression vector pVAX I. We immunized Kunming mice intramuscularly. After immunization, we evaluated the immune response using a lymphoproliferative assay, cytokine and antibody measurements, and the survival times of mice challenged lethally. The results showed that mice immunized with pVAX-ROP16 developed a high level of specific antibody responses against T. gondii ROP16 expressed in Escherichia coli, a strong lymphoproliferative response, and significant levels of gamma interferon (IFN-γ), interleukin-2 (IL-2), IL-4, and IL-10 production compared with results for other mice immunized with either empty plasmid or phosphate-buffered saline, respectively. The results showed that pVAX-ROP16 induces significant humoral and cellular Th1 immune responses. After lethal challenge, the mice immunized with pVAX-ROP16 showed a significantly (P < 0.05) prolonged survival time (21.6 ± 9.9 days) compared with control mice, which died within 7 days of challenge. Our data demonstrate, for the first time, that ROP16 triggers a strong humoral and cellular response against T. gondii and that ROP16 is a promising vaccine candidate against toxoplasmosis, worth further development.
Recombinant pseudorabies virus (PRV) Bartha-K61 vaccine strains expressing Schistosoma japonicum 26kDa glutathione S-transferase (Sj26GST) and fatty acid binding protein (SjFABP), designated as rPRV/Sj26GST, rPRV/SjFABP and rPRV/Sj26GST-SjFABP, were constructed and evaluated for their ability to protect mice and sheep against S. japonicum challenge. Animals were given 2 intramuscular immunizations 3 weeks apart, and challenged with S. japonicum cercariae 4 weeks later. All mice vaccinated with recombinant virus developed specific anti-SWAP (soluble worm antigen preparation) antibody, splenocyte proliferative response and production of IFN-gamma and IL-2. Injection of rPRV/Sj26GST-SjFABP significantly increased levels of antibody, splenocyte proliferative response and production of IFN-gamma, compared with rPRV/Sj26GST and rPRV/SjFABP. These recombinant viruses have been shown to be safe for sheep. Challenge experiments showed worms and egg burdens were significantly reduced in animals immunized with recombinant PRVs. Most importantly, rPRV/Sj26GST-SjFABP dramatically enhanced protection with worm reduction and hepatic reduction of 39.3% and 45.5% respectively in mice, and 48.5% and 51.2% in sheep, while rPRV/Sj26GST and rPRV/SjFABP provided corresponding protection of only up to 23.7% and 27.2% in mice, and 29.0% and 35.5% in sheep. These results indicate that the multivalent vaccine for S. japonicum can produce significant specific immunity and protection, and that PRV Bartha-K61 is an effective live vector for an animal schistosomiasis japonica vaccine.
Toxoplasma gondii is a protozoan parasite causing toxoplasmosis to almost one-third of population all over the world. One of the most efficient ways to control this disease is immunization. However, so far, there is no effective vaccine available against this pathogen. Recently, a baculovirus pseudotype with vesicular stomatitis virus G protein (Bac-VSV-G) was found to efficiently transduce and express transgenes on mammalian cells, so it was considered as an excellent expressing vector. In this study, the value of Bac-VSV-G in delivering T. gondii antigen was investigated. T. gondii SAG1 gene was cloned into Bac-VSV-G, and recombinant baculovirus BV-G-SAG1 was obtained. Indirect immunofluorescence test showed BV-G-SAG1 was efficiently transduced and expressed in pig kidney cells. Then BALB/c mice were immunized with BV-G-SAG1 at different doses (1 x 10(8), 1 x 10(9), and 1 x 10(10)PFU/mouse) and challenged with T. gondii RH strain tachyzoites after immunization. The levels of specific T. gondii antibody, interferon (IFN)-gamma, IL-4, IL-10 expression and release, and the survival rate of treated mice were evaluated. Compared with the mice immunized with DNA vaccine (pcDNA/SAG1) encoding the same gene, BV-G-SAG1 induced higher levels of specific T. gondii antibody and (IFN)-gamma expression with dose-dependent manner and the survival rate of mice with BV-G-SAG1 was significantly improved. These results indicated that pseudotype baculovirus-mediated gene delivery can be utilized as an alternative strategy to develop new generation of vaccines against T. gondii infection.
Infection with the intracellular protozoan parasite Toxoplasma gondii causes serious public health problems and is of great economic importance worldwide. Microneme proteins which are responsible for adhesion and invasion have been implicated as vaccine candidates. In this study, we constructed a DNA vaccine expressing microneme protein 6 (MIC6) of T. gondii, and evaluated the immune response it induced in Kunming mice. The gene sequence encoding MIC6 was inserted into the eukaryotic expression vector pVAXI. We immunized Kunming mice intramuscularly. After immunization, we evaluated the immune response using lymphoproliferative assay, cytokine and antibody measurements, and the survival times of mice challenged lethally. The results showed that the group immunized with pVAX-MIC6 developed a high level of specific antibody responses against T. gondii lysate antigen (TLA), a strong lymphoproliferative response, and significant levels of IFN-gamma, IL-2, IL-4 and IL-10 production, compared with the other groups immunized with empty plasmid or phosphate-buffered saline, respectively. These results demonstrate that pVAX-MIC6 induces significant humoral and cellular Th1 immune responses. After lethal challenge, the mice immunized with the pVAX-MIC6 showed an increased survival time (13.3+/-1.2 days) compared with control mice died within 7 days of challenge. Our data demonstrate, for the first time, that MIC6 triggered a strong humoral and cellular response against T. gondii, and that the antigen is a potential vaccine candidate against toxoplasmosis, worth further development.
