With Chitosan and PLGA as the Delivery Vehicle, Toxoplasma gondii Oxidoreductase-Based DNA Vaccines Decrease Parasite Burdens in Mice.

Toxoplasma gondii (T. gondii) is an intracellular parasitic protozoan that can cause serious public health problems. However, there is no effectively preventive or therapeutic strategy available for human and animals. In the present study, we developed a DNA vaccine encoding T. gondii oxidoreductase from short-chain dehydrogenase/reductase family (TgSDRO-pVAX1) and then entrapped in chitosan and poly lactic-co-glycolic acid (PLGA) to improve the efficacy. When encapsulated in chitosan (TgSDRO-pVAX1/CS nanospheres) and PLGA (TgSDRO-pVAX1/PLGA nanospheres), adequate plasmids were loaded and released stably. Before animal immunizations, the DNA vaccine was transfected into HEK 293-T cells and examined by western blotting and laser confocal microscopy. Th1/Th2 cellular and humoral immunity was induced in immunized mice, accompanied by modulated secretion of antibodies and cytokines, promoted the maturation and MHC expression of dendritic cells, and enhanced the percentages of CD4+ and CD8+ T lymphocytes. Immunization with TgSDRO-pVAX1/CS and TgSDRO-pVAX1/PLGA nanospheres conferred significant immunity with lower parasite burden in the mice model of acute toxoplasmosis. Furthermore, our results also lent credit to the idea that TgSDRO-pVAX1/CS and TgSDRO-pVAX1/PLGA nanospheres are substitutes for each other. In general, the current study proposed that TgSDRO-pVAX1 with chitosan or PLGA as the delivery vehicle is a promising vaccine candidate against acute toxoplasmosis.

Development of a Coccidiosis Disease Challenge Model Using a Commercially Available Live Oocyst Vaccine.

With growing cross-disciplinary collaboration among researchers, it is increasingly important to record detailed methodology to prevent the repetition of preliminary experiments. The purpose of this paper is to explain the development of a coccidiosis challenge model for the investigation of dietary interventions to coccidiosis in broiler chickens. The objectives are to select a dose of mixed species coccidial vaccine and evaluate the suitability (ability to produce a consistent, marked change) of selected response variables important to nutritional studies at different times postinfection (PI). Coccivac-B and Coccivac-B52 (Merck Animal Health) were evaluated as the source of coccidia in three trials. Trials 1 and 2 were randomized complete block designs with four doses (0, 10, 20, or 30 times (×) label dose) of Coccivac-B administered to 12 replicate cages of six birds by repeater pipette (Trial 1) or gavaging needle (Trial 2). Trial 3 used a completely randomized design with 0× or 30× label dose of Coccivac-B52 administered by gavaging needle to six replicate cages of six birds. Birds were gavaged at 15 days of age, and response criteria were evaluated 7 days PI in all trials and again at 10 days PI in Trials 1 and 2. All means are reported in order of increasing coccidia dose with significance accepted at P ≤ 0.05. Broiler performance was not affected by coccidia in Trials 1 or 3 but grew poorer with increasing dose from 0 to 7 days PI in Trial 2 (body weight gain, 465, 421, 388, 365 g; feed to gain, 1.37, 1.47, 1.52, 1.58). As coccidia dose increased, nitrogen corrected apparent metabolizable energy decreased (Trial 1, 3387, 3318, 3267, 3170 kcal kg-1; Trial 2, 3358, 2535, 2422, 2309 kcal kg-1; Trial 3, not measured), while relative weight, length, and content for intestinal sections increased (Trials 1through 3). Gross lesion (duodenum, jejunum/ileum, ceca) and oocyst count scores (jejunum/ileum, ceca) increased with dose; however, gross scoring often suggested infection in unchallenged birds, a finding unsupported by oocyst count scores. At 7 days PI there was no correlation between midgut gross lesion score and midgut oocyst count score (r = 0.06, P = 0.705), but cecal scores were weakly correlated (r = 0.55, P < 0.001). Administering coccidia via repeater pipette (Trial 1) resulted in respiratory distress in some birds, while use of the gavaging needle (Trials 2 and 3) successfully induced intestinal damage in chickens without resulting in coccidia related mortality. Thirty times the label dose at 7 days PI resulted in the greatest number of response variables that produced a consistent, marked change. Therefore, consideration should be given to these conditions when designing future coccidiosis challenge models using vaccines as a source of coccidia.

