Immunoglobulin and T cell receptor repertoire changes induced by a prototype vaccine against Chagas disease in naïve rhesus macaques.

BACKGROUND: A vaccine against Trypanosoma cruzi, the agent of Chagas disease, would be an excellent additional tool for disease control. A recombinant vaccine based on Tc24 and TSA1 parasite antigens was found to be safe and immunogenic in naïve macaques. METHODS: We used RNA-sequencing and performed a transcriptomic analysis of PBMC responses to vaccination of naïve macaques after each vaccine dose, to shed light on the immunogenicity of this vaccine and guide the optimization of doses and formulation. We identified differentially expressed genes and pathways and characterized immunoglobulin and T cell receptor repertoires. RESULTS: RNA-sequencing analysis indicated a clear transcriptomic response of PBMCs after three vaccine doses, with the up-regulation of several immune cell activation pathways and a broad non-polarized immune profile. Analysis of the IgG repertoire showed that it had a rapid turnover with novel IgGs produced following each vaccine dose, while the TCR repertoire presented several persisting clones that were expanded after each vaccine dose. CONCLUSIONS: These data suggest that three vaccine doses may be needed for optimum immunogenicity and support the further evaluation of the protective efficacy of this vaccine.

A conserved motif in the immune-subdominant RAP-1 related antigen of Babesia bovis contains a B-cell epitope recognized by antibodies from protected cattle.

Introduction: Babesia bovis, a tick-borne apicomplexan parasite causing bovine babesiosis, remains a significant threat worldwide, and improved and practical vaccines are needed. Previous studies defined the members of the rhoptry associated protein-1 (RAP-1), and the neutralization-sensitive rhoptry associated protein-1 related antigen (RRA) superfamily in B. bovis, as strong candidates for the development of subunit vaccines. Both RAP-1 and RRA share conservation of a group of 4 cysteines and amino acids motifs at the amino terminal end (NT) of these proteins. Methods and results: Sequence comparisons among the RRA sequences of several B. bovis strains and other Babesia spp parasites indicate a high level of conservation of a 15-amino acid (15-mer) motif located at the NT of the protein. BlastP searches indicate that the 15-mer motif is also present in adenylate cyclase, dynein, and other ATP binding proteins. AlphaFold2 structure predictions suggest partial exposure of the 15-mer on the surface of RRA of three distinct Babesia species. Antibodies in protected cattle recognize a synthetic peptide representing the 15-mer motif sequence in iELISA, and rabbit antibodies against the 15-mer react with the surface of free merozoites in immunofluorescence. Discussion and conclusion: The presence of the 15-mer-like regions in dynein and ATP-binding proteins provides a rationale for investigating possible functional roles for RRA. The demonstrated presence of a surface exposed B-cell epitope in the 15-mer motif of the B. bovis RRA, which is recognized by sera from protected bovines, supports its inclusion in future subunit epitope-based vaccines against B. bovis.

Working towards the development of vaccines and chemotherapeutics against neosporosis-With all of its ups and downs-Looking ahead.

Neospora caninum is an apicomplexan and obligatory intracellular parasite, which is the leading cause of reproductive failure in cattle and affects other farm and domestic animals, but also induces neuromuscular disease in dogs of all ages. In cattle, neosporosis is an important health problem, and has a considerable economic impact. To date there is no protective vaccine or chemotherapeutic treatment on the market. Immuno-prophylaxis has long been considered as the best control measure. Proteins involved in host cell interaction and invasion, as well as antigens mediating inflammatory responses have been the most frequently assessed vaccine targets. However, despite considerable efforts no effective vaccine has been introduced to the market to date. The development of effective compounds to limit the effects of vertical transmission of N. caninum tachyzoites has emerged as an alternative or addition to vaccination, provided suitable targets and safe and efficacious drugs can be identified. Additionally, the combination of both treatment strategies might be interesting to further increase protectivity against N. caninum infections and to decrease the duration of treatment and the risk of potential drug resistance. Well-established and standardized animal infection models are key factors for the evaluation of promising vaccine and compound candidates. The vast majority of experimental animal experiments concerning neosporosis have been performed in mice, although in recent years the numbers of experimental studies in cattle and sheep have increased. In this review, we discuss the recent findings concerning the progress in drug and vaccine development against N. caninum infections in mice and ruminants.