An effective vaccine of animals can block transmission of Toxoplasma gondii to humans. In this study, mice have been protected against lethal T. gondii challenge by a prime-boost vaccination strategy using DNA vaccine pVAX/TgSAG1 and recombinant pseudorabies virus rPRV/TgSAG1, both expressing the major immunodominant surface antigen of T. gondii (TgSAG1). High levels of splenocyte proliferative responses and significant levels of IFN-gamma resulted, with strong cytotoxic T lymphocyte (CTL) responses in vitro. After lethal challenge, prime-boost vaccinated mice showed an increased survival time (15.4+/-5.0 days) and a 40% survival rate compared with controls who all died within 11 days of challenge. Results of the present study indicated that this novel immunization strategy is useful in enhancing immune protection in mice against lethal T. gondii infection, which would provide foundation for the development of effective vaccines against T. gondii.
A 3-yr field trial was conducted on 8 commercial swine farms in Illinois to determine the effectiveness of a feline Toxoplasma gondii vaccine in reducing the exposure of swine to T. gondii. A vaccine consisting of live bradyzoites of the mutant T-263 strain, capable of preventing oocyst shedding by cats, was used in this study. Each farm was visited 3 times in 1994, 3 times in 1995, and once in 1996. Cats were trapped and inoculated with the T-263 oral vaccine during 1994 and 1995. On each visit, the following samples were collected: blood from pigs, cats, and mice for detection of serum antibodies to T. gondii, feces from cats to detect oocysts, and heart and brain tissues from rodents to determine the presence of T. gondii tissue cysts. The modified agglutination test (MAT), with a positive titer set at the 1:25 dilution, was used to determine serum antibodies. At first capture, 72.6% (61/84) of juvenile cats and 32.6% (31/95) of adult cats had no detectable antibodies (seronegative), indicating no prior exposure to T. gondii when they received their first vaccine. Of these first-time seronegative cats, 58.1% (18/31) of adult and 45.9% (28/61) of juvenile cats were recaptured and received a second dose of vaccine. Changes in the prevalence of T. gondii infection were evaluated from the prevaccination (1992, 1993) to the postvaccination (1996) period. Eleven cats (5%) were detected shedding oocysts between 1994 and 1996, of which 10 (90.1%) shed during 1994. The last detection of oocyst shedding by cats was during the first farm visit in 1995. There was a significant decrease in T. gondii seroprevalence for finishing pigs (P
BALB/c mice were intramuscularly immunized with low doses (25-50microg) of DNA cocktail containing plasmids encoding the full-length SAG1/P30 and the 196-561 terminal sequence of ROP2 genes. This immunization resulted in a Th1-type response with predominance of IgG2a and a specific T-cell proliferation with high levels of interferon-gamma (IFN-gamma) secretion, whereas no IL-4 was detected. Moreover, DNA cocktail immunization induced a long-lasting protection against a lethal challenge with the highly virulent Toxoplasma gondii RH strain, whereas low doses of single genes were not protective. These results support further investigations to achieve a multigene anti-T. gondii DNA vaccine.
We have obtained the complete DNA sequence of pseudorabies virus (PRV), an alphaherpesvirus also known as Aujeszky's disease
virus or suid herpesvirus 1, using sequence fragments derived from six different strains (Kaplan, Becker, Rice, Indiana-Funkhauser,
NIA-3, and TNL). The assembled PRV genome sequence comprises 143,461 nucleotides. As expected, it matches the predicted gene
arrangement, genome size, and restriction enzyme digest patterns. More than 70 open reading frames were identified with homologs
in related alphaherpesviruses; none were unique to PRV. RNA polymerase II transcriptional control elements in the PRV genome,
including core promoters, splice sites, and polyadenylation sites, were identified with computer prediction programs. The
correlation between predicted and experimentally determined transcription start and stop sites was excellent. The transcriptional
control architecture is characterized by three key features: core transcription elements shared between genes, yielding divergent
transcripts and a large number of coterminal transcripts; bifunctional transcriptional elements, yielding head-to-tail transcripts;
and short repetitive sequences that could function as insulators against improperly terminated transcripts. Many of these
features are conserved in the alphaherpesvirus subfamily and have important implications for gene array analyses.