Artículo regular­Desarrollo de un modelo de desafío para coccidiosis utilizando una vacuna de ooquistes vivos disponible comercialmente. Con la creciente colaboración interdisciplinaria entre investigadores, es cada vez más importante registrar la metodología detallada para evitar la repetición de experimentos preliminares. El propósito de este artículo es explicar el desarrollo de un modelo de desafío de coccidiosis para la investigación de intervenciones dietéticas para coccidiosis en pollos de engorde. Los objetivos son seleccionar una dosis de vacuna coccidial de especies mixtas y evaluar la idoneidad (capacidad de producir un cambio marcado y consistente) de las variables de respuesta seleccionadas que son importantes para los estudios nutricionales en diferentes momentos posteriores a la infección (PI). Las vacunas Coccivac-B o Coccivac B-52 (Merck Animal Health) se evaluaron como fuente de coccidias en tres ensayos. Los ensayos 1 y 2 fueron diseños de bloques completamente aleatorios con cuatro dosis (0, 10, 20 o 30 veces (×) la dosis indicada en la etiqueta) de Coccivac-B administradas a 12 jaulas repetidas de seis aves mediante una pipeta repetidora (ensayo 1) o por sonda oral. (Prueba 2). El ensayo 3 utilizó un diseño completamente aleatorio con una dosis de etiqueta de 0 × o 30 × de Coccivac-B52 administrada con una sonda oral en seis jaulas repetidas de seis aves. Las aves fueron inoculadas por sonda a los 15 días de edad y los criterios de respuesta se evaluaron a los 7 días postinoculación en todos los ensayos y nuevamente a los 10 días postinoculación en los ensayos 1 y 2. Todos los promedios se reportan en orden de dosis crecientes de coccidias con significancia aceptada en P ≤ 0.05. El rendimiento de los pollos de engorde no se vio afectado por las coccidias en los Ensayos 1 o 3, pero empeoró al aumentar la dosis de los cero a 7 días después de la inoculación en el Ensayo 2 (aumento de peso corporal, 465, 421, 388, 365 g; alimento para ganar, 1.37, 1.47, 1.52, 1.58). A medida que aumentaba la dosis de coccidia, la energía metabolizable de nitrógeno aparente y corregida disminuyó (Prueba 1, 3387, 3318, 3267, 3170 kcal kg-1; Prueba 2, 3358, 2535, 2422, 2309 kcal kg-1; Prueba 3, no medida), mientras que el peso relativo, la longitud y el contenido de las secciones intestinales aumentaron (ensayos 1 a 3). La lesión macroscópica (duodeno, yeyuno/íleon, ciego) y las puntuaciones del recuento de oocistos (yeyuno/íleon, ciego) aumentaron con la dosis; sin embargo, la puntuación bruta a menudo sugirió infección en aves no desafiadas, un hallazgo que no está respaldado por las puntuaciones del recuento de ooquistes. A los 7 días después de la infección no hubo correlación entre la puntuación de la lesión macroscópica del intestino medio y la puntuación del recuento de oocistos del intestino medio (r= 0,06, P= 0,705), pero las puntuaciones cecales se correlacionaron débilmente (r = 0.55, P <0.001). La administración de coccidias a través de una pipeta repetidora (Ensayo 1) provocó dificultad respiratoria en algunas aves, mientras que el uso de la sonda oral (Ensayos 2 y 3) indujo con éxito el daño intestinal en los pollos sin dar como resultado mortalidad relacionada con los coccidias. Treinta veces la dosis de la etiqueta a los 7 días después de la infección resultó en el mayor número de variables de respuesta que produjeron un cambio marcado y consistente. Por lo tanto, deben tenerse en cuenta estas condiciones al diseñar futuros modelos de exposición a la coccidiosis que utilicen vacunas como fuente de coccidias.

Optimizing a Multi-Component Intranasal Entamoeba Histolytica Vaccine Formulation Using a Design of Experiments Strategy.

Amebiasis is a neglected tropical disease caused by Entamoeba histolytica. Although the disease burden varies geographically, amebiasis is estimated to account for some 55,000 deaths and millions of infections globally per year. Children and travelers are among the groups with the greatest risk of infection. There are currently no licensed vaccines for prevention of amebiasis, although key immune correlates for protection have been proposed from observational studies in humans. We previously described the development of a liposomal adjuvant formulation containing two synthetic TLR ligands (GLA and 3M-052) that enhanced antigen-specific fecal IgA, serum IgG2a, a mixed IFNγ and IL-17A cytokine profile from splenocytes, and protective efficacy following intranasal administration with the LecA antigen. By applying a statistical design of experiments (DOE) and desirability function approach, we now describe the optimization of the dose of each vaccine formulation component (LecA, GLA, 3M-052, and liposome) as well as the excipient composition (acyl chain length and saturation; PEGylated lipid:phospholipid ratio; and presence of antioxidant, tonicity, or viscosity agents) to maximize desired immunogenicity characteristics while maintaining physicochemical stability. This DOE/desirability index approach led to the identification of a lead candidate composition that demonstrated immune response durability and protective efficacy in the mouse model, as well as an assessment of the impact of each active vaccine formulation component on protection. Thus, we demonstrate that both GLA and 3M-052 are required for statistically significant protective efficacy. We also show that immunogenicity and efficacy results differ in female vs male mice, and the differences appear to be at least partly associated with adjuvant formulation composition.