Ontological representation, modeling, and analysis of parasite vaccines.

BACKGROUND: Pathogenic parasites are responsible for multiple diseases, such as malaria and Chagas disease, in humans and livestock. Traditionally, pathogenic parasites have been largely an evasive topic for vaccine design, with most successful vaccines only emerging recently. To aid vaccine design, the VIOLIN vaccine knowledgebase has collected vaccines from all sources to serve as a comprehensive vaccine knowledgebase. VIOLIN utilizes the Vaccine Ontology (VO) to standardize the modeling of vaccine data. VO did not model complex life cycles as seen in parasites. With the inclusion of successful parasite vaccines, an update in parasite vaccine modeling was needed. RESULTS: VIOLIN was expanded to include 258 parasite vaccines against 23 protozoan species, and 607 new parasite vaccine-related terms were added to VO since 2022. The updated VO design for parasite vaccines accounts for parasite life stages and for transmission-blocking vaccines. A total of 356 terms from the Ontology of Parasite Lifecycle (OPL) were imported to VO to help represent the effect of different parasite life stages. A new VO class term, 'transmission-blocking vaccine,' was added to represent vaccines able to block infectious transmission, and one new VO object property, 'blocks transmission of pathogen via vaccine,' was added to link vaccine and pathogen in which the vaccine blocks the transmission of the pathogen. Additionally, our Gene Set Enrichment Analysis (GSEA) of 140 parasite antigens used in the parasitic vaccines identified enriched features. For example, significant patterns, such as signal, plasma membrane, and entry into host, were found in the antigens of the vaccines against two parasite species: Plasmodium falciparum and Toxoplasma gondii. The analysis found 18 out of the 140 parasite antigens involved with the malaria disease process. Moreover, a majority (15 out of 54) of P. falciparum parasite antigens are localized in the cell membrane. T. gondii antigens, in contrast, have a majority (19/24) of their proteins related to signaling pathways. The antigen-enriched patterns align with the life cycle stage patterns identified in our ontological parasite vaccine modeling. CONCLUSIONS: The updated VO modeling and GSEA analysis capture the influence of the complex parasite life cycles and their associated antigens on vaccine development.

Harnessing RNA Technology to Advance Therapeutic Vaccine Antigens against Chagas Disease.

Chagas disease (CD) (American trypanosomiasis caused by Trypanosoma cruzi) is a parasitic disease endemic in 21 countries in South America, with increasing global spread. When administered late in the infection, the current antiparasitic drugs do not prevent the onset of cardiac illness leading to chronic Chagasic cardiomyopathy. Therefore, new therapeutic vaccines or immunotherapies are under development using multiple platforms. In this study, we assessed the feasibility of developing an mRNA-based therapeutic CD vaccine targeting two known T. cruzi vaccine antigens (Tc24─a flagellar antigen and ASP-2─an amastigote antigen). We present the mRNA engineering steps, preparation, and stability of the lipid nanoparticles and evaluation of their uptake by dendritic cells, as well as their biodistribution in c57BL/J mice. Furthermore, we assessed the immunogenicity and efficacy of two mRNA-based candidates as monovalent and bivalent vaccine strategies using an in vivo chronic mouse model of CD. Our results show several therapeutic benefits, including reductions in parasite burdens and cardiac inflammation, with each mRNA antigen, especially with the mRNA encoding Tc24, and Tc24 in combination with ASP-2. Therefore, our findings demonstrate the potential of mRNA-based vaccines as a therapeutic option for CD and highlight the opportunities for developing multivalent vaccines using this approach.

Protective efficacy of recombinant Toxoplasma gondii dense granule protein 15 against toxoplasmosis in C57BL/6 mice.