Vaccine approaches to infectious diseases are widely applied and appreciated. Amongst them, vectors based on recombinant viruses have shown great promise and play an important role in the development of new vaccines. Many viruses have been investigated for their ability to express proteins from foreign pathogens and induce specific immunological responses against these antigens in vivo. Generally, gene-based vaccines can stimulate potent humoral and cellular immune responses and viral vectors might be an effective strategy for both the delivery of antigen-encoding genes and the facilitation and enhancement of antigen presentation. In order to be utilized as a vaccine carrier, the ideal viral vector should be safe and enable efficient presentation of required pathogen-specific antigens to the immune system. It should also exhibit low intrinsic immunogenicity to allow for its re-administration in order to boost relevant specific immune responses. Furthermore, the vector system must meet criteria that enable its production on a large-scale basis. Several viral vaccine vectors have thus emerged to date, all of them having relative advantages and limits depending on the proposed application, and thus far none of them have proven to be ideal vaccine carriers. In this review we describe the potential, as well as some of the foreseeable obstacles associated with viral vaccine vectors and their use in preventive medicine.
We have generated recombinant adenoviruses encoding three genetically modified surface antigens (SAGs) of the parasite Toxoplasma gondii, that is, AdSAG1, AdSAG2, and AdSAG3. Modifications included the removal of their glycosylphosphatidylinositol (GPI) anchoring motifs and, in some cases, the exchange of the native signal peptide for influenza virus hemagglutinin signal sequence. Adenovirus immunization of BALB/c mice elicited potent antibody responses against each protein, displaying a significant bias toward a helper T cell type 1 (Th1) profile in animals vaccinated with AdSAG1. Furthermore, the presence of parasite-specific IFN-gamma-producing T cells was analyzed by proliferation assays and enzyme-linked immunospot assays in the same animals. Splenocytes from immunized mice secreted IFN-gamma after in vitro stimulation with tachyzoite lysate antigen or with a fraction enriched for membrane-purified GPI-anchored proteins (F3) from the T. gondii tachyzoite surface. Epitopes recognized by CD8+ T cells were identified in SAG1 and SAG3, but not SAG2, sequences, although this protein also induced a specific response. We also tested the capacity of the immune responses detected to protect mice against a challenge with live T. gondii parasites. Although no protection was observed against tachyzoites of the highly virulent RH strain, a significant reduction in cyst loads in the brain was observed in animals challenged with the P-Br strain. Thus, up to 80% of the cysts were eliminated from animals vaccinated with a mixture of the three recombinant viruses. Because adenoviruses seemed capable of inducing Th1-biased protective immune responses against T. gondii antigens, other parasite antigens should be tested alone or in combination with those described here to further develop a protective vaccine against toxoplasmosis.
The first part of this work presents the sequence of the first 20 NH2 terminus residues obtained from P30, the major surface Ag of Toxoplasma gondii, purified by HPLC. A synthetic peptide (P30 48-67) has been prepared both in linear form and as a multiple antigenic peptide (MAP) construct. Immunization of mice and rats with the P30 48-67 MAP in the presence of IFA induces high levels of IgG antibodies that recognize both the linear peptide and the MAP construct in ELISA, and P30 in Western blots of NP-40-extracted tachyzoite Ag. Because these sera are negative in immunofluorescence assays with whole tachyzoites, it seems that IgG antibodies induced by P30 48-67 MAP, although recognizing the denatured structure, are unable to recognize the native protein. However, the protective effect of both constructs has then been studied in mice and nude rats. Whereas immunization of mice with the monomeric peptide does not confer any protection against oral infection with 1200 cysts of T. gondii 76K strain (mortality within 11 days), 40% of mice immunized with the MAP construct survived up to 75 days after infection. Nude rats were passively transferred with 5 x 10(4) T lymphocytes from P30 48-67 MAP-immunized Fischer rats before infection with 5 x 10(4) RH strain tachyzoites. They survived up to 40 days after infection and raised an intense IgG antibody response against P30, whereas nude rats transferred with control lymphocytes died within 21 days. This shows that immunization with P30 48-67 MAP also induces an efficient T cell immune response. The present work confirms the recently demonstrated role of P30 in protective immunity and shows the interest of peptide octameric constructions as inducers of partially protective immune responses in toxoplasmosis, as already demonstrated in schistosomiasis.
The presence of the pseudorabies virus (PRV) genome in infected hosts has previously been studied by standard hybridization techniques, which showed the viral genome to be present at very low levels in infected tissues. The recently introduced polymerase chain reaction (PCR) procedure provides an alternative and rapid means of amplifying small quantities of specific DNA sequences. We applied this technique to a study of pigs infected by PRV. The sequence selected for amplification consisted of 222 base pairs lying in the gene coding for the glycoprotein gp50. We used a pair of 20-mer oligonucleotides flanking this sequence as primer and a cloned Stu-Nde fragment containing the sequence as target DNA. To avoid the tedious DNA extraction procedure we performed PCR directly on disrupted cells and detected specific amplification after 25 cycles of PCR with the thermostable Taq DNA polymerase. Amplified products were detected by gel electrophoresis directly. Nasal samples from experimentally and naturally infected pigs were tested by this PCR technique. When compared with tissue culture and serological tests, detection by gel electrophoresis of PCR amplified fragments provided excellent specificity and sensitivity. We concluded that PCR amplification will be a valuable tool for rapid diagnosis of PRV infection in pigs, taking less than 1 h to complete.
