Muhammad Haseeb

Background: Haemonchus contortus (H. contortus), a nematode with global prevalence, poses a major threat to the gastrointestinal health of sheep and goats. In an effort to combat this parasite, a nanovaccine was created using a recombinant ADP-ribosylation factor 1 (ARF1) antigen encapsulated within poly lactic-co-glycolic acid (PLGA). This study aimed to assess the effectiveness of this nanovaccine in providing protection against H. contortus infection. Methods: Fifteen goats were randomly divided into three groups. The experimental group received two doses of the PLGA encapsulated rHcARF1 (rHcARF1-PLGA) nanovaccine on days 0 and 14. Fourteen days after the second immunization, both the experimental and positive control groups were challenged with 8000 infective larvae (L3) of H. contortus, while the negative control group remained unvaccinated and unchallenged. At the end of the experiment on the 63rd day, all animals were humanly euthanized. Results: The results showed that the experimental group had significantly higher levels of sera IgG, IgA, and IgE antibodies, as well as increased concentrations of cytokines, such as IL-4, IL-9, IL-17, and TGF-β, compared to the negative control group after immunization. Following the L3 challenge, the experimental group exhibited a 47.5% reduction in mean eggs per gram of feces (EPG) and a 55.7% reduction in worm burden as compared to the positive control group. Conclusions: These findings indicate that the nanovaccine expressing rHcARF1 offers significant protective efficacy against H. contortus infection in goats. The results also suggest the need for more precise optimization of the antigen dose or a reassessment of the vaccination regimen. Additionally, the small sample size limits the statistical rigor and the broader applicability of the findings.

Haemonchus contortus is a gastrointestinal parasite that adversely impacts small ruminants, resulting in a notable reduction in animal productivity. In the current investigation, we developed a nanovaccine by encapsulating the recombinant protein rHcES-15, sourced from the excretory/secretory products of H. contortus, within biodegradable poly (D, L-lactide-co-glycolide) (PLGA) nanoparticles (NPs). The development of this nanovaccine involved the formulation of PLGA NPs using a modified double emulsion solvent evaporation technique. Scanning electron microscopy (SEM)verified the successful encapsulation of rHcES-15 within PLGA NPs, exhibiting a size range of 350–400 nm. The encapsulation efficiency (EE) of the antigen in the nanovaccine was determined to be 72%. A total of forty experimental mice were allocated into five groups, with the nanovaccine administered on day 0 and the mice euthanized at the end of the 14-day trial. The stimulation index (SI) from the mice subjected to the nanovaccine indicated heightened lymphocyte proliferation (*** p < 0.001) and a noteworthy increase in anti-inflammatory cytokines (IL-4, IL-10, and IL-17). Additionally, the percentages of T-cells (CD4⁺, CD8⁺) and dendritic cell phenotypes (CD83⁺, CD86⁺) were significantly elevated (** p < 0.01, *** p < 0.001) in mice inoculated with the nanovaccine compared to control groups and the rHcES-15 group. Correspondingly, higher levels of antigen-specific serum immunoglobulins (IgG1, IgG2a, IgM) were observed in response to the nanovaccine in comparison to both the antigenic (rHcES-15) and control groups (* p < 0.05, ** p < 0.01). In conclusion, the data strongly supports the proposal that the encapsulation of rHcES-15 within PLGA NPs effectively triggers immune cells in vivo, ultimately enhancing the antigen-specific adaptive immune responses against H. contortus. This finding underscores the promising potential of the nanovaccine, justifying further investigations to definitively ascertain its efficacy.