Toxoplasma gondii is a pervasive protozoan parasite that is responsible for significant zoonoses. A wide array of vaccines using different effector molecules of T. gondii have been studied worldwide to control toxoplasmosis. None of the existing vaccines are sufficiently effective to confer protective immunity. Among the different Toxoplasma-derived effector molecules, T. gondii dense granule protein 15 from the type II strain (GRA15 (II)) was recently characterized as an immunomodulatory molecule that induced host immunity via NF-κB. Therefore, we assessed the immunostimulatory and protective efficacy of recombinant GRA15 (II) (rGRA15) against T. gondii infection in a C57BL/6 mouse model. We observed that rGRA15 treatment increased the production of IL-12p40 from mouse peritoneal macrophages in vitro. Immunization of mice with rGRA15 induced the production of anti-TgGRA15-specific IgG, IgG1 and IgG2c antibodies. The rGRA15-sensitized spleen cells from mice inoculated with the same antigen strongly promoted spleen cell proliferation and IFN-γ production. Immunization with rGRA15 significantly enhanced the survival rate of mice and dramatically decreased parasite burden in mice challenged with the Pru (type II) strain. These results suggested that rGRA15 triggered humoral and cellular immune responses to control infection. However, all of the immunized mice died when challenged with the GRA15-deficient Pru strain or the RH (type I) strain. These results suggest that GRA15 (II)-dependent immunity plays a crucial role in protection against challenge infection with the type II strain of T. gondii. This study is the first report to show GRA15 (II) as a recombinant vaccine antigen against Toxoplasma infection.

Comparing the effect of phytobiotic, coccidiostat, toltrazuril, and vaccine on the prevention and treatment of coccidiosis in broilers.

This study compared 2 herbal anticoccidiosis drugs (water-soluble and feed-additive drugs) with monensin coccidiostat, toltrazuril (TTZ, anticoccidiosis drug), and Livacox Q (anticoccidiosis vaccine) in terms of their effects on the prevention and treatment of coccidiosis in broilers. In this study, 280 Ross 308 broiler chickens (a mix of both genders) were used in a completely randomized design with 7 treatments and 5 replications each including 8 chickens per replicate. On d 21 of rearing, all experimental groups, except for the negative control group (NC), were challenged with a mixed suspension of common strains of Eimeria, and the intended indices were assessed, including performance indices, number of oocysts per gram (OPG) of feces, intestinal injuries, and the total number of intestinal bacteria. In addition, the NC and the group receiving the monensin had greater body weight gain (BWG) (P < 0.05). At the end of week 6, the monensin group had the highest feed intake (FI), while the water soluble medicine treatment resulted in the lowest feed intake (P < 0.05). Regarding the lesion scores on day 28, the highest and lowest rates of jejunal injuries were observed in the positive control group (PC), the monensin and vaccine group respectively. The rate of oocysts excretion (oocysts per gram of feces = OPG) on different days was higher in the PC group, and the use of monensin could further reduce excretion compared to the other groups (P > 0.05). Based on a comparison of the population of lactic acid bacteria between the NC and both medicinal plant treated groups, the use of these products could increase the population of these types of bacteria. Moreover, the population of Escherichia coli was less considerable in the NC and herbal powder groups (P < 0.05). Overall, similar to commercial medicines, the herbal medicines used in this project can be effective in the prevention and treatment of coccidiosis and can improve profitability in broiler rearing centers by improving intestinal health.

Vaccination with parasite-specific TcTASV proteins combined with recombinant baculovirus as a delivery platform protects against acute and chronic Trypanosoma cruzi infection.

Chagas' is a neglected disease caused by the eukaryotic kinetoplastid parasite, Trypanosoma cruzi. Currently, approximately 8 million people are infected worldwide, most of whom are in the chronic phase of the disease, which involves cardiac, digestive, or neurologic manifestations. There is an urgent need for a vaccine because treatments are only effective in the initial phase of infection, which is generally underdiagnosed. The selection and combination of antigens, adjuvants, and delivery platforms for vaccine formulations should be designed to trigger mixed humoral and cellular immune responses, considering that T. cruzi has a complex life cycle with both intracellular and bloodstream circulating parasite stages in vertebrate hosts. Here, we report the effectiveness of vaccination with a T. cruzi-specific protein family (TcTASV), employing both recombinant proteins with aluminum hydroxide and a recombinant baculovirus displaying a TcTASV antigen at the capsid. Vaccination stimulated immunological responses by producing lytic antibodies and antigen-specific CD4+ and CD8+ IFNÉ£ secreting lymphocytes. More than 90% of vaccinated animals survived after lethal challenges with T. cruzi, whereas all control mice died before 30 days post-infection. Vaccination also induced a strong decrease in chronic tissue parasitism and generated immunological memory that allowed vaccinated and infected animals to control both the reactivation of the infection after immunosuppression and a second challenge with T. cruzi. Interestingly, inoculation with wild-type baculovirus partially protected the mice against T. cruzi. In brief, we demonstrated for the first time that the combination of the baculovirus platform and the TcTASV family provides effective protection against Trypanosoma cruzi, which is a promising vaccine for Chagas disease.