The cDNA coding for human tissue plasminogen activator (tPA) was cloned downstream from the promoter for pseudorabies virus (PRV) glycoprotein and flanked by downstream PRV DNA. After co-transfection with PRV DNA, this plasmid recombined to insert the tPA cDNA into the viral genome. In cells infected by this recombinant virus, tPA was detected by immunoprecipitation analysis and by enzymatic activity. Since it has a wide host range but does not infect humans, PRV is a possible vaccine vector for genes from animal pathogens.
To evaluate the contribution of glycoprotein E (gE), thymidine kinase (TK), and the US3-encoded protein kinase (PK) in the induction of protective immunity to pseudorabies virus (PRV), we intranasally inoculated pigs, the natural host of this virus, with mutant PRV strains in which the genes encoding these proteins were inactivated. Both single and double mutants were constructed. Of these proteins, gE has previously been demonstrated to induce antibodies (in mice and pigs), which require complement to neutralize the virus, and helper T cell responses (in mice). PK and TK have thus far not been reported to induce B or T cell responses. All mutants had a strongly reduced virulence for pigs in comparison with wild-type (wt) PRV. After primary infection, most virus was excreted by wt PRV-inoculated animals. Animals inoculated with gE-PK- and gE-TK- double mutants excreted less virus than animals inoculated with gE-, PK-, and TK- single mutants. After challenge infection with the virulent PRV strain NIA-3, no virus was excreted by wt PRV- and PK- mutant-immunized animals, indicating complete protective immunity. Only one of seven gE- and two of seven TK- mutant-immunized animals excreted virus after the challenge inoculation. In contrast, most animals immunized with the gE-PK- or gE-TK- double mutants excreted virus after the challenge inoculation. Daily mean virus excretion after challenge infection was inversely correlated with daily mean virus excretion after primary infection. In most animals, lack of virus excretion was associated with lack of secondary antibody responses, probably attributable to inadequate stimulation of memory B cells as a consequence of early elimination of viral antigen. Thus, inactivation of gE, TK, and PK all affected the immunogenicity of PRV and the effect of gE and TK and gE and PK inactivation appeared synergistic. We found no simple correlation between in vitro growth properties of the mutants and their immunogenic capacity. Strains lacking PK reached lower end titers in vitro than the other mutants. The most likely explanation for the lower protective capacity of some of the mutants appears their reduced replicative capacity in some cells or tissues in vivo, rather than a loss of particular epitopes.
GRA4 is a dense granule protein of Toxoplasma gondii that is a candidate for vaccination against this parasite. We have inserted the entire coding sequence of GRA4 into an eukaryotic expression vector to determine whether DNA immunization can elicit protective immune response to T. gondii. Susceptible C57BL/6 mice were then vaccinated intramuscularly with GRA4 DNA and orally challenged with a lethal dose of 76 K T. gondii strain cysts. Immunization with pGRA4 resulted in a 62% survival of C57BL/6 infected mice. Mice immunized with GRA4 DNA developed high levels of serum anti-GRA4 immunoglobulin G antibodies as well as a cellular immune response, as assessed by splenocyte proliferation, in response to recombinant GRA4 protein restimulation in vitro. The cellular immune response was associated with IFN-gamma and IL-10 synthesis, suggesting a modulated Th1-type response. Splenocyte proliferation was strongly enhanced and protection slightly higher by inoculation with GRA4 DNA combined with a granulocyte-macrophage colony-stimulating factor expressing vector. This is the first report that demonstrates the establishment of a DNA vaccine-induced protective immunity against the acute phase of T. gondii infection.
Toxoplasmosis is one of the more common parasitic zoonoses world-wide. Its causative agent, Toxoplasma gondii, is a facultatively heteroxenous, polyxenous protozoon that has developed several potential routes of transmission within and between different host species. If first contracted during pregnancy, T. gondii may be transmitted vertically by tachyzoites that are passed to the foetus via the placenta. Horizontal transmission of T. gondii may involve three life-cycle stages, i.e. ingesting infectious oocysts from the environment or ingesting tissue cysts or tachyzoites which are contained in meat or primary offal (viscera) of many different animals. Transmission may also occur via tachyzoites contained in blood products, tissue transplants, or unpasteurised milk. However, it is not known which of these routes is more important epidemiologically. In the past, the consumption of raw or undercooked meat, in particular of pigs and sheep, has been regarded as a major route of transmission to humans. However, recent studies showed that the prevalence of T. gondii in meat-producing animals decreased considerably over the past 20 years in areas with intensive farm management. For example, in several countries of the European Union prevalences of T. gondii in fattening pigs are now <1%. Considering these data it is unlikely that pork is still a major source of infection for humans in these countries. However, it is likely that the major routes of transmission are different in human populations with differences in culture and eating habits. In the Americas, recent outbreaks of acute toxoplasmosis in humans have been associated with oocyst contamination of the environment. Therefore, future epidemiological studies on T. gondii infections should consider the role of oocysts as potential sources of infection for humans, and methods to monitor these are currently being developed. This review presents recent epidemiological data on T. gondii, hypotheses on the major routes of transmission to humans in different populations, and preventive measures that may reduce the risk of contracting a primary infection during pregnancy.
