Small ruminant lentivirus capsid protein (SRLV-p25) antigenic structural prediction and immunogenicity to recombinant SRLV-rp25-coupled to immunostimulatory complexes based on glycyrrhizinic acid

Abstract

Small Ruminant Lentiviruses (SRLV) infect sheep and goats resulting in significant economic losses. This study evaluated for the first time the predicted conformational structure of the SRLV-capsid-protein 25 (SRLV-p25) and analysed the antigenicity of recombinant protein (SRLV-rp25) in mice by coupling to an immunostimulatory complexes based on glycyrrhizinic acid liposomes (GAL) and tested plasma from goats and sheep naturally infected. Analysis in silico and conformational structure of SRLV-p25 (genotype B-FESC-752) showed similar characteristics to other lentiviral capsids. Efficient