In the Americas, P. vivax is the predominant causative species of malaria, a debilitating and economically significant disease. Due to the complexity of the malaria parasite life cycle, a vaccine formulation with multiple antigens expressed in various parasite stages may represent an effective approach. Based on this, we previously designed and constructed a chimeric recombinant protein, PvRMC-1, composed by PvCyRPA, PvCelTOS, and Pvs25 epitopes. This chimeric protein was strongly recognized by naturally acquired antibodies from exposed population in the Brazilian Amazon. However, there was no investigation about the induced immune response of PvRMC-1. Therefore, in this work, we evaluated the immunogenicity of this chimeric antigen formulated in three distinct adjuvants: Stimune, AddaVax or Aluminum hydroxide (Al(OH)3) in BALB/c mice. Our results suggested that the chimeric protein PvRMC-1 were capable to generate humoral and cellular responses across all three formulations. Antibodies recognized full-length PvRMC-1 and linear B-cell epitopes from PvCyRPA, PvCelTOS, and Pvs25 individually. Moreover, mice\'s splenocytes were activated, producing IFN-γ in response to PvCelTOS and PvCyRPA peptide epitopes, affirming T-cell epitopes in the antigen. While aluminum hydroxide showed notable cellular response, Stimune and Addavax induced a more comprehensive immune response, encompassing both cellular and humoral components. Thus, our findings indicate that PvRMC-1 would be a promising multistage vaccine candidate that could advance to further preclinical studies.

OBJECTIVE: To prepare and characterize the mouse polyclonal antibody against the dense granule protein 24 (GRA24) of Toxoplasma gondii, and explore its preliminary applications.
METHODS: The GRA24 coding sequences of different T. gondii strains were aligned using the MEGA-X software, and the dominant peptide of the GRA24 protein was analyzed with the Protean software. The base sequence encoding this peptide was amplified using PCR assay and ligated into the pET-28a vector, and the generated GRA24 truncated protein was transformed into Escherichia coli BL21. After induction by isopropyl-beta-D-thiogalactopyranoside (IPTG), the expression and purification of the recombinant GRA24 protein was analyzed using sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE). BALB/c mice were immunized by subcutaneous injection with the purified recombinant GRA24 truncated protein to generate the polyclonal antibody, and the titer of the polyclonal antibody was measured using enzyme linked immunosorbent assay (ELISA). The specificity of the polyclonal antibody was tested using Western blotting, and the intracellular localization of the polyclonal antibody was investigated using immunofluorescence assay (IFA).
RESULTS: SDS-PAGE showed successful construction of the recombinant expression plasmid, and Coomassie brilliant blue staining showed the generation of the high-purity recombinant GRA24 truncated protein. ELISA measured that the titer of the polyclonal antibody against the GRA24 truncated protein was higher than 1:208 400, and Western blotting showed that the polyclonal antibody was effective to recognize the endogenous GRA24 proteins of different T. gondii strains and specifically recognize the recombinant GRA24 truncated protein. Indirect IFA showed that the GRA24 protein secreted 16 hour following T. gondii invasion in host cells.
CONCLUSIONS: The polyclonal antibody against the T. gondii GRA24 protein has been successfully prepared, which has a widespread applicability, high titers and a high specificity. This polyclonal antibody is available for Western blotting and IFA, which provides the basis for investigating the function of the GRA24 protein.
［摘要］ 目的 制备并鉴定鼠抗刚地弓形虫致密颗粒蛋白24 (dense granule protein 24, GRA24) 多克隆抗体, 并探索其初 步应用。方法 利用MEGA-X软件比对弓形虫不同虫株GRA24编码区序列, 使用Protean软件分析GRA24蛋白优势肽 段, 通过PCR反应扩增编码该肽段的碱基序列, 并连接至pET-28a载体中。将获得的GRA24截短蛋白原核表达质粒转化 于大肠埃希菌BL21感受态细胞中, 异丙基-β-D-硫代半乳糖苷 (isopropyl-beta-D-thiogalactopyranoside, IPTG) 诱导后采用 十二烷基硫酸钠聚丙烯酰胺凝胶电泳 (sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE) 检测蛋白表达 与纯化。使用纯化的GRA24截短蛋白皮下注射免疫BALB/c小鼠获得GRA24截短蛋白多克隆抗体, 采用酶联免疫吸附 试验 (enzyme-linked immunosorbent assay, ELISA) 检测抗体效价, 采用Western blotting检测抗体特异性, 并将该抗体应用 于免疫荧光试验 (immunofluorescence assay, IFA) 。结果 SDS-PAGE结果表明成功构建重组质粒, 考马斯亮蓝染色结果 显示获得高纯度重组GRA24截短蛋白。ELISA结果显示, GRA24 截短蛋白多克隆抗体效价在 1:208 400 以上; Western blotting检测发现, 该抗体可识别弓形虫不同虫株内源性 GRA24 蛋白, 特异性识别重组 GRA24 截短蛋白; 间接IFA检测发 现, 弓形虫入侵宿主细胞 16 h 后分泌的GRA24蛋白定位于宿主细胞核中。结论 成功制备广适性、高效价、强特异性的 抗弓形虫 GRA24 多克隆抗体, 可应用于 Western blotting 与 IFA, 为进一步研究 GRA24 功能奠定了基础。.