Toxoplasmosis is caused by infection with the protozoan parasite Toxoplasma gondii. In the United States, approximately 85% of women of childbearing age are susceptible to acute infection with T. gondii. Acute infections in pregnant women may cause serious health problems when the organism is transmitted to the fetus (congenital toxoplasmosis), including mental retardation, seizures, blindness, and death. An estimated 400 to 4000 cases of congenital toxoplasmosis occur in the U.S. each year. Manifestations of congenital toxoplasmosis may not become apparent until the second or third decade of life. Serologic tests are used to diagnose acute infection in pregnant women, but false-positive tests occur frequently, therefore, serologic diagnosis must be confirmed at a reference laboratory before treatment with potentially toxic drugs should be considered. Much of congenital toxoplasmosis can be prevented by educating women of childbearing age and pregnant women to avoid eating raw or undercooked meat, to avoid cross-contamination of other foods with raw or undercooked meat, and to use proper cat-litter and soil-related hygiene.
In order to investigate whether a recombinant viral vaccine composed of a feline herpesvirus type 1 (FHVI) vector and an immunogenic antigen of Toxoplasma gondii can induce a protective immunity in cats, a recombinant FHV1 expressing ROP2 antigen of T. gondii was prepared. We introduced a DNA fragment encoding 1-538 amino acid residues of ROP2 precursor into FHV1 genome under the control of a cytomegalovirus promoter. The recombinant FHV1 (FHV/ROP2) successfully expressed a 59 kDa antigen that was recognized by anti-ROP2 antibodies. Vaccination of cats with FHV/ROP2 induced serum IgG recognizing the native antigen. Moreover the antibodies inhibited the in vitro invasion of tachyzoites. The vaccination also accelerated the IgG response after T. gondii infection and reduced brain parasite load in cats.
The need for a vaccine for human toxoplasmosis is briefly discussed. Recent progress in knowledge of the protective immune response generated by Toxoplasma gondii and the current status of development of a vaccine for toxoplasmosis are highlighted.
The aim of this study was to test the protective immunity against Toxoplasma gondii in mice induced by a chimeric protein rSAG1/2. The PCR-amplified SAG2 fragment (558 bp) digested with the restriction enzyme XhoI was inserted into the XhoI site of plasmid pGEX-6p-1, termed pGexSAG2. The PCR-amplified SAG1 fragment (1,008 bp) digested with restriction enzymes EcoRI and XhoI was cloned into the EcoRI/ XhoI sites of a separate plasmid pGEX-6p-1, termed pGexSAG1. The SAG2 fragment (HindIII/HindIII) excised from pGexSAG2 was inserted into the HindIII site of pGexSAG1 and a chimeric vector constructed, pGexSAG1/2. The fusion proteins GST-SAG1/2, GST-SAG1 and GST-SAG2 were expressed in BL21 Star (DE3) Escherichia coli and purified by GSTrap FF columns. After removing the GST tag, the recombinant proteins rSAG1/2, rSAG1 and rSAG2 were independently collected and injected into different groups of mice to evaluate their protective capability. The highest proliferation of splenocytes stimulated with tachyzoite sonicate antigen (TsoAg) was observed in BALB/c mice which had received two intraperitoneal injections of rSAG1/2. Maximum production of IFN-gamma was also found in the culture supernatants of TsoAg-stimulated splenocytes from rSAG1/2-immunized mice. Finally, 73% (11/15) of mice immunized with rSAG1/2 survived at least 28 days after a lethal challenge of 1 x 10(3) live tachyzoites which killed all non-immunized mice within 10 days. Moderate survival rates were observed in mice immunized with either rSAG1 (60%) or rSAG2 (53%). These results show that the chimeric protein rSAG1/2 can elicit a Th1-associated protection against T. gondii infections in mice.
In the present study, the immunogenicity and protective efficacy of a suicidal DNA vaccine (pSFVC1.5) incorporating Semliki Forest virus (SFV) replicon and expressing glycoprotein C (gC) of pseudorabies virus (PrV) was investigated and compared with a conventional plasmid DNA vaccine (pcDC) encoding the same antigen. In vitro, pSFVC1.5 could express gC protein and induce apoptosis of the transfected cells. After immunization in BALB/c mice, the gC-specific ELISA antibodies and neutralizing antibodies induced by pSFVC1.5 were relatively lower than those obtained in mice immunized with pcDC. However, mice immunized with pSFVC1.5 could confer more efficient protection than pcDC (100 and 62.5%, respectively) when challenged with the field PrV at 4 weeks after secondary immunization. Further analyses of cell-mediated immune responses showed that pSFVC1.5 induced stronger lymphocyte proliferative responses and higher levels of IFN-gamma, suggesting pSFVC1.5 could induce an enhanced Th1-type immune response. Collectively these results indicated that suicidal DNA vaccine is an alternative strategy to conventional DNA vaccine and can be considered a promising approach for the development of an efficacious vaccine against PrV.
