Background and Aim: Bovine interleukin 15 (bIL15) is a potential immunotherapy that can block the spread of bovine leukemia virus (BLV). However, immune checkpoints that maintain body homeostasis may reduce their effectiveness. Thus, an analysis of the effectiveness of bIL15 while blocking negative immune regulators is necessary. We aimed to obtain recombinant bIL15 (rbIL15) and determine its percentage using monoclonal antibodies against bovine cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed cell death ligand 1 (PD-L1). To achieve this goal, peripheral blood mononuclear cells (PBMCs) from healthy and BLV+ cattle were treated with bIL15 using a CTLA-4– and PD-L1–blocking algorithm. Materials and Methods: The codon-optimized bIL15 gene was synthesized under de novo conditions using polymerase chain reaction (PCR). The synthesized gene was cloned into pET28 and transformed into electrocompetent Escherichia coli BL21 cells; rbIL15 was purified using metal affinity chromatography and analyzed using sodium dodecyl-sulfate– polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. The expression of the Bcl2, STAT3, and STAT5 genes was studied using qualitative PCR. An enzyme-linked immunosorbent assay (ELISA) was used to analyze interferon (IFN)-γ production by rbIL15-treated mononuclear cells. Results: Analysis of rbIL15 using SDS-PAGE and western blotting revealed a specific product weighing 24 kDa. The optimal conditions for rbIL15 induction were 0.2 mM isopropyl-β-D-1-galactopyranoside and 37°C. When rbIL15 was added to PBMCs from healthy cattle, the Bcl2, STAT3, and STAT5 genes were expressed. ELISA of the culture medium of rbIL15-treated PBMCs revealed IFN-γ production. When PBMCs from healthy cows were treated with rbIL15, CTLA-4, and PD-L1 blockade together, they did not produce more IFN-γ than the rbIL15 group. Using PBMCs from BLV+ cattle, combination treatment increased IFN-γ production. Conclusion: The biological activity of rbIL15 is characterized by the induction of transcription factors and the production of IFN-γ. Using rbIL15 with CTLA-4 and PD-L1 blockade in PBMCs from healthy and BLV+ cows led to the production of a transcription factor and cytokine. The results demonstrate the possibility of using this method to improve immunity and immunological memory in patients with chronic viral infections. Keywords: bovine interleukin-15, bovine leukemia virus, cytotoxic T-lymphocyte antigen 4, monoclonal antibody, programmed cell death ligand 1.

Background and Aim: Bovine babesiosis, caused by Babesia bovis, poses significant economic challenges to Kazakhstan’s cattle industry. Early and accurate detection is crucial for interrupting transmission cycles, particularly in regions lacking advanced diagnostic infrastructure. This study aimed to develop a rapid lateral flow immunoassay (LFIA) using a recombinant C-terminal fragment of the recombinant rhoptry-associated protein 1 (rRap1) antigen for the serodiagnosis of bovine babesiosis. Materials and Methods: A C-terminal fragment (amino acids 345–480) of the B. bovis Rap1 gene was codon optimized and expressed in Escherichia coli. The recombinant protein was purified using metal-affinity chromatography and validated through sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blotting, and nanoflow liquid chromatography-tandem mass spectrometry. A diagnostic evaluation was performed using enzyme-linked immunosorbent assay (ELISA) and LFIA on sera from 102 uninfected and 15 infected cattle, all of which had been pre-tested using polymerase chain reaction. Colloidal gold-protein G conjugates were prepared for LFIA, and test conditions were optimized for antigen concentration and serum dilution. Assay performance was compared with previously published LFIAs. Results: A 21-kDa rRap1 protein was successfully expressed and demonstrated high specificity to positive control sera. ELISA and LFIA both detected antibodies in 13 of 15 infected samples (sensitivity 86.6%). Specificity was 90.1% for ELISA and 88.2% for LFIA. Receiver operating characteristic analysis showed an area under the curve of 0.83, and Cohen’s Kappa indicated fair-to-moderate agreement between ELISA and LFIA. The LFIA exhibited comparable performance to assays based on merozoite surface antigen 1 or spherical body protein antigens, marking the first successful use of a B. bovis Rap1 C-terminal fragment for LFIA-based field diagnostics in Kazakhstan. Conclusion: The developed rRap1-based LFIA is a promising, field-deployable diagnostic tool for bovine babesiosis, offering rapid results without the need for laboratory equipment. Despite slightly lower sensitivity than ELISA, its simplicity, cost-effectiveness, and specificity support its use in large-scale epidemiological surveillance. Further validation in diverse field conditions and cattle populations is recommended to refine sensitivity and broaden applicability. Keywords: Babesia bovis, bovine babesiosis, enzyme-linked immunosorbent assay, lateral flow immunoassay, rapid diagnostics, recombinant rhoptry-associated protein 1, serodiagnosis.