BACKGROUND: Abiotic factors play a significant role in the evolution of Leishmania infantum infection due to its vectorial nature. This study aims to assess the evolution in the detection of new L. infantum infection cases in Valdeorras (Ourense, Northwestern Spain) over a 20-year period and how different climatic variables and preventive measures may have affected it.
METHODS: Indirect immunofluorescence antibody tests (IFAT) were performed on serum samples collected from dogs attending the \'Servicios Veterinarios de Sil\' veterinary clinic (Valdeorras, Northwestern Spain) between May 2003 and April 2023 to detect L. infantum exposure. The percentage of new cases of L. infantum infection was calculated from May of one year to April of the following year. Climatic conditions in the region, global sales of ectoparasiticides and the number of vaccines against L. infantum delivered in the veterinary clinic from 2003 to 2022 were recorded. Statistical analyses were conducted to determine the associations between these factors and the percentage of new cases of L. infantum infection.
RESULTS: A total of 2909 dogs were assessed, and 3785 IFAT tests were performed between May 2003 and April 2023. The mean percentage of new seropositive cases over the 20-year period studied was 21.65 ± 10.8%, with a decline from the beginning to the end of the period studied. The percentage was significantly higher between May 2003 and April 2008 compared with the other periods (May 2008 to April 2013, May 2013 to April 2018 and May 2018 to April 2023). There was a positive correlation between the percentage of new cases of L. infantum infection and the maximum relative humidity in winter. Conversely, there was a negative correlation between the percentage of new cases and sales of ectoparasiticides and vaccination against L. infantum.
CONCLUSIONS: This study is one of the longest evaluations of the evolution of L. infantum infection in a fixed location and its association with external factors including climatic conditions and preventive measures. The results confirm that Valdeorras is a high-risk area for L. infantum infection. The use of ectoparasiticides and vaccines against L. infantum has been shown to play a significant role in preventing L. infantum infection, highlighting the crucial role of veterinarians in the fight against this disease.

BACKGROUND: Toxoplasmosis is a zoonotic parasitic disease caused by Toxoplasma gondii (T. gondii). It has a wide host range and is capable of vertical transmission in pregnant women, which may lead to undesirable pregnancy outcomes such as congenital malformations, miscarriage, premature birth and stillbirth. This study investigated the seroprevalence of T. gondii infection among pregnant women attending the antenatal clinic at Namwala District Hospital in Southern Zambia.
METHODS: This was a cross-sectional study where blood was collected, and the serum was tested for Toxoplasma IgG and IgM. A questionnaire was administered to participants on demographic characteristics and risk factors. Data were entered in Microsoft Excel and exported to STATA version 14 for analysis.
RESULTS: A total of 401 women were enrolled in the study from 3 March to 5 August 2021. The seroprevalence of Toxoplasma IgG was 4.2% (n=17), while the seroprevalence of Toxoplasma IgM was 0.7% (n=3). The median age was 27 (IQR: 24-30) years, and a larger proportion had primary-level education (n=223, 55.6%). The majority (81.6%) of the women were married. None of the risk factors investigated in this study were significant for T. gondii infection.
CONCLUSIONS: There was a low seroprevalence of T. gondii infection among pregnant women in the Namwala district of Southern Province, Zambia, and regular screening may not be warranted in this population. Continued research on toxoplasmosis is recommended to understand its epidemiology across Zambia.