The bivalent genetic engineering vaccine of Japanese encephalitis (JE) and Aujeszkj disease (AD) was developed to provide a novel approach to prevent and control these two diseases. NS1 gene of Japanese encephalitis virus (JEV) SA14-14-2 strain was produced by reverse transcriptase-mediated PCR (RT-PCR) and was cloned into vector pUSK to form recombinant plasmid (designed as pUSK-NS1). A co-transfection experiment was performed in porcine kidney (PK-15) cells with pUSK-NS1 and the genome of the vector virus (PRV TK(-)/gG(-)/LacZ(+) mutant). By plaque purification, PCR detection and southern hybridization, recombinant pseudorabies virus (PRV) expressing NS1 protein of JEV was acquired and named TK(-)/gG(-)/NS1(+). Western blot analysis and ELISA demonstrated the NS1 protein expression. To evaluate the recombinant virus's potential application, we characterized the safety and immune responses in Balb/c mice and swine. The safety test indicated that, when receiving the recombinant virus at a concentration of 10(6.0)pfu, no virulence of the recombinant virus to the mice, piglets and pregnant sows was observed. The vaccinated animals could acquire protective immunity against lethal challenge of the virulent PRV Ea strain and develop a good humoral and cellular immune response against JEV. The above results revealed that the recombinant virus could be a suitable candidate vaccine strain for developing a novel genetic vaccine to combat pseudorabies and Japanese encephalitis in the pig industry.
Toxoplasma gondii is the aetiological agent of toxoplasmosis and is the most frequent and best known of the parasitic diseases. In the United States, a serological survey from the Third National Health and Nutrition Examination Survey found that an estimated 23% of adolescents and adults have laboratory evidence of infection with T. gondii. Although toxoplasmosis is asymptomatic or shows self-limited symptoms in adults, in pregnant women infections can cause severe health problems to the fetus if the parasites are transmitted. Also, in immunodeficient patients, chronic infection with T. gondii can reactivate and produce encephalitis, which is frequently lethal. In addition, in veterinary medicine, T. gondii infection is of economic importance due to abortion and neonatal loss in sheep and goats. Recently, the development of vaccines against toxoplasmosis has progressed considerably. The live attenuated S48 strain of Toxoplasma has been broadly used for veterinary purposes. DNA vaccines containing the full-length of SAG1/P30, ROP2 or ROP1 genes have proved to be a promising candidate to induce protection against toxoplasmosis. Viral vectors have proved to be the best candidates for vaccination in different diseases. A recombinant Herpes virus carrying the ROP2 gene is able to induce protective immunity in cats. In the present work we describe the potential of the MVA ROP2 recombinant vaccinia virus as a vaccine against toxoplasmosis. MVA ROP2 induces antibodies against the ROP2 protein in similar amount and types as the thermo-sensible strain ts-4 of T. gondii, which is able to fully protect mice against challenge with the virulent RH strain of T. gondii. Also, the life-span of mice is increased in MVA ROP2 vaccinated animals. We conclude that MVA ROP2 vaccine can possibly generate an immune response, which could be useful in protection against toxoplasmosis.
Toxoplasma gondii is a protozoan parasite that infects up to a third of the world's population. Infection is mainly acquired by ingestion of food or water that is contaminated with oocysts shed by cats or by eating undercooked or raw meat containing tissue cysts. Primary infection is usually subclinical but in some patients cervical lymphadenopathy or ocular disease can be present. Infection acquired during pregnancy may cause severe damage to the fetus. In immunocompromised patients, reactivation of latent disease can cause life-threatening encephalitis. Diagnosis of toxoplasmosis can be established by direct detection of the parasite or by serological techniques. The most commonly used therapeutic regimen, and probably the most effective, is the combination of pyrimethamine with sulfadiazine and folinic acid. This Seminar provides an overview and update on management of patients with acute infection, pregnant women who acquire infection during gestation, fetuses or infants who are congenitally infected, those with ocular disease, and immunocompromised individuals. Controversy about the effectiveness of primary and secondary prevention in pregnant women is discussed. Important topics of current and future research are presented.
An outbreak of Aujeszky's disease (AD) occurred in a herd of domestic animals that led to the death of seven cattle, three goats, three sheep, two cats and one dog, all of them with CNS signs. The animals were not in direct contact with swine. The ADV was detected in the tissue of affected animals by celi culture methods and PCR. Genome strains of ADV were characterized by restriction endonuclease analysis using BamH I. The results indicated that the strains of virus were identical and belonged to the type genome I of AD. Compared with vaccine and isolated strains obtained from the pig in the same region, considerable differences in DNA patterns were detected. Interestingly, the strains isolated from the dead animals were similar to Buk T-900 reference strains.