Evaluation of the immunoprotective effect of the recombinant Eimeria intestinalis rhoptry protein 25 and rhoptry protein 30 on New Zealand rabbits.

BACKGROUND: Rabbit coccidiosis is a parasitism caused by either one or multiple co-infections of Eimeria species. Among them, Eimeria intestinalis is the primary pathogen responsible for diarrhea, growth retardation, and potential mortality in rabbits. Concerns regarding drug resistance and drug residues have led to the development of recombinant subunit vaccines targeting Eimeria species as a promising preventive measure. The aim of this study was to assess the immunoprotective efficacy of recombinant subunit vaccines comprising EiROP25 and EiROP30 (rhoptry proteins (ROPs)) against E. intestinalis infection in rabbits. METHODS: Cloning, prokaryotic expression, and protein purification were performed to obtain EiROP25 and EiROP30. Five groups of fifty 35-day-old Eimeria-free rabbits were created (unchallenged control group, challenged control group, vector protein control group, rEiROP25 group, and rEiROP30 group), with 10 rabbits in each group. Rabbits in the rEiROP25 and rEiROP30 groups were immunized with the recombinant proteins (100 µg per rabbit) for primary and booster immunization (100 µg per rabbit) at a two-week intervals, and challenged with 7 × 104 oocysts per rabbit after an additional two-week interval. Two weeks after the challenge, the rabbits were euthanized for analysis. Weekly collections of rabbit sera were made to measure changes in specific IgG and cytokine level. Clinical symptoms and pathological changes after challenge were observed and recorded. At the conclusion of the animal experiment, lesion scores, the relative weight increase ratio, the oocyst reduction rate, and the anticoccidial index were computed. RESULTS: Rabbits immunized with rEiROP25 and rEiROP30 exhibited relative weight gain ratios of 56.57% and 72.36%, respectively. Oocysts decreased by 78.14% and 84.06% for the rEiROP25 and rEiROP30 groups, respectively. The anticoccidial indexes were 140 and 155. Furthermore, there was a noticeable drop in intestinal lesions. After the primary immunization with rEiROP25 and rEiROP30, a week later, there was a notable rise in specific IgG levels, which remained elevated for two weeks following challenge (P < 0.05). Interleukin (IL)-2 levels increased markedly in the rEiROP25 group, whereas IL-2, interferon gamma (IFN-γ), and IL-4 levels increased substantially in the rEiROP30 group (P < 0.05). CONCLUSION: Immunization of rabbits indicated that both rEiROP25 and rEiROP30 are capable of inducing an increase in specific antibody levels. rEiROP25 triggered a Th1-type immune protection response, while rEiROP30 elicited a Th1/Th2 mixed response. EiROP25 and EiROP30 can generate a moderate level of immune protection, with better efficacy observed for EiROP30. This study provides valuable insights for the promotion of recombinant subunit vaccines targeting rabbit E. intestinalis infection.

Comparison of the immune response and protection against the experimental Toxoplasma gondii infection elicited by immunization with the recombinant proteins BAG1, ROP8, and BAG1-ROP8.

Toxoplasmosis is one of the most dangerous zoonotic diseases, causing serious economic losses worldwide due to abortion and reproductive problems. Vaccination is the best way to prevent disease; thus, it is imperative to develop a candidate vaccine for toxoplasmosis. BAG1 and ROP8 have the potential to become vaccine candidates. In this study, rTgBAG1, rTgROP8, and rTgBAG1-rTgROP8 were used to evaluate the immune effect of vaccines in each group by detecting the humoral and cellular immune response levels of BABL/c mice after immunization and the ability to resist acute and chronic infection with Toxoplasma gondii (T. gondii). We divided the mice into vaccine groups with different proteins, and the mice were immunized on days 0, 14, and 28. The protective effects of different proteins against T. gondii were analysed by measuring the cytokines, serum antibodies, splenocyte proliferation assay results, survival time, and number and diameter of brain cysts of mice after infection. The vaccine groups exhibited substantially higher IgG, IgG1, and IgG2a levels and effectively stimulated lymphocyte proliferation. The levels of IFN-γ and IL-2 in the vaccine group were significantly increased. The survival time of the mice in each vaccine group was prolonged and the diameter of the cysts in the vaccine group was smaller; rTgBAG1-rTgROP8 had a better protection. Our study showed that the rTgBAG1, rTgROP8, and rTgBAG1-rTgROP8 recombinant protein vaccines are partial but effective approaches against acute or chronic T. gondii infection. They are potential candidates for a toxoplasmosis vaccine.

Protective effect against toxoplasmosis in BALB/C mice vaccinated with recombinant Toxoplasma gondii CDPK3, GRA35, and ROP46 protein cocktail vaccine.

Toxoplasma gondii (T. gondii) is one of the most common pathogenic protozoa in the world, and causes toxoplasmosis, which in varying degrees causes significant economic losses and poses a serious public health challenge globally. To date, the development of an effective vaccine for human toxoplasmosis remains a challenge. Given that T.gondii calcium-dependent protein kinase 3 (CDPK3), dense granule protein 35 (GRA35) and rhoptry organelle protein 46 (ROP46) play key roles during Toxoplasma gondii invasion of host cells, we developed a protein vaccine cocktail including these proteins and validated its protective efficacy. The specific protective effects of vaccine on mice were analyzed by measuring serum antibodies, cytokines, splenocyte proliferation, the percentage of CD4+ and CD8+ T-lymphocytes, survival rate, and parasite cyst burden. The results showed that mice vaccinated with a three-protein cocktail produced the highest levels of immune protein antibodies to IgG, and high levels of IFN-γ, IL-2, IL-4, and IL-10 compared to other mice vaccinated with two proteins. In addition, CD4+ and CD8+ T cell percentages were significantly elevated. Compared to the control groups, mice vaccinated with the three-protein cocktail survived significantly longer after acute infection with T. gondii and had significantly fewer cysts after chronic infection. These results demonstrated that a cocktail vaccine of TgCDPK3, TgGRA35, and TgROP46 can effectively induce cellular and humoral immune responses with good protective effects in mice, indicating its potential as vaccine candidates for toxoplasmosis.

Immunoprotective effects of DNA vaccine against Eimeria tenella based on EtAMA3 and EtRON2L2.

Eimeria tenella is the most pathogenic and harmful intestinal parasitic protozoan. Recombinant DNA vaccines open options for promising strategies for preventing avian coccidiosis, replacing chemical drugs and live oocyst vaccines. Two important antigenic proteins, EtAMA3 (also known as SporoAMA1) and EtRON2L2, act together to promote the invasion of E. tenella sporozoites. In this study, a recombinant DNA vaccine, designated pcDNA3.1(+)-AR, was constructed based on EtAMA3DII, EtRON2L2D3, and EtRON2L2D4. Chickens were intramuscularly immunized with different doses (25, 50, or 100 µg) of pcDNA3.1(+)-AR to evaluate its immunoprotective effects in vivo. The chickens in the 50 µg and 100 µg groups had higher cytokine concentrations (interleukin 2, interferon-gamma, and interleukin 10), and lesion scores (81.9% and 67.57%, respectively) and relative oocyst production (47% and 19%, respectively) reduced compared with the unchallenged group, indicating partial protection against E. tenella. These results suggest that pcDNA3.1(+)-AR is a promising vaccine candidate against avian coccidiosis.

Memory-like NK Cells Are a Critical Component of Vaccine-Induced Immunity to Trypanosoma cruzi Infection.

Chagas disease by Trypanosoma cruzi infection is a major public health issue. The available therapeutic agents have limited efficacy and significant side effects. A reliable vaccine would reduce the threat of T. cruzi infections and prevent Chagas disease. Understanding the immune response to this infection would improve vaccine design. We previously demonstrated that adoptively transferred NK cells from mice immunized with highly attenuated T. cruzi, GFP-DDDHA strain, provided potent protection in naive recipients against secondary lethal challenge with various wild-type (WT) strains. To understand the importance of NK cells in protecting mice against T. cruzi infection, we performed an in-depth characterization of NK cell phenotype, responses, and memory-like traits during acute infections due to GFP-DDDHA and WT strains and in immunized mice during a recall response to a WT lethal challenge. NK cells robustly expanded and became more mature and cytolytic during the GFP-DDDHA strain immunization. NK cells in immunized mice responded more robustly after WT lethal challenge than during an acute primary WT infection. In addition, protection by immunization with the GFP-DDDHA strain is significantly weakened in NK cell-deficient mice and did not prevent parasitemia from WT lethal challenge, indicating that NK cells with memory-like traits were a critical component for early control of WT lethal challenge. Prior T. cruzi vaccine development studies have not included studies of this rapid NK response. These findings provide insights into overcoming existing challenges in developing a safe and effective vaccine to prevent this infection.

Intranasal delivery of LaAg vaccine improves immunity of aged mice against visceral Leishmaniasis.

There are no approved vaccines yet for human visceral leishmaniasis (VL), the most severe form of the leishmaniasis clinical manifestations that is fatal in over 95 % of untreated cases. It is well-accepted that immunological changes during aging have deleterious impact on the efficacy of vaccines and response to infections. In this work, we compared the response of young and aged mice to intranasal vaccination with killed Leishmania amazonensis promastigote antigens (LaAg) that were then challenged with L. infantum infection, a species that causes visceral leishmaniasis. Intranasal vaccination with LaAg induced a similar reduction in parasitism and hepatosplenomegaly in both young and aged mice compared to their unvaccinated counterparts. Following infection, there was also a less prominent inflammatory profile particularly in the vaccinated aged group, with lower production of TNF-α and nitrite compared to the respective unvaccinated group. Interestingly, the LaAg intranasal vaccination promoted increased production of IFN-γ that was observed in both young- and aged vaccinated groups. Additionally, CD4+ and CD8+T cells from both vaccinated groups presented decreased expression of the inhibitory receptors PD-1 and KLRG1 compared to their unvaccinated controls. Interestingly, a strong positive correlation was observed between the expression of both inhibitory receptors PD-1 and KLRG1 and parasitism, which was more conspicuous in the unvaccinated-aged mice than in the others. Overall, this study helps define new strategies to improve vaccine effectiveness and provides a perspective for prophylactic alternatives against leishmaniasis.

Molecular characterization and immune protective efficacy of 3 Eimeria tenella antigens.

Avian coccidiosis caused by Eimeria is a serious parasitic disease that poses a threat to the poultry industry. Currently, prevention and treatment mainly rely on the administration of anticoccidials and live oocyst vaccines. However, the prevalence of drug resistance and the inherent limitations of live vaccines have driven the development of novel vaccines. In this study, the surface protein (Et-SAG14), a previously annotated rhoptry protein (Eten5-B), and a gametocyte phosphoglucomutase (Et-PGM1) were characterized and the vaccine potential of the recombinant proteins were evaluated. Et-SAG14 was dispersed in the form of particles in the sporozoite and merozoite stages, whereas Et-PGM1 was distributed in the apical part of the sporozoite and merozoite stages. The previously annotated rhoptry Eten5-B was found not to be located in the rhoptry but distributed in the cytoplasm of sporozoites and merozoites. Immunization with rEten5-B significantly elevated host interferon gamma (IFN-γ) and interleukin 10 (IL-10) transcript levels and exhibited moderate anticoccidial effects with an anticoccidial index (ACI) of 161. Unexpectedly, both recombinant Et-SAG14 and Et-PGM1 immunization significantly reduced host IFN-γ and IL-10 transcription levels, and did not show protection against E. tenella challenge (ACI < 80). These results suggest that the rEten5-B protein can trigger immune protection against E. tenella and may be a potential and effective subunit vaccine for the control of coccidiosis in poultry.

Protective efficacy of the oral vaccine Tc24:Co1 produced in Schizochytrium sp. against Trypanosoma cruzi infection in a mouse model.

Trypanosoma cruzi parasite - causal Chagas disease agent - affects about 7 million people; no vaccine is available, and current medications have not been entirely effective. Multidisciplinary efforts are necessary for developing clinical vaccine prototypes. Thus, this research study aims to assess the expressed and whole-cell administration protection of the oral vaccine prototype Tc24:Co1 using Schizochytrium sp. microalga. High recombinant protein expression yields (675 µg/L) of algal culture were obtained. Additionally, Schizochytrium sp.-Tc24:Co1 resulted stable at 4 °C for up to six months and at 25 °C for three months. After receiving four oral doses of the vaccine, the mice showed a significant humoral immune response and a parasitemia reduction associated with a lack of heart inflammatory damage compared with the unvaccinated controls. The Schizochytrium sp.-Tc24:Co1 vaccine demonstrates to be promising as a prototype for further development showing protective effects against a T. cruzi challenge in a mouse model.

Protective efficacy of multiepitope vaccines constructed from common antigens of Eimeria species in chickens.

Clinical avian coccidiosis is typically caused by coinfection with several Eimeria species. Recombinant protein and DNA vaccines have shown promise in controlling coccidiosis. On this basis, DNA vaccines that encode multiple epitopes from different Eimeria species may provide broad protection against coinfections. In this study, we designed a fusion gene fragment, 14EGT, that contained concentrated T-cell epitopes from four common antigens of Eimeria species (14-3-3, elongation factor 2, glyceraldehyde-3-phosphate dehydrogenase, and transhydrogenase). The multiepitope DNA vaccine pVAX1-14EGT and recombinant protein vaccine pET-32a-14EGT (r14EGT) were then created based on the 14EGT fragment. Subsequently, cellular and humoral immune responses were measured in vaccinated chickens. Vaccination-challenge trials were also conducted, where the birds were vaccinated with the 14EGT preparations and later exposed to single or multiple Eimeria species to evaluate the protective efficacy of the vaccines. According to the results, vaccination with 14EGT preparations effectively increased the proportions of CD4+ and CD8+ T cells and the levels of Th1 and Th2 hallmark cytokines. The levels of serum IgG antibodies were also significantly increased. Animal vaccination trials revealed alleviated enteric lesions, weight loss, and oocyst output compared to those of the control groups. The preparations were found to be moderately effective against single Eimeria species, with the anticoccidial index (ACI) ranging from 160 to 180. However, after challenge with multiple Eimeria species, the protection provided by the 14EGT preparations was not satisfactory, with ACI values of 142.18 and 146.41. Collectively, the results suggest that a multiepitope vaccine that encodes the T-cell epitopes of common antigens derived from Eimeria parasites could be a potential and effective strategy to control avian coccidiosis.

Immunisation with Neospora caninum subunits rsNcSAG4 and rsNcGRA1 (NcSAG4 and NcGRA1 epitopes construct) in BALB/c mice: the profile of the immune response and controlling the vertical transmission.

Neospora caninum is an apicomplexan protozoan that causes neosporosis, which has a high economic impact on cattle herds with no available vaccine. During infection, the secretion of dense granules and the expression of surface antigens play an important role in hosting immunomodulation. However, some epitopes of those antigens are immunogenic, and using these fractions could improve the subunit antigens in vaccine design. This study evaluates the recombinant peptides rsNcGRA1 and rsNcSAG4 derived from NcGRA1 and NcSAG4 native antigens as vaccine candidates produced by a fermentative process in the yeast culture system of Komagataella phaffii strain Km71, confirmed by colony PCR, SDS-PAGE, and western blotting. The assay was conducted in BALB/c mice using the peptides at low (25 µg) and standard (50 µg) dosages in monovalent and combined administrations at three time points with saponin as an adjuvant assessing the immunogenicity by antibodies response and cytokine production. We challenge the females after pregnancy confirmation using 2 × 105 NC-1 tachyzoites previously propagated in Vero cells. We assessed the chronic infection in dams and vertical transmission in the offspring by PCR and histopathology. Mice, especially those immunised with combined peptides and monovalent rsNcGRA1 at a standard dose, controlling the chronic infection in dams with the absence of clinical manifestations, showed an immune response with induction of IgG1, a proper balance between Th1/Th2 cytokines and reduced vertical transmission in the pups. In contrast, dams inoculated with a placebo vaccine showed clinical signs, low-scored brain lesions, augmented chronic infection with 80% positivity, 31% mortality in pups, and 81% vertical transmission. These findings indicate that rsNcGRA1 peptides in monovalent and combined with rsNCSAG4 at standard dose are potential vaccine candidates and improve the protective immune response against neosporosis in mice.

Intranasal immunisation with recombinant Toxoplasma gondii uridine phosphorylase confers resistance against acute toxoplasmosis in mice.

Toxoplasmosis is caused by Toxoplasma gondii, which infects all warm-blooded animals, including humans. Currently, control measures for T. gondii infection are insufficient due to the lack of effective medications or vaccines. In this paper, recombinant T. gondii uridine phosphorylase (rTgUPase) was expressed in Escherichia coli and purified via Ni2+-NTA agarose. rTgUPase was inoculated intranasally into BALB/c mice, and the induced immune responses were evaluated by mucosal and humoral antibody and cytokine assays and lymphoproliferative measurements. Moreover, the protective effect against the T. gondii RH strain infection was assessed by calculating the burdens of tachyzoites in the liver and brain and by recording the survival rate and time. Our results revealed that mice immunised with 30 µg rTgUPase produced significantly higher levels of secretory IgA (sIgA) in nasal, intestinal, vaginal and vesical washes and synthesised higher levels of total IgG, IgG1 and, in particular, IgG2a in their blood sera. rTgUPase immunisation increased the production of IFN-gamma, interleukin IL-2 and IL-4, but not IL-10 from isolated mouse spleen cells and enhanced splenocyte proliferation in vitro. rTgUPase-inoculated mice were effectively protected against infection with the T. gondii RH strain, showing considerable reduction of tachyzoite burdens in liver and brain tissues after 30 days of infection, and a 44.29% increase in survival rate during an acute challenge. The above findings show that intranasal inoculation with rTgUPase provoked mucosal, humoral and cellular immune responses and indicate that rTgUPase might serve as a promising vaccine candidate for protecting against toxoplasmosis.

Title: L'immunisation intranasale avec l'uridine phosphorylase recombinante de Toxoplasma gondii confère une résistance contre la toxoplasmose aiguë chez la souris. Abstract: La toxoplasmose est causée par Toxoplasma gondii, qui infecte tous les animaux à sang chaud, y compris les humains. Actuellement, les mesures de contrôle de l'infection à T. gondii sont insuffisantes en raison du manque de médicaments ou de vaccins efficaces. Dans cet article, l'uridine phosphorylase recombinante de T. gondii (rTgUPase) a été exprimée dans Escherichia coli et purifiée via de l'agarose Ni2+-NTA. La rTgUPase a été inoculée par voie intranasale à des souris BALB/c et les réponses immunitaires induites ont été évaluées par des dosages d'anticorps et de cytokines muqueuses et humorales et par des mesures de lymphoprolifération. De plus, l'effet protecteur contre l'infection par la souche RH de T. gondii a été évalué en calculant la charge de tachyzoïtes dans le foie et le cerveau et en enregistrant le taux et la durée de survie. Nos résultats ont révélé que les souris immunisées avec 30 µg de rTgUPase produisaient des taux significativement plus élevés d'IgA sécrétoires (sIgA) dans les lavages nasaux, intestinaux, vaginaux et vésicaux et synthétisaient des taux plus élevés d'IgG totales, d'IgG1 et, en particulier, d'IgG2a dans leur sérum sanguin. L'immunisation par la rTgUPase a augmenté la production d'IFN-gamma, d'interleukine IL-2 et IL-4, mais pas d'IL-10 à partir de cellules de rate de souris isolées et a amélioré la prolifération des splénocytes in vitro. Les souris inoculées par la rTgUPase ont été efficacement protégées contre l'infection par la souche RH de T. gondii, montrant une réduction considérable de la charge de tachyzoïtes dans les tissus hépatiques et cérébraux après 30 jours d'infection et une augmentation de 44,29 % du taux de survie lors d'une épreuve aiguë. Les résultats ci-dessus montrent que l'inoculation intranasale de rTgUPase provoque des réponses immunitaires muqueuses, humorales et cellulaires et indiquent que la rTgUPase pourrait servir de candidat vaccin prometteur pour la protection contre la toxoplasmose.