Immunomics-Guided Antigen Discovery for Praziquantel-Induced Vaccination in Urogenital Human Schistosomiasis.

Despite the enormous morbidity attributed to schistosomiasis, there is still no vaccine to combat the disease for the hundreds of millions of infected people. The anthelmintic drug, praziquantel, is the mainstay treatment option, although its molecular mechanism of action remains poorly defined. Praziquantel treatment damages the outermost surface of the parasite, the tegument, liberating surface antigens from dying worms that invoke a robust immune response which in some subjects results in immunologic resistance to reinfection. Herein we term this phenomenon Drug-Induced Vaccination (DIV). To identify the antigenic targets of DIV antibodies in urogenital schistosomiasis, we constructed a recombinant proteome array consisting of approximately 1,000 proteins informed by various secretome datasets including validated proteomes and bioinformatic predictions. Arrays were screened with sera from human subjects treated with praziquantel and shown 18 months later to be either reinfected (chronically infected subjects, CI) or resistant to reinfection (DIV). IgG responses to numerous antigens were significantly elevated in DIV compared to CI subjects, and indeed IgG responses to some antigens were completely undetectable in CI subjects but robustly recognized by DIV subjects. One antigen in particular, a cystatin cysteine protease inhibitor stood out as a unique target of DIV IgG, so recombinant cystatin was produced, and its vaccine efficacy assessed in a heterologous Schistosoma mansoni mouse challenge model. While there was no significant impact of vaccination with adjuvanted cystatin on adult worm numbers, highly significant reductions in liver egg burdens (45-55%, P<0.0001) and intestinal egg burdens (50-54%, P<0.0003) were achieved in mice vaccinated with cystatin in two independent trials. This study has revealed numerous antigens that are targets of DIV antibodies in urogenital schistosomiasis and offer promise as subunit vaccine targets for a drug-linked vaccination approach to controlling schistosomiasis.

Evaluation of the PEΔIII-LC3-KDEL3 Chimeric Protein of Entamoeba histolytica-Lectin as a Vaccine Candidate against Amebic Liver Abscess.

Entamoeba histolytica is an intestinal parasite that causes dysentery and amebic liver abscess. E. histolytica has the capability to invade host tissue by union of virulence factor Gal/GalNAc lectin; this molecule induces an adherence-inhibitory antibody response as well as to protect against amebic liver abscess (ALA). The present work showed the effect of the immunization with PEΔIII-LC3-KDEL3 recombinant protein. In vitro, this candidate vaccine inhibited adherence of E. histolytica trophozoites to HepG2 cell monolayer, avoiding the cytolysis, and in a hamster model, we observed a vaccine-induced protection against the damage to tissue liver and the inhibition of uncontrolled inflammation. PEΔIII-LC3-KDEL3 reduced the expression of TNF-α, IL-1ß, and NF-κB in all immunized groups at 4- and 7-day postinfection. The levels of IL-10, FOXP3, and IFN-γ were elevated at 7 days. The immunohistochemistry assay confirmed this result, revealing an elevated quantity of +IFN-γ cells in the liver tissue. ALA formation in hamsters immunized was minimal, and few trophozoites were identified. Hence, immunization with PEΔIII-LC3-KDEL3 herein prevented invasive amebiasis, avoided an acute proinflammatory response, and activated a protective response within a short time. Finally, this recombinant protein induced an increase of serum IgG.

Assessment of Babesia bovis 6cys A and 6cys B as components of transmission blocking vaccines for babesiosis.

BACKGROUND: Babesia bovis reproduces sexually in the gut of its tick vector Rhipicephalus microplus, which involves expression of 6cys A and 6cys B proteins. Members of the widely conserved 6cys superfamily are candidates for transmission blocking vaccines (TBV), but intricacies in the immunogenicity of the 6cys proteins in the related Plasmodium parasites required the identification of transmission blocking domains in these molecules for vaccine design. Hereby, the immunogenic efficacy of recombinant (r) B. bovis 6cys A and B proteins as a TBV formulation was studied. METHODS: The immunogenicity of r6cys A and 6cys B proteins expressed in a eukaryotic system was evaluated in a cattle immunization trial (3 immunized and 3 control calves). A B. bovis sexual stage induction in vitro inhibition assay to assess the ability of antibodies to block the production of sexual forms by the parasite was developed. RESULTS: Immunized cattle generated antibodies against r6cys A and r6cys B that were unable to block sexual reproduction of the parasite in ticks. Additionally, these antibodies also failed in recognizing native 6cys A and 6cys B and peptides representing 6cys A and 6cys B functional domains and in inhibiting the development of sexual forms in an in vitro induction system. In contrast, rabbit antibodies generated against synthetic peptides representing predicted B-cell epitopes of 6cys A and 6cys B recognized recombinant and native forms of both 6cys proteins as well as peptides representing 6cys A and 6cys B functional domains and were able to neutralize development of sexual forms of the parasite in vitro. CONCLUSIONS: These data, combined with similar work performed on Plasmodium 6cys proteins, indicate that an effective 6cys protein-based TBV against B. bovis will require identifying and targeting selected regions of proteins containing epitopes able to reduce transmission.