Pseudorabies virus (PRV) has been developed as a vaccine vector for expressing foreign immunogens. Porcine reproductive and respiratory syndrome (PRRS), caused by porcine reproductive and respiratory syndrome virus (PRRSV), continues to be a major problem to the pork industry worldwide. Many vaccine strategies have been developed to control the disease but most of them turn out to be unsuccessful. The objective of this research was to explore the feasibility of PRV-based vector vaccine in protection against PRRSV. A live attenuated vaccine-based PRV recombinant expressing the envelope protein GP5 of PRRSV was generated using recombinant DNA techniques. The Bartha-K61-derived recombinant virus, named rPRV-GP5, was shown to express PRRSV GP5 efficiently. Sixteen healthy piglets were assigned to one of four groups (one to four, four pigs per group). Animals in Groups 1 and 2 were each inoculated intramuscularly and intranasally with 10(7.0) PFU of rPRV-GP5 and its parent Bartha-K61, respectively; Group 3 were vaccinated intramuscularly with one-dose of PRRS inactivated vaccine; Group 4 was served as non-vaccinated control. One month later, all animals were all challenged with 10(6.5) TCID(50) of virulent PRRSV CH-1a. All animals in Groups 1 and 3 remained clinically healthy before and after challenge, with only a short period of fever (no more than 41 degrees C and 3 days), mild and gradually improving lung and kidney lesions, and short-term viremia (2 and 3 week, respectively) in spite of no detectable anti-PRRSV antibody before challenge. On the other hand, all animals in the other two groups showed evident clinical signs with higher temperatures (more than 41 degrees C) after challenge, and severe lung, kidney and spleen lesions and extended viremia (4 weeks). The results indicate that the rPRV-GP5 is safe for vaccinates and able to confer significant protection against clinical disease and reduce pathogenic lesions induced by PRRSV challenge in vaccinated pigs.
Two severe porcine infectious diseases, pseudorabies (PR) and transmissible gastroenteritis (TGE) caused by pseudorabies virus (PRV) and transmissible gastroenteritis virus (TGEV) respectively often result in serious economic loss in animal husbandry worldwide. Vaccination is the important prevention means against both infections. To achieve a PRV genome-based virus live vector, aiming at further TGEV/PRV bivalent vaccine development, a recombinant plasmid pUG was constructed via inserting partial PK and full-length gG genes of PRV strain Bartha K-61 amplified into pUC119 vector. In parallel, another recombinant pHS was generated by introducing a fragment designated S1 encoding the major antigen sites of S gene from TGEV strain TH-98 into a prokaryotic expression vector pP(RO)EX HTc. The SV40 polyA sequence was then inserted into the downstream of S1 fragment of pHS. The continuous region containing S1fragment, SV40 polyA and four single restriction enzyme sites digested from pHS was subcloned into the downstream of gG promoter of pUG. In addition, a LacZ reporter gene was introduced into the universal transfer vector named pUGS-LacZ. Subsequently, a PRV genome-based virus live vector was generated via homologous recombination. The functionally effective vector was purified and partially characterized. Moreover, the potential advantages of this system are discussed.
Porcine reproductive and respiratory syndrome virus (PRRSV) infection still remains today as the most significant health threat to swine and poses a challenge to current vaccination strategies. To develop a new generation of vaccine against PRRSV, a live attenuated pseudorabies virus (PRV) was used as vaccine vector to express the two major membrane-associated proteins (GP5 or M) of PRRSV in various forms. Four PRV recombinants, rPRV-GP5 (expressing native GP5), rPRV-GP5m (expressing GP5m, a modified GP5), rPRV-GP5-M (co-expressing GP5 and M proteins), rPRV-GP5m-M (co-expressing GP5m and M proteins) were generated. Mouse immunized with all these recombinants developed comparable PRV-specific humoral immune responses and provided complete protection against a lethal PRV challenge. However, the highest level of PRRSV-specific neutralizing antibodies and lymphocyte proliferative responses was observed in mice immunized with rPRV-GP5m-M. The immunogenicity and protective efficiency of rPRV-GP5m-M were further evaluated in the piglets. Compared to commercial PRRSV killed vaccine, detectable PRRSV-specific neutralizing antibody and higher lymphocyte proliferative responses could be developed in piglets immunized with rPRV-GP5m-M before virus challenge. Furthermore, more efficient protection against a PRRSV challenge was obtained in piglets immunized with rPRV-GP5m-M, as showed by the balanced body-temperature fluctuation, shorter-term viremia, lower proportion of virus load in nasal and oropharyngeal scrapings and tissues, and milder lung lesions. These data indicate that the recombinant rPRV-GP5m-M is a promising candidate bivalent vaccine against both PRV and PRRSV infection.
The heavy incidence and severe or lethal damages of toxoplasmosis clearly indicate the need for the development of a more effective vaccine. In the present study, we constructed a multiantigenic DNA vaccine, eukaryotic plasmid pcDNA3.1-SAG1-ROP2, expressing surface protein SAG1 and rhoptry protein ROP2 of Toxoplasma gondii, and examined the expression ability of the DNA vaccine in HeLa cells by Western blot. Afterwards, we investigated the efficacy of pcDNA3.1-SAG1-ROP2 with or without co-administration of a plasmid encoding murine interleukin-12 (pIL-12) as a genetic adjuvant to protect Bagg albino/c mice against toxoplasmosis. After T. gondii RH strain challenge, mice immunized with pcDNA3.1-SAG1-ROP2 displayed significant high survival rates. Moreover, the protection was markedly enhanced by pIL-12 co-administration. The results of lymphocyte proliferation assay, cytokine, and antibody determinations show that mice immunized with pcDNA3.1-SAG1-ROP2 elicited stronger humoral and Th1-type cellular immune responses than those immunized with single-gene plasmids, empty plasmid, or phosphate-buffered saline. Furthermore, co-immunization with IL-12 genes resulted in a dramatic enhancement of these responses. Our study indicates that the introduction of multiantigenic DNA vaccine is more powerful and efficient than single-gene vaccine, and the co-delivery of pIL-12 further enhanced the potency of multiantigenic DNA vaccine.