Background/Objectives: Human adenoviruses (HAdVs) are widely used as vectors for vaccines and gene therapy; however, pre-existing immunity can reduce their efficacy. Therefore, rapid and accessible serological methods are required to assess antibody levels against adenoviruses. Lateral flow immunochromatographic assay (LFIA) sensitivity depends on the label and antigen. In this study, we aimed to develop and evaluate LFIA systems based on gold nanoparticles (GNPs) and quantum dots (QDs) using a recombinant HAdV hexon protein. Methods: A recombinant HAdV hexon protein fragment (rhHAdV, 35 kDa; amino acids A120-R316) was expressed in Escherichia coli and purified using Ni²⁺ affinity chromatography. Protein identity was confirmed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis, Western blotting, and liquid chromatography–tandem mass spectrometry analyses. Two LFIA formats were developed using Protein G-conjugated GNPs (GNP-G) and QDs (QD-G). Agreement with the reference ELISA was evaluated using 90 human serum samples. Results: The rhHAdV antigen demonstrated reactivity with anti-adenovirus-positive human sera in ELISA. Antibody detection was achieved at serum dilutions of up to 1:300 and 1:1000 for the GNP- and QD-based LFIA, respectively. The GNP-G and QD-G LFIA formats demonstrated positive percent agreements of 96.9% and 100%, respectively, while both assays showed a negative percent agreement of 98.2% compared with the reference ELISA. Conclusions: The findings demonstrate the feasibility of a QD-based LFIA for qualitative detection of antibodies reactive with the recombinant HAdV-B3 hexon fragment.

Background/Objectives: Human adenoviruses (HAdVs) are widely used as vectors for vaccines and gene therapy; however, pre-existing immunity can reduce their efficacy. Therefore, rapid and accessible serological methods are required to assess antibody levels against adenoviruses. Lateral flow immunochromatographic assay (LFIA) sensitivity depends on the label and antigen. In this study, we aimed to develop and evaluate LFIA systems based on gold nanoparticles (GNPs) and quantum dots (QDs) using a recombinant HAdV hexon protein. Methods: A recombinant HAdV hexon protein fragment (rhHAdV, 35 kDa; amino acids A120–R316) was expressed in Escherichia coli and purified using Ni²⁺ affinity chromatography. Protein identity was confirmed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, western blotting, and liquid chromatography–tandem mass spectrometry analyses. Two LFIA formats were developed using Protein G-conjugated GNPs (GNP-G) and QDs (QD-G). Analytical sensitivity was evaluated using serial dilutions of positive serum samples. Diagnostic performance was assessed using 90 human serum samples and compared with that of a commercial enzyme-linked immunosorbent assay (ELISA). Results: The rhHAdV antigen demonstrated high immunoreactivity in ELISA. Antibody detection was achieved at serum dilutions of up to 1:300 and 1:1000 for the GNP- and QD-based LFIA, respectively. Both LFIA formats showed high specificity (98.2%). Sensitivity was 96.9% and 100% for GNP-G and QD-G, respectively; ROC analysis demonstrated excellent diagnostic accuracy, with AUC values of 0.976 and 0.991, respectively. Conclusions: The findings of this study highlight the potential of QD-based LFIA as an advanced tool for rapid serodiagnostics and large-scale immunological monitoring.

Bovine babesiosis is a disease caused by parasites of the genus Babesia. One of the main Babesia genera causing disease in cattle, according to the International Office of Epizootics (OIE), is Babesia bovis. Recovered cattle become permanent latent carriers and can be a source of tick infestation and spread of infection. Despite susceptibility and specificity, diagnostic methods such as enzyme-linked immunosorbent assays (ELISA) and indirect fluorescent antibody assays (IFA) have some disadvantages. These methods require a long analysis time, the use of specialized equipment and trained specialists. At the same time, immunochromatographic tests (ICA) are widely used in veterinary practice today. ICA is characterized by a short analysis time, ease of performance and interpretation of results. The main components of the ICA test system are monoclonal antibodies. As a result of the studies, three mAbs against the recombinant Rap1 antigen of B. bovis were obtained. The resulting mAbs have high diagnostic characteristics. A conjugate of colloidal gold with mAbs is capable of detecting the Rap1 antigen of the causative agent of babesiosis in cattle.

Interleukin-15 (IL-15) is a promising cytokine for immunotherapy of human cancers and bovine viral infections. IL-15 indirectly has a negative effect on tumor and virus-infected cells by activating the signaling pathways of proliferation and activation of natural killer (NK) and memory CD8+ T cells. IL-15 therapy may have widespread clinical use, particularly when combined with chimeric antigen receptor (CAR) T or CAR NK cells. Additionally, the use of IL-15 in combination with checkpoint inhibitors and other therapies holds promise as a treatment for cancer and viral infections. Understanding the biological characteristics of IL-15 is crucial for developing effective therapeutic strategies. This study aimed to explore recent advances in IL-15 research, focusing on its antitumor mechanisms within the tumor microenvironment, advances in IL-15-based therapies for bovine viral infections, and its integration with other treatment approaches, including monoclonal antibodies.