Systems Biology Analysis of the Radiation-Attenuated Schistosome Vaccine Reveals a Role for Growth Factors in Protection and Hemostasis Inhibition in Parasite Survival.

In spite of several decades of research, an effective vaccine against schistosomiasis remains elusive. The radiation-attenuated (RA) cercarial vaccine is still the best model eliciting high protection levels, although the immune mechanisms have not yet been fully characterized. In order to identify genes and pathways underlying protection we investigated patterns of gene expression in PBMC and skin draining Lymph Nodes (LN) from mice using two exposure comparisons: vaccination with 500 attenuated cercariae versus infection with 500 normal cercariae; one versus three doses. Vaccinated mice were challenged with 120 normal parasites. Integration of PBMC and LN data from the infected group revealed early up-regulation of pathways associated with Th2 skewing and polarization of IgG antibody profiles. Additionally, hemostasis pathways were downregulated in infected mice, correlating with platelet reduction, potentially a mechanism to assist parasite migration through capillary beds. Conversely, up regulation of such mechanisms after vaccination may explain parasite blockade in the lungs. In contrast, a single exposure to attenuated parasites revealed early establishment of a Th1 bias (signaling of IL-1, IFN-γ; and Leishmania infection). Genes encoding chemokines and their receptors were more prominent in vaccinated mice, indicating an enhanced capacity for inflammation, potentially augmenting the inhibition of intravascular migration. Increasing the vaccinations from one to three did not dramatically elevate protection, but there was a clear shift towards antibody-mediated effectors. However, elements of the Th1 bias were still evident. Notable features after three vaccinations were markers of cytotoxicity (including IL-6 and NK cells) together with growth factors and their receptors (FGFR/VEGF/EGF) and the apoptosis pathway. Indeed, there is evidence for the development of anergy after three vaccinations, borne out by the limited responses detected in samples after challenge. We infer that persistence of a Th1 response puts a limit on expression of antibody-mediated mechanisms. This feature may explain the failure of multiple doses to drive protection towards sterile immunity. We suggest that the secretions of lung stage parasites would make a novel cohort of antigens for testing in protection experiments.

Research Note: The administration schedule of coccidia is a major determinant in broiler necrotic enteritis models.

A reliable and reproducible in vivo experimental model is an essential tool to study the pathogenesis of broiler necrotic enteritis and to evaluate control methods. Most current in vivo models use Eimeria as predisposing factor. Nevertheless, most models only result in a limited number of animals with intestinal necrosis. This research describes the necrotic enteritis incidence and severity using 2 previously described experimental models varying in the time point and frequency of Eimeria administration: single late and early repeated Eimeria administration models. In an in vivo model in which Clostridium perfringens is administered at 3 consecutive days between day 18 and 20 of age, birds belonging to the single late Eimeria administration regimen received a single administration of a tenfold dose of a live attenuated Eimeria vaccine on the second day of C. perfringens challenge. Broilers belonging to the early repeated administration regimen were inoculated with the same Eimeria vaccine 4 and 2 d before the start of the C. perfringens challenge. Early repeated coccidial administration resulted in a significant increase in average necrotic lesion score (value 3.26) as compared with a single late Eimeria administration regimen (value 1.2). In addition, the number of necrotic enteritis-positive animals was significantly higher in the group that received the early repeated coccidial administration. Single Eimeria administration during C. perfringens challenge resulted in a skewed distribution of lesion scoring with hardly any birds in the high score categories. A more centered distribution was obtained with the early repeated Eimeria administration regimen, having observations in every lesion score category. These findings allow better standardization of a subclinical necrotic enteritis model and reduction of the required numbers of experimental animals.

ASCVac-1, a Multi-Peptide Chimeric Vaccine, Protects Mice Against Ascaris suum Infection.

