Razi Vaccine & Serum Research Institute

Equine herpesvirus type 1 (EHV-1) is a globally prevalent equine pathogen responsible for severe respiratory, neurological, and reproductive disorders. Accurate and ultrasensitive detection of EHV-1 is critical for timely disease management. In this study, we report the development of the first G-quadruplex-forming aptamer specifically designed for EHV-1 detection. The aptamer was generated using an in silico approach, and its G-quadruplex conformation was confirmed using circular dichroism (CD) spectroscopy and crystal violet fluorescence assays. Binding affinity and specificity were assessed using a comprehensive panel of analytical techniques, including colorimetric assays, enzyme-linked apta-sorbent assay (ELASA), surface plasmon resonance (SPR), CD spectroscopy, and fluorescence analysis. The aptamer exhibited a high binding affinity in the picomolar range, as determined by SPR. In both colorimetric and ELASA platforms, it enabled the detection of as few as 10 viral particles per milliliter, compared to the 1000 viral particles per milliliter required by conventional PCR. ELASA results demonstrated excellent diagnostic performance, yielding an area under the curve of 0.96. Importantly, this aptamer-based method eliminates the need for DNA extraction, primers, or gel electrophoresis. These findings underscore the aptamer's strong potential as a cost-effective, rapid, and user-friendly point-of-care diagnostic tool for EHV-1, especially in low-resource or field settings.

The multiplicity of infection sources along with the genetically homogenous nature of Mycobacterium avium subsp. paratuberculosis (MAP) necessitates the application of high-discriminative fingerprinting techniques to establish epidemiological links and control of paratuberculosis in ruminants. The present study aimed to develop a simple Multiple-Locus Variable-Number Tandem-Repeat Analysis (MLVA) assay suitable for low-budget laboratory settings.

A bioinformatics screening was conducted on three annotated MAP genomes (K10, MAP4, and MAP-JIII-386) leading to the detection of 7 potential polymorphic variable-number tandem repeats (VNTRs). A MAP collection of the three aforementioned strains, 30 field isolates and 2 laboratory strains from Iran plus 59 global Mycobacterium avium complex strains were subsequently genotyped at laboratory or in situ. Findings were validated against the French INRAE MLVA typing system (Thibault's scheme) dedicated to typing Mycobacterium avium subsp. paratuberculosis, hominissuis, avium, and silvaticum.

Fourty-one MLVA types were detected by our suggested MLVA scheme, compared to 45 types found by Thibault's scheme. The calculated discriminatory power of the MLVA for the former and the latter was 0.93 and 0.95, respectively.

The suggested MLVA scheme discriminated between isolates with identical types determined by Thibault's scheme. Noting the fact that strains grouped in INMV types 2, 3, 4, 5, 33, 39, 68, 82, 131, 219, 220, New1 and New 25 were further resolved into sub-groups, we suggest considering VNTR 1067 (DI = 0.71), MAP 4356 (DI = 0.68), MATR 7 (DI = 0.53), MAP 3824 (DI = 0.56) and MATR 6 (DI = 0.49), in any future upgrading of the current version of the Thibault's scheme.

Human papillomavirus (HPV) is the leading cause of cervical cancer worldwide. The pathogenesis of HPV is mainly dependent on its E7 and E6 proteins. Up to now, different adjuvants have been used to enhance the efficacy of the immune response against these two proteins. In this study, Flagellin (FLA) was used as adjuvant to test adjuvant activity and also see whether its orientation of attachment can affect the immune response pattern. The E7d-FLA and FLA-E7d in pET28a vector were constructed and then the recombinant proteins were expressed in E. coli BL21 (DE3) bacteria under IPTG induction. The expression of recombinant E7d-FLA and FLA-E7d proteins is confirmed by SDS-PAGE and western blot. Then, recombinant fusion proteins were purified using a nickel-nitrilotriacetic acid (Ni-NTA) column. The recombinant proteins were checked for endotoxin contamination and then quantified by Bradford. Eight-to-ten-week-old male Balb/C mice were immunized subcutaneously with 10 µg recombinant E7d-FLA, FLA-E7d and HPV16E7d vaccine on days 0, 14 and 28. In addition, PBS and FLA groups were considered as control group. Then, spleen cells were harvested to assess lymphocyte proliferation and IFN-γ, IL-4 and IL-17 cytokines. In addition, mice sera were used for specific total IgG and IgG1, IgG2a, IgG2b and IgM antibodies assessment by ELISA. The results show that E7d-FLA is more potent in the induction of lymphocyte proliferation, CTL response and specific total IgG, IgG2a and IgG2b response, while the FLA-E7d vaccine was associated with more IFN-γ, and IL-17 cytokine response. The results of this study proved the ability of FLA as an adjuvant in fusion with E7d in the induction of cellular and humoral immune responses. In addition, it also emphasizes that antigen-adjuvant orientation can affect the immune response strength and polarization against HPV E7d vaccine candidate. HIGHLIGHTS: Flagellin is attached to HPV-16 E7d at the C- or N-terminus to create E7d-FLA and FLA-E7d candidate vaccines. The E7d-FLA vaccine showed a significant increase in lymphocyte proliferation, CTL response and IgG response versus FLA-E7d vaccine. The FLA-E7d vaccine is associated with a significant increase in IFN-γ and IL-17 cytokines response versus E7d-FLA vaccine. It seems that that antigen-adjuvant orientation is an important parameter in the strength and polarization of immune response in HPV E7d vaccine candidate.