UNASSIGNED: The integration of diagnostic methods holds promise for advancing the surveillance of malaria transmission in both endemic and non-endemic regions. Serological assays emerge as valuable tools to identify and delimit malaria transmission, serving as a complementary method to rapid diagnostic tests (RDT) and thick smear microscopy. Here, we evaluate the potential of antibodies directed against peptides encompassing the entire amino acid sequence of the PvMSP-1 Sal-I strain as viable serological biomarkers for P. vivax exposure.
UNASSIGNED: We screened peptides encompassing the complete amino acid sequence of the Plasmodium vivax Merozoite Surface Protein 1 (PvMSP-1) Sal-I strain as potential biomarkers for P. vivax exposure. Here, immunodominant peptides specifically recognized by antibodies from individuals infected with P. vivax were identified using the SPOT-synthesis technique followed by immunoblotting. Two 15-mer peptides were selected based on their higher and specific reactivity in immunoblotting assays. Subsequently, peptides p70 and p314 were synthesized in soluble form using SPPS (Solid Phase Peptide Synthesis) and tested by ELISA (IgG, and subclasses).
UNASSIGNED: This study unveils the presence of IgG antibodies against the peptide p314 in most P. vivax-infected individuals from the Brazilian Amazon region. In silico B-cell epitope prediction further supports the utilization of p314 as a potential biomarker for evaluating malaria transmission, strengthened by its amino acid sequence being part of a conserved block of PvMSP-1. Indeed, compared to patients infected with P. falciparum and uninfected individuals never exposed to malaria, P. vivax-infected patients have a notably higher recognition of p314 by IgG1 and IgG3.

BACKGROUND: Chicken coccidiosis is a protozoan disease that leads to considerable economic losses in the poultry industry. Live oocyst vaccination is currently the most effective measure for the prevention of coccidiosis. However, it provides limited protection with several drawbacks, such as poor immunological protection and potential reversion to virulence. Therefore, the development of effective and safe vaccines against chicken coccidiosis is still urgently needed.
METHODS: In this study, a novel oral vaccine against Eimeria tenella was developed by constructing a recombinant Lactobacillus plantarum (NC8) strain expressing the E. tenella RON2 protein. We administered recombinant L. plantarum orally at 3, 4 and 5 days of age and again at 17, 18 and 19 days of age. Meanwhile, each chick in the commercial vaccine group was immunized with 3 × 102 live oocysts of coccidia. A total of 5 × 104 sporulated oocysts of E. tenella were inoculated in each chicken at 30 days. Then, the immunoprotection effect was evaluated after E. tenella infection.
RESULTS: The results showed that the proportion of CD4+ and CD8+ T cells, the proliferative ability of spleen lymphocytes, inflammatory cytokine levels and specific antibody titers of chicks immunized with recombinant L. plantarum were significantly increased (P < 0.05). The relative body weight gains were increased and the number of oocysts per gram (OPG) was decreased after E. tenella challenge. Moreover, the lesion scores and histopathological cecum sections showed that recombinant L. plantarum can significantly relieve pathological damage in the cecum. The ACI was 170.89 in the recombinant L. plantarum group, which was higher than the 150.14 in the commercial vaccine group.
CONCLUSIONS: These above results indicate that L. plantarum expressing RON2 improved humoral and cellular immunity and enhanced immunoprotection against E. tenella. The protective efficacy was superior to that of vaccination with the commercial live oocyst vaccine. This study suggests that recombinant L. plantarum expressing the RON2 protein provides a promising strategy for vaccine development against coccidiosis.

UNASSIGNED: Plasmodium knowlesi, a simian malaria species, is now known to infect humans. Due to disadvantages in the current diagnosis methods, many efforts have been placed into developing new methods to diagnose the disease. This study assessed the ability of the PkRAP-1 sandwich enzyme-linked immunosorbent (ELISA) to detect P knowlesi antigens in whole blood specimens.
METHODS: Western blot assay was conducted to evaluate the ability of raised mouse and rabbit anti-PkRAP-1 polyclonal antibodies to bind to the native proteins in P. knowlesi lysate. The polyclonal antibodies were then used in sandwich ELISA to detect P. knowlesi. In the sandwich ELISA, mouse and rabbit polyclonal antibodies were used as the capture and detection antibodies, respectively. The limit of detection (LOD) of the assay was determined using P. knowlesi A1H1 culture and purified recombinant PkRAP-1.
RESULTS: Western blot results showed positive reactions towards the proteins in P. knowlesi lysate. The LOD of the assay from three technical replicates was 0.068% parasitaemia. The assay performance in detecting P. knowlesi was 83% sensitivity and 70% specificity with positive and negative predictive values of 74% and 80%, respectively. The anti-PkRAP-1 polyclonal antibodies did not cross-react with P. falciparum and healthy samples, but P. vivax by detecting all 12 samples.
UNASSIGNED: PkRAP-1 has the potential as a biomarker for the development of a new diagnostic tool for P. knowlesi detection. Further studies need to be conducted to establish the full potential of the usage of anti-PkRAP-1 antibodies for P. knowlesi detection.