Control of human ascariasis, the most prevalent neglected tropical disease globally affecting 450 million people, mostly relies on mass drug administration of anthelmintics. However, chemotherapy alone is not efficient due to the high re-infection rate for people who live in the endemic area. The development of a vaccine that reduces the intensity of infection and maintains lower morbidity should be the primary target for infection control. Previously, our group demonstrated that immunization with crude Ascaris antigens in mice induced an IgG-mediated protective response with significant worm reduction. Here, we aimed to develop a multipeptide chimera vaccine based on conserved B-cell epitopes predicted from 17 common helminth proteomes using a bioinformatics algorithm. More than 480 B-cell epitopes were identified that are conserved in all 17 helminths. The Ascaris-specific epitopes were selected based on their reactivity to the pooled sera of mice immunized with Ascaris crude antigens or infected three times with A. suum infective eggs. The top 35 peptides with the strongest reactivity to Ascaris immune serum were selected to construct a chimeric antigen connected in sequence based on conformation. This chimera, called ASCVac-1, was produced as a soluble recombinant protein in an Escherichia coli expression system and, formulated with MPLA, was used to immunize mice. Mice immunized with ASCVac-1/MPLA showed around 50% reduced larvae production in the lungs after being challenged with A. suum infective eggs, along with significantly reduced inflammation and lung tissue/function damage. The reduced parasite count and pathology in infected lungs were associated with strong Th2 immune responses characterized by the high titers of antigen-specific IgG and its subclasses (IgG1, IgG2a, and IgG3) in the sera and significantly increased IL-4, IL-5, IL-13 levels in lung tissues. The reduced IL-33 titers and stimulated eosinophils were also observed in lung tissues and may also contribute to the ASCVac-1-induced protection. Taken together, the preclinical trial with ASCVac-1 chimera in a mouse model demonstrated its significant vaccine efficacy associated with strong IgG-based Th2 responses, without IgE induction, thus reducing the risk of an allergic response. All results suggest that the multiepitope-based ASCVac-1 chimera is a promising vaccine candidate against Ascaris sp. infections.

Comparative investigation of IFN-γ-producing T cells in chickens and turkeys following vaccination and infection with the extracellular parasite Histomonas meleagridis.

The re-emerging disease histomonosis is caused by the protozoan parasite Histomonas meleagridis that affects chickens and turkeys. Previously, protection by vaccination with in vitro attenuated H. meleagridis has been demonstrated and an involvement of T cells, potentially by IFN-γ production, was hypothesized. However, comparative studies between chickens and turkeys on H. meleagridis-specific T cells were not conducted yet. This work investigated IFN-γ production within CD4+, CD8α+ and TCRγδ+ (chicken) or CD3ε+CD4-CD8α- (turkey) T cells of spleen and liver from vaccinated and/or infected birds using clonal cultures of a monoxenic H. meleagridis strain. In infected chickens, re-stimulated splenocytes showed a significant increase of IFN-γ+CD4+ T cells. Contrariwise, significant increments of IFN-γ-producing cells within all major T-cell subsets of the spleen and liver were found for vaccinated/infected turkeys. This indicates that the vaccine in turkeys causes more intense systemic immune responses whereas in chickens protection might be mainly driven by local immunity.

Leishmanial CpG DNA nanovesicles: A propitious prophylactic approach against visceral leishmaniasis.

Unmethylated CpG motifs with phosphothioate backbone trigger TLR9 to elicit innate immune response characterized by the production of Th1 cytokines. The use of CpG DNA as an adjuvant has established its role in potentiating the humoral and cell mediated vaccine specific immune response. However, none of the synthetic oligodeoxynucleotides (ODNs) know and used till date are associated with the parasite itself. Our group identified a novel CG rich sequence of 14 base pairs from Leishmania donovani genome (Ld CpG ODN) and established it as a TLR9 agonist. The present study was designed to ascertain the adjuvanticity of Ld CpG ODN with soluble leishmanial antigen in experimental model of L. donovani. During the study Schizophyllan (SPG), a fungal polymer was used for encapsulating Ld CpG ODN for efficient endosomal delivery. The synthesized nanovehicles were of nearly 100 nm and localized within endosomes as confirmed by confocal microscopy. Immunization studies displayed the superior ability of synthesized nanovehicles co-administered with parasite antigen in augmenting innate immune response in comparison to ODN, nanoparticles or soluble antigen alone. The response included generation of ROS, NO and iNOS expression followed by proinflammatory cytokine milieu with reduced parasitic load within liver, spleen and bone marrow. These immune-tailored particles in combination with parasitic antigens elicited significant generation of cell mediated response owing to the presence of high levels of CD8+ T-cells and lymphocyte proliferation. Moreover, vaccination regime with synthesized adjuvant also activated humoral immunity by escalating the levels of IgG2 followed by reduced levels of anti-leishmanial IgG and IgG1 antibodies. The findings support the efficacy of Ld CpG ODN as a potential adjuvant against visceral leishmaniasis.

Cruzipain and Its Physiological Inhibitor, Chagasin, as a DNA-Based Therapeutic Vaccine Against Trypanosoma cruzi.

Chagas disease caused by the protozoan parasite Trypanosoma cruzi is endemic in 21 Latin American countries and the southern United States and now is spreading into several other countries due to migration. Despite the efforts to control the vector throughout the Americas, currently, there are almost seven million infected people worldwide, causing ~10,000 deaths per year, and 70 million people at risk to acquire the infection. Chagas disease treatment is restricted only to two parasiticidal drugs, benznidazole and nifurtimox, which are effective during the acute and early infections but have not been found to be as effective in chronic infection. No prophylactic or therapeutic vaccine for human use has been communicated at this moment. Here, we evaluate in a mouse model a therapeutic DNA vaccine combining Cruzipain (Cz), a T. cruzi cysteine protease that proved to be protective in several settings, and Chagasin (Chg), which is the natural Cz inhibitor. The DNAs of both antigens, as well as a plasmid encoding GM-CSF as adjuvant, were orally administrated and delivered by an attenuated Salmonella strain to treat mice during the acute phase of T. cruzi infection. The bicomponent vaccine based on Salmonella carrying Cz and Chg (SChg+SCz) was able to improve the protection obtained by each antigen as monocomponent therapeutic vaccine and significantly increased the titers of antigen- and parasite-specific antibodies. More importantly, the bicomponent vaccine triggered a robust cellular response with interferon gamma (IFN-γ) secretion that rapidly reduced the parasitemia during the acute phase and decreased the tissue damage in the chronic stage of the infection, suggesting it could be an effective tool to ameliorate the pathology associated to Chagas disease.

Protection against Toxoplasma gondii cysts in pigs immunized with rROP2 plus Iscomatrix.

This study aimed to evaluate the humoral immune response in pigs immunized intranasally and intramuscularly with recombinant Toxoplasma gondii rROP2 protein in combination with the adjuvant Iscomatrix. Twelve mixed breed pigs divided into three groups (n=4) were used, G1 received recombinant ROP2 proteins (200 µg/dose) plus Iscomatrix, G2 received PBS plus Iscomatrix, and G3 as the control group. The intranasal (IN) and intramuscular (IM) routes were used. Animals were challenged orally with VEG strain oocysts and treated on day three after challenge. Fever, anorexia, and prostration were the clinical signs observed in all animals. All the G1 animals produced antibodies above the cut-off on the day of the challenge, while the G2 and G3 remained below the cut-off. Better partial protection against parasitemia and cyst tissue formation was observed in G1 than G3. The protection factors against tissue cyst formation were 40.0% and 6.1% for G1 and G2, respectively, compared to G3. In conclusion, there were not systemic antibody responses in pigs with IN immunization with rROP2+Iscomatrix; however, after IM immunization, those animals produced higher titers than animal controls. We associated these results with partial protection obtained against parasitemia and tissue cysts formation.

Mucosal Delivery of a Self-destructing Salmonella-Based Vaccine Inducing Immunity Against Eimeria.

A programmed self-destructive Salmonella vaccine delivery system was developed to facilitate efficient colonization in host tissues that allows release of the bacterial cell contents after lysis to stimulate mucosal, systemic, and cellular immunities against a diversity of pathogens. Adoption and modification of these technological improvements could form part of an integrated strategy for cost-effective control and prevention of infectious diseases, including those caused by parasitic pathogens. Avian coccidiosis is a common poultry disease caused by Eimeria. Coccidiosis has been controlled by medicating feed with anticoccidial drugs or administering vaccines containing low doses of virulent or attenuated Eimeria oocysts. Problems of drug resistance and nonuniform administration of these Eimeria resulting in variable immunity are prompting efforts to develop recombinant Eimeria vaccines. In this study, we designed, constructed, and evaluated a self-destructing recombinant attenuated Salmonella vaccine (RASV) lysis strain synthesizing the Eimeria tenella SO7 antigen. We showed that the RASV lysis strain χ11791(pYA5293) with a ΔsifA mutation enabling escape from the Salmonella-containing vesicle (or endosome) successfully colonized chicken lymphoid tissues and induced strong mucosal and cell-mediated immunities, which are critically important for protection against Eimeria challenge. The results from animal clinical trials show that this vaccine strain significantly increased food conversion efficiency and protection against weight gain depression after challenge with 105E. tenella oocysts with concomitant decreased oocyst output. More importantly, the programmed regulated lysis feature designed into this RASV strain promotes bacterial self-clearance from the host, lessening persistence of vaccine strains in vivo and survival if excreted, which is a critically important advantage in a vaccine for livestock animals. Our approach should provide a safe, cost-effective, and efficacious vaccine to control coccidiosis upon addition of additional protective Eimeria antigens. These improved RASVs can also be modified for use to control other parasitic diseases infecting other animal species.

Influence of coccidiosis vaccination on nutrient utilization of corn, soybean meal, and distillers dried grains with solubles in broilers.

