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.

Intestinal changes and performance parameters in ticks feeding on calves immunized with subunits of immunogens against Rhipicephalus microplus.

We describe the intestinal changes and biological parameters of the tick species Rhipicephalus microplus exposed to the immune response of calves vaccinated with two subunits of immunogens. The first group of Bos taurus calves was immunized with a synthetic peptide (SBm7462), whereas the second group received an inoculum for synthetic control. The third group was immunized with a recombinant peptide (rSBm7462); an inoculum was injected into a fourth group of calves for recombinant control. Each formulation was administered to these calves during three times at intervals of 30 days. At 21 days after the last immunization, the calves were challenged using a total of 4500 larvae per animal. Indirect ELISA was realized to identify the kinetics of IgGs from samples of calves studied. Naturally detaching ticks were collected for analyses of biological performance and histological changes in the midgut. We dissected randomly detached ticks. The midgut of each of these ticks was removed and processed routinely for histology, stained with hematoxylin-eosin (H&E) and slow Giemsa. Slides were also subjected to immunohistochemistry. The antibody response showed significant induction of high-affinity IgGs in calves immunized with both peptides in comparison to calves of the control groups. Histological changes included damage of the intestinal epithelium in ticks fed on immunized hosts and intense immunostaining in midgut cells, using the serum of calves immunized with recombinant peptide. There were significant differences in all biological performing parameters of ticks detached from vaccinated calves in comparison with ticks of the control groups. We identified reductions of 87.7 and 93.5% in engorged ticks detached from calves immunized with a synthetic and recombinant peptides, respectively, a 28 and 8.60% lower egg mass in groups immunized with synthetic and recombinant peptides, respectively, and a 38.4% reduction of the value of nutrient index/tick in the group immunized with the recombinant peptide. Our findings show that the immune response induced by small peptides in cattle can modify the digestion and metabolism of ticks fed on vaccinated animals, resulting in changes in tick performance.

Bovine immunisation with a recombinant peptide derived from synthetic SBm7462® (Bm86 epitope construct) immunogen for Rhipicephalus microplus control.

RHIPICEPHALUS MICROPLUS: is the most important ectoparasite of livestock in tropical and subtropical areas around the world. Research focused on developing an efficient vaccine for cattle tick control is a high priority. The aim of this study was to evaluate the rSBm7462® peptide (Bm86-B and T cell epitopes) regarding its properties of immunogenicity, protective effect in cattle and efficacy against R. microplus. This peptide was produced by a fermentative process in the yeast culture system of Komagataella (Pichia) pastoris strain Km 71. The vaccination assay was conducted in a tick-free area using non-splenectomised Holstein Friesian calves, separated into immunisation and control groups. These animals individually received the recombinant peptide and the inoculum without peptide using saponin as an adjuvant at three time points. The calves were challenged 21 days after the last immunisation using 4500 larvae per animal. An indirect ELISA was used to identify the IgG kinetics of serum samples from the calves studied. The qPCR was performed to determine the cytokine gene expression from the total RNA of the cultured PBMCs. Histomorphometry of the germinal centres (GCs) was performed measuring slides with haematoxylin-eosin staining of surgically removed lymph nodes from immunised calves. The antibody response showed a significant induction of high-affinity IgGs in calves immunised with the recombinant peptide in comparison to the controls. The kinetics of antibodies in immunised calves showed a significant increase during the experiment. This increase in high-affinity IgGs correlated with a gradual increase of the GC diameter following each peptide vaccination. Cytokine expression profiles demonstrating an adaptive immune response in calves immunised with rSBm7462® confirmed the T-dependent response. Vaccine efficacy was calculated at 72.4 % following the analysis and fecundity of collected adult female ticks, compared between control and vaccinated groups. These findings demonstrate that this new recombinant peptide is an option for control of R. microplus infestations.