UNASSIGNED: Trypanosoma cruzi is a protozoan parasite that causes the tropical ailment known as Chagas disease, which has its origins in South America. Globally, it has a major impact on health and is transported by insect vector that serves as a parasite. Given the scarcity of vaccines and the limited treatment choices, we conducted a comprehensive investigation of core proteomics to explore a potential reverse vaccine candidate with high antigenicity.
UNASSIGNED: To identify the immunodominant epitopes, T. cruzi core proteomics was initially explored. Consequently, the vaccine sequence was engineered to possess characteristics of non-allergenicity, antigenicity, immunogenicity, and enhanced solubility. After modeling the tertiary structure of the human TLR4 receptor, the binding affinities were assessed employing molecular docking and molecular dynamics simulations (MDS).
UNASSIGNED: Docking of the final vaccine design with TLR4 receptors revealed substantial hydrogen bond interactions. A server-based methodology for immunological simulation was developed to forecast the effectiveness against antibodies (IgM + IgG) and interferons (IFN-g). The MDS analysis revealed notable levels of structural compactness and binding stability with average RMSD of 5.03 Aring;, beta-factor 1.09e+5 Å, Rg is 44.7 Aring; and RMSF of 49.50 Aring;. This is followed by binding free energies calculation. The system stability was compromised by the complexes, as evidenced by their corresponding Gibbs free energies of -54.6 kcal/mol.
UNASSIGNED: Subtractive proteomics approach was applied to determine the antigenic regions of the T cruzi. Our study utilized computational techniques to identify B- and T-cell epitopes in the T. cruzi core proteome. In current study the developed vaccine candidate exhibits immunodominant features. Our findings suggest that formulating a vaccine targeting the causative agent of Chagas disease should be the initial step in its development.

This study documents the presence of anti-Neospora caninum antibodies and their association with certain risk factors in 2 deer species from the central region of Veracruz State, Mexico. A total of 90 blood samples, 20 from temazate deer (Mazama temama) and 70 from white-tailed deer (Odocoileus virginianus), were taken from 3 farms, and serum samples were subjected to ELISA indirect test to detect N. caninum antibodies; the association between the serological status and the possible risk factors was then estimated. The overall presence of anti-N. caninum antibodies was 57.7% (52/90; 95% CI 46.9-67.9), with positive animals identified on all farms; in white-tailed deer it was 57% and in temazate deer 60%. Prevalence was higher in females than males. Adult animals had a higher prevalence than young ones. The risk analysis identified the age in the adult animal category (odds ratio 5.8) as being associated with the presence of anti-N. caninum antibodies. These results provide evidence of the significant contamination of oocysts in the environment and allow us to estimate the contribution of deer to the sylvatic cycle.

Resistance to clinical malaria takes years to develop even in hyperendemic regions and sterilizing immunity has rarely been observed. To evaluate the maturation of the host response against controlled repeat exposures to P. falciparum (Pf) NF54 strain-infected mosquitoes, we systematically monitored malaria-naïve participants through an initial exposure to uninfected mosquitoes and 4 subsequent homologous exposures to Pf-infected mosquitoes over 21 months (n = 8 males) (ClinicalTrials.gov# NCT03014258). The primary outcome was to determine whether protective immunity against parasite infection develops following repeat CHMI and the secondary outcomes were to track the clinical signs and symptoms of malaria and anti-Pf antibody development following repeat CHMI. After two exposures, time to blood stage patency increases significantly and the number of reported symptoms decreases indicating the development of clinical tolerance. The time to patency correlates positively with both anti-Pf circumsporozoite protein (CSP) IgG and CD8 + CD69+ effector memory T cell levels consistent with partial pre-erythrocytic immunity. IFNγ levels decrease significantly during the participants\' second exposure to high blood stage parasitemia and could contribute to the decrease in symptoms. In contrast, CD4-CD8 + T cells expressing CXCR5 and the inhibitory receptor, PD-1, increase significantly after subsequent Pf exposures, possibly dampening the memory response and interfering with the generation of robust sterilizing immunity.