Two experiments were conducted to determine the impact of coccidiosis vaccination on the apparent ileal digestibility (AID) of nutrients and ileal digestible energy (IDE) in commonly used feed ingredients in broilers. Eight experimental treatments based on a factorial arrangement of coccidiosis vaccination (control with in-feed diclazuril [CTL] or vaccinated [VAC]) and 4 different diets were administered to male Cobb 500 broilers in floor pens containing 12 birds per pen. For the vaccinated group, a 3× dose of a live coccidiosis vaccine was given via oral gavage on the day of hatch. Experimental diets consisted of a basal diet and 3 test diets in which 30% of the basal diet was replaced with either corn, soybean meal (SBM), or distillers dried grains with solubles (DDGS) to allow for calculation of nutrient digestibility of individual ingredients by difference. Broilers were fed a common diet from 0 to 7 D and experimental diets from 7 to 12 D. On day 12, blood and ileal digesta were collected to measure plasma carotenoids and determine AID of nitrogen, ether extract, IDE (experiments 1 and 2), and amino acids (AA) (experiment 2). Vaccination increased (P < 0.05) excreta oocyst counts and decreased (P < 0.05) plasma carotenoids when compared with CTL birds. Interactive effects (P < 0.05) were observed for AID of nitrogen (experiment 1) which was reduced by vaccination in birds fed the corn diet and increased for birds fed DDGS. No differences (P > 0.05) in IDE were observed between VAC and CTL birds in either experiment, whereas vaccination decreased (P < 0.05) AID of ether extract independently of diet. Interactive effects (P < 0.05) were observed for AA digestibility, whereby digestibility of all AA was reduced by VAC in corn diets but generally increased AA digestibility of DDGS diets, with minimal impact on SBM diets. In conclusion, the impact of coccidiosis vaccination on nutrient and energy digestibility varied among ingredients; however, digestibility was minimally impacted or improved with DDGS.

Immune response and protective efficacy of recombinant Enterococcus faecalis displaying dendritic cell-targeting peptide fused with Eimeria tenella 3-1E protein.

Avian coccidiosis causes significant economic losses on the global poultry breeding industry. Exploration of new-concept vaccines against coccidiosis has gradually become a research hotspot. In this study, an Enterococcus faecalis strain (MDXEF-1) showing excellent performance isolated from chicken intestinal tract was used as a vector to deliver Eimeria target protein. The plasmid pTX8048-SP-DCpep-NAΔ3-1E-CWA harboring dendritic cell-targeting peptide (DCpep) fusion with Eimeria tenella NAΔ3-1E gene (3-1E protein-coding gene without start codon ATG and terminator codon TAA) was electrotransformed into MDXEF-1 to generate the recombinant bacteria MDXEF-1/pTX8048-SP-DCpep-NAΔ3-1E-CWA in which NAΔ3-1E protein was covalently anchored to the surface of bacteria cells by cell wall anchor (CWA) sequence. The expression of target fusion protein DCpep-NAΔ3-1E-CWA was detected by Western blot. Each chicken was immunized 3 times at 2-wk intervals with live E. faecalis expressing DCpep-NAΔ3-1E fusion protein (DCpep-NAΔ3-1E group), live E. faecalis expressing NAΔ3-1E protein (NAΔ3-1E group), and live E. faecalis containing empty vector only. The 3 immunized groups were then challenged with homologous E. tenella sporulated oocyst after immunizations, and the immune response and protective efficacy in each group were evaluated. The results showed that serum IgG levels, secretory IgA levels in cecal lavage, proportion of CD4+ and CD8α+ cells in peripheral blood, and mRNA expression levels of IL-2 and IFN-γ in the spleen were significantly higher in chickens in the DCpep-NAΔ3-1E group than in chickens of the NAΔ3-1E group (P < 0.05). Oral immunization to chickens with live E. faecalis expressing DCpep-NAΔ3-1E offered more protective efficacy against homologous challenge including significant improved body weight gain, increased oocyst decrease ratio, and reduced average lesion scores in cecum compared with chickens with live E. faecalis expressing NAΔ3-1E protein. These results suggest that recombinant E. faecalis expressing dendritic cell-targeting peptide fusion with E. tenella 3-1E protein could be a potential approach for prevention of Eimeria infection.

Effects of stage of broiler embryo development on coccidiosis vaccine injection accuracy, and subsequent oocyst localization and hatchling quality.

Control of coccidiosis in broiler chickens continues to pose challenges to commercial poultry producers, especially in an era of increased consumer demand for antibiotic-free broiler production. As a result, coccidiosis vaccines are now commonly used in rotation programs to achieve effective coccidiosis control. Inovocox EM1 vaccine (EM1) is a coccidiosis vaccine that allows for earlier immune acquisition through oocyst cycling, which reduces the effects of wild-type coccidia. The EM1 vaccine is administered to embryonated broiler hatching eggs between 18 and 19 D of incubation (doi). In the U.S., commercial broiler hatcheries vaccinate embryonated eggs at either 18.5 or 19 doi. However, it is unclear whether a difference in embryo age at the time of in ovo injection can impact the actual site of vaccine delivery. In addition, it is unclear where oocysts eventually become localized within the embryo following the in ovo injection of EM1. Therefore, the objective of this study was to determine the effects of stage of embryonic development on the actual deposition site of the EM1 vaccine oocysts when they are in ovo injected and to subsequently investigate the movement and eventual location of EM1 oocysts after in ovo injection. Because all eggs were injected at the same time, a 12-h difference in set time was a means to derive 18.5 and 19.0 incubation age of injection (IAN) treatments. The experimental design was a 3 injection treatment (noninjected, diluent-injected, and vaccine-injected) × 2 IAN factorial. There was a significant main effect of IAN on site of vaccine oocysts delivery, and subsequent hatching chick quality. Qualitative histological evaluation revealed the oral uptake of vaccine oocysts through the amnion, with their subsequent presence in the gizzard and intestinal lumen by 24 to 36 h postinjection. In conclusion, physiological development influenced the site of injection, and oocysts imbibed along with the amniotic fluid in late stage broiler embryos are subsequently transported to the gastrointestinal tract.

Drug-cured experimental Trypanosoma cruzi infections confer long-lasting and cross-strain protection.

BACKGROUND: The long term and complex nature of Chagas disease in humans has restricted studies on vaccine feasibility. Animal models also have limitations due to technical difficulties in monitoring the extremely low parasite burden that is characteristic of chronic stage infections. Advances in imaging technology offer alternative approaches that circumvent these problems. Here, we describe the use of highly sensitive whole body in vivo imaging to assess the efficacy of recombinant viral vector vaccines and benznidazole-cured infections to protect mice from challenge with Trypanosoma cruzi. METHODOLOGY/PRINCIPAL FINDINGS: Mice were infected with T. cruzi strains modified to express a red-shifted luciferase reporter. Using bioluminescence imaging, we assessed the degree of immunity to re-infection conferred after benznidazole-cure. Those infected for 14 days or more, prior to the onset of benznidazole treatment, were highly protected from challenge with both homologous and heterologous strains. There was a >99% reduction in parasite burden, with parasites frequently undetectable after homologous challenge. This level of protection was considerably greater than that achieved with recombinant vaccines. It was also independent of the route of infection or size of the challenge inoculum, and was long-lasting, with no significant diminution in immunity after almost a year. When the primary infection was benznidazole-treated after 4 days (before completion of the first cycle of intracellular infection), the degree of protection was much reduced, an outcome associated with a minimal T. cruzi-specific IFN-γ+ T cell response. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that a protective Chagas disease vaccine must have the ability to eliminate parasites before they reach organs/tissues, such as the GI tract, where once established, they become largely refractory to the induced immune response.

Current use and management of commercial fish vaccines in Korea.

The aquaculture industry in Korea has grown rapidly since the 1960s, and it is a major food source. However, the expansion of aquaculture systems has increased the chances of infectious disease outbreaks, and vaccination plays an important role in commercial fish farming. This is the first comprehensive review of commercial fish vaccines in Korea. It not only provides an overview of commercially available fish vaccines and their associated approval processes and laws, but also some perspectives on research advances regarding fish vaccines in Korea. In Korea, fish vaccines are approved only after their safety and effectiveness have been verified according to the Pharmaceutical Affairs Act, and after approval, each vaccine lot must pass the national evaluation criteria. As of the end of 2019, 29 vaccines were approved for 10 fish pathogens, including both single and combination vaccines containing more than two inactivated pathogens. The approved fish vaccines consist of 2 immersion vaccines, as well as 1 intramuscular and 26 intraperitoneal vaccines, which require syringe injection. All the 29 vaccines are manufactured as formalin-inactivated vaccines; 1 is an adjuvant vaccine and 28 are non-adjuvant vaccines; 25 are bacterial vaccines, 2 are viral vaccines, 1 is a parasite vaccine, and 1 is a parasite and bacterial vaccine. In terms of the target fish species, 27 vaccines are used in the olive flounder (Paralichthys olivaceus), 1 in the starry flounder (Platichthys stellatus), and 1 in the red seabream (Pagrus major), striped beakfish (Oplegnathus fasciatus), and amberjack (Seriola quinqueradiata). This imbalance exists mostly because the olive flounder is the main farmed fish species in Korea. In 2018, 67.71 million vaccine doses were distributed following satisfactory performance in the national evaluation. They were used to vaccinate approximately 80.6% of farmed olive flounders.

Establishment of a Standardized Vaccine Protocol for the Analysis of Protective Immune Responses During Experimental Trypanosome Infections in Mice.

To date, trypanosomosis control in humans and animals is achieved by a combination of parasitological screening and treatment. While this approach has successfully brought down the number of reported T. b. gambiense Human African Trypanosomosis (HAT) cases, the method does not offer a sustainable solution for animal trypanosomosis (AT). The main reasons for this are (i) the worldwide distribution of AT, (ii) the wide range of insect vectors involved in transmission of AT, and (iii) the existence of a wildlife parasite reservoir that can serve as a source for livestock reinfection. Hence, in order to control livestock trypanosomosis the only viable long-term solution is an effective antitrypanosome vaccination strategy. Over the last decades, multiple vaccine approaches have been proposed. Despite repeated reports of promising experimental approaches, none of those made it to a field applicable vaccine format. This failure can in part be attributed to flaws in the experimental design that favor a positive laboratory result. This chapter provides a vaccine protocol that should allow for a proper outcome prediction in experimental anti-AT vaccine approaches.