The protozoan parasites Eimeria spp. Toxoplasma gondii and Neospora caninum are significant causes of disease in livestock worldwide and T. gondii is also an important human pathogen. Drugs have been used with varying success to help control aspects of these diseases and commercial vaccines are available for all three groups of parasites. However, there are issues with increasing development of resistance to many of the anti-coccidial drugs used to help control avian eimeriosis and public concerns about the use of drugs in food animals. In addition there are no drugs available that can act against the tissue cyst stage of either T. gondii or N. caninum and thus cure animals or people of infection. All three groups of parasites multiply within the cells of their host species and therefore cell mediated immune mechanisms are thought to be an important component of host protective immunity. Successful vaccination strategies for both Eimeria and Toxoplasma have relied on using a live vaccination approach using attenuated parasites which allows correct processing and presentation of antigen to the host immune system to stimulate appropriate cell mediated immune responses. However, live vaccines can have problems with safety, short shelf-life and large-scale production; therefore there is continued interest in devising new vaccines using defined recombinant antigens. The major challenges in devising novel vaccines are to select relevant antigens and then present them to the immune system in an appropriate manner to enable the induction of protective immune responses. With all three groups of parasites, vaccine preparations comprising antigens from the different life cycle stages may also be advantageous. In the case of Eimeria parasites there are also problems with strain-specific immunity therefore a cocktail of antigens from different parasite strains may be required. Improving our knowledge of the different parasite transmission routes, host-parasite relationships, disease pathogenesis and determining the various roles of the host immune response being at times host-protective, parasite protective and in causing immunopathology will help to tailor a vaccination strategy against a particular disease target. This paper discusses current vaccination strategies to help combat infections with Eimeria, Toxoplasma and Neospora and recent research looking towards developing new vaccine targets and approaches.
To determine Toxoplasma gondii antibodies in cancer patients, 267 cancer patients were studied using ELISA and higher positivity rates of T. gondii IgG were detected than the control. The positivity rates of T. gondii IgG in nasopharyngeal carcinoma, rectal cancer groups were significantly higher than the other cancer groups, but the differences in IgM positivity rates were not significant, demonstrating that there is a likely association between T. gondii infection and some kinds of cancer, especially nasopharyngeal carcinoma and rectal cancer.
The dense granule antigen 4 (GRA4) is known as an immundominant antigen of Toxoplasma gondii and, therefore, is considered as a vaccine candidate. For further evaluation of its vaccine effect, a recombinant plasmid and vaccinia virus, both expressing GRA4, were constructed, and a heterologous prime-boost vaccination regime was performed in a mouse model. The mice immunized with the heterologous prime-boost vaccination regime showed a high level of specific antibody response against GRA4 and a significantly high level of gamma interferon (IFN-gamma) production and survived completely against a subsequent challenge infection with a lethal dose of T. gondii. In addition, the formation of cysts was inhibited in the mice vaccinated with the heterologous prime-boost vaccination regime. These results demonstrate that the heterologous prime-boost vaccination regime using DNA and a vaccinia virus, both expressing GRA4, could induce both humoral and cellular immune responses and provide effective protection against lethal acute and chronic T. gondii infections in mice.
Toxoplasma gondii is an obligate intracellular parasite, capable of infecting a variety of mammals and birds. Development of vaccine against T. gondii would be of great medical and veterinary value. In this study, the DNA sequence encoding ROP2 from T. gondii was cloned into the muticopy mycobacterial expression vector, pMV262, under the control of the Bacillus Calmette-Guerin (BCG) hsp60 promoter, and electroporated into BCG. Following selection of kanamycin, the recombinant BCG/pMV262-ROP2 was constructed and the expression of ROP2 was confirmed by Western blotting. The BALB/c mice inoculated with the BCG/pMV262-ROP2 developed specific immune responses against ROP2 protein, and there was an obvious delay in the mortality curve than the control (P<0.05). These results indicated that M. bovis BCG is an adequate vector to express and present antigens of T. gondii, and it may be used to further study the induction of protective immunity in other animals.
Pseudorabies (PR), foot-and-mouth disease (FMD), and porcine parvovirus disease are three important infectious diseases in swine worldwide. The gene-deleted pseudorabies virus (PRV) has been used as a live-viral vector to develop multivalent genetic engineering vaccine. In this study, a recombinant PRV, which could co-express protein precursor P1-2A of FMDV and VP2 protein of PPV, was constructed using PRV TK(-)/gE(-)/LacZ(+) mutant as the vector. After homologous recombination and plaque purification, recombinant virus PRV TK(-)/gE(-)/P1-2A-VP2 was acquired and identified. Immunogenicity, safety of the recombinant PRV and its protection against PRV were confirmed in a mouse model by indirect ELISA and serum neutralization test. The results show that the recombinant PRV is a candidate vaccine strain to develop a novel trivalent vaccine against PRV, FMDV and PPV in swine.