Haemonchus contortus (H. contortus) is a gastrointestinal parasite affecting small ruminants, leading to a significant decline in animal productivity. In this study, we developed a nanovaccine by encapsulating the recombinant protein rHcES-15, derived from the excretory/secretory products of H. contortus, within biodegradable poly (D, L-lactide-co-glycolide) (PLGA) nanoparticles (NPs). To construct the nanovaccine, PLGA NPs were prepared using a modified double emulsion solvent evaporation technique. Scanning electron microscopy (SEM) illustrated successful encapsulation of rHcES-15 within PLGA NPs, with a size ranging between 350-400 nm. The encapsulation efficiency (EE) of the antigen in the nanovaccine was determined to be 72%. A total of forty experimental mice were divided into five groups, receiving the nanovaccine on day 0 and being humanely sacrificed at the end of the 14-day trial. The stimulation index (SI) from mice vaccinated with the nanovaccine indicated an amplified lymphocyte proliferation and a significant increase in anti-inflammatory cytokines (IL-4, IL-10, and IL-17). Furthermore, the percentages of T-cells (CD4+, CD8+) and dendritic cell phenotypes (CD83+, CD86+) were substantially upregulated in mice immunized with the nanovaccine compared to control groups and the rHcES-15 group. Similarly, higher levels of antigen-specific serum immunoglobulins (IgG1, IgG2a, IgM) were observed in response to the nanovaccine compared to both the antigenic (rHcES-15) and control groups. In conclusion, the data strongly supports the notion that encapsulation of rHcES-15 within PLGA NPs effectively stimulates immune cells in vivo, ultimately augmenting antigen-specific adaptive immune responses against H. contortus. This discovery highlights the promising potential of the nanovaccine, justifying additional investigations to ascertain its efficacy finally.

Eimeria maxima (E. maxima) are an intracellular apicomplexan protozoan that causes intestinal coccidiosis in chickens. The purpose of this research was to develop a novel delivery approach for recombinant E. maxima (rEm) 14-3-3 antigen to elicit enhanced immunogenic protection using poly (D, L-lactide-co-glycolide) (PLGA) and chitosan (CS) nanoparticles (NPs) against E. maxima challenge. The morphologies of prepared antigen-loaded NPs (PLGA/CS-rEm14-3-3 NPs) were visualized by a scanning electron microscope. The rEm14-3-3 and PLGA/CS-rEm14-3-3 NPs–immunized chicken-induced changes of serum cytokines, IgY-antibody level, and T-lymphocyte subsets and protective efficacies against E. maxima challenge were evaluated. The results revealed that encapsulated rEm14-3-3 in PLGA and CS NPs presented spherical morphology with a smooth surface. The chickens immunized with only rEm14-3-3 and PLGA/CS-rEm14-3-3 NPs elicited a significant (p+/CD3⁺, CD8⁺/CD3⁺ T-cells, and provoked sera IgY-antibody immune response compared to control groups (PBS, pET-32a, PLGA, and CS). Whereas, PLGA-rEm14-3-3 NP–immunized chicken provoked a higher level of IFN- γ production and IgY-antibody response rather than CS-rEm14-3-3 and bare antigen, relatively. The animal experiment results ratified that PLGA-rEm14-3-3 NP–immunized chicken significantly alleviated the relative body weight gain (%), decreased lesion score, and enhanced oocyst decrease ratio compared to CS-rEm14-3-3 NPs and only rEm14-3-3. The anti-coccidial index of the chicken vaccinated with the PLGA-rEm14-3-3 NPs was (180.1) higher than that of the Cs-rEm14-3-3 NPs (167.4) and bare antigen (165.9). Collectively, our statistics approved that PLGA NPs might be an efficient antigen carrier system (Em14-3-3) to act as a nanosubunit vaccine that can improve protective efficacies in chicken against E. maxima challenge.

Trichinellosis is a foodborne zoonotic disease caused by Trichinella spp., including Trichinella spiralis. In the present study, T. spiralis membrane-associated progesterone receptor component-2 (Ts-MAPRC2) gene was cloned and characterized using protein sequencing analysis. Furthermore, the expression, purification, immunoblot assay, binding ability with progesterone antibody, and immunofluorescence assay were performed. A direct effect of progesterone (P4) and mifepristone (RU486) on the Ts-MAPRC2 gene was determined using in vitro cell culture that showed different expression levels at all developmental stages (muscle larvae (ML), female adult worm (F-AL), male adult worm (M-AL), and newborn larvae (NBL)). Subsequently, the in vitro phenotypic effects of P4, RU486, and rTs-MAPRC2-Ab on F-AL and ML stages were measured. Later, the in vivo phenotypic effect and relative mRNA expression of mifepristone on the F-AL stage were studied. Our results revealed that the Ts-MAPRC2 gene is critical to maintaining pregnancy in the female adult worm (F-AL) of T. spiralis. The 300 ng/mL of P4 and 100 ng/mL of RU486 showed downregulation of the Ts-MAPRC2 gene in F-AL (p ≤ 0.05). This plays an important role in abortion and possibly decreases the worm burden of T. spiralis in the host. Only 30 ng/mL P4 showed significant upregulation in F-AL (p ≤ 0.05). The current study provides new insights regarding the antihormone (P4 and RU486) drug design and vaccine therapy of recombinant (rTs-MAPRC2) protein as well as their combined effects to control T. spiralis infection.

ADP-ribosylation factor 1 (HcARF1) is one of the Haemonchus contortus (H. contortus) excretory/secretory proteins involved in modulating the immune response of goat peripheral blood mononuclear cells (PBMC). Here, we evaluated the immunogenic potential of recombinant HcARF1 (rHcARF1) against H. contortus infection in Institute of Cancer Research (ICR) mice. Briefly, rHcARF1 was entrapped in poly (D, L-lactide-co-glycolide) (PLGA) and chitosan (CS) nanoparticles (NP) and injected into mice as a vaccine. Fifty-six ICR mice were assigned randomly into seven groups, with eight animals in each group, and they were vaccinated subcutaneously. At the end of the experiment (14th day), the blood and the spleen were collected from euthanized mice to detect lymphocyte proliferation, cytokine analysis, and the production of antigen-specific antibodies. Scanning electron microscope was used to determine the size, morphology, and zeta potential of nanoparticles. Flow cytometry was performed, which presented the increase percentages of CD4⁺ T cells (CD3e⁺CD4⁺), CD8⁺ T cells (CD3e⁺CD8⁺) and dendritic cells (CD11c⁺CD83⁺, CD11c⁺CD86⁺) in mice vaccinated with rHcARF1+PLGA NP. Immunoassay analysis show raised humoral (Immunoglobulin (Ig)G1, IgG2a, IgM) and cell-mediated immune response (Interleukin (IL)-4, IL-12, and IL-17, and Interferon (IFN)-γ) induced by rHcARF1+PLGA NP. Experimental groups that were treated with the antigen-loaded NP yield higher lymphocyte proliferation than the control groups. Based on these results, we could propose that the rHcARF1 encapsulated in NP could stimulate a strong immune response in mice rather than administering alone against the infection of H. contortus.

Methyltransferases (MTFs) are broad range of enzymes, which are ubiquitously expressed in diverse organisms ranging from bacteria to animals. MTFs proteins have been associated with various biological/cellular processes including transcriptional regulation, subcellular protein and RNA localization, signal transduction and DNA-damage repair. However, the role of MTFs in immune mechanism during host–parasite interaction has not been addressed yet. An open reading frame (764 bp) of methyltransferase-type 12 gene of H. contortus denoted as HcMTF-12, was successfully cloned using reverse transcriptase-polymerase chain reaction (RT-PCR) followed by prokaryotic expression in Escherichia coli BL21 (DE3 strain). The recombinant HcMTF-12 protein (rHcMTF-12) was about 47 kDa along with a fusion vector protein of 18 kDa. Immunoblot results identified the native protein MTF-12 with antibodies produced in rats against rHcMT-12, whereas rHcMTF-12 protein was recognized with sera of goat experimentally infected with H. contortus. Immunohistochemical analysis revealed that the native MTF-12 protein was mainly located in the periphery (cuticle) of parasite sections as well as within the pharynx and intestinal region. An immunofluorescence assay validated that rHcMTF-12 attached to the surface of goat PBMCs. Furthermore, the cytokines transcription of IL-2, IFN-γ and IL-4 transcripts of PBMCs incubated with rHcMTF-12 were enhanced in a dose-dependent manner. The secretion of TGF-β1 and IL-10 was significantly decreased. However, IL-6 production was not significantly different as compared to the control groups. Moreover, the migration activity and nitric oxide (NO) production by PBMCs were induced considerably, whereas the proliferation of PBMCs cells was negatively affected when incubated with the rHcMTF-12 protein. Our findings suggest that HcMTF-12 significantly mediated the functions of PBMCs, and it might be a potential candidate for therapeutic interventions against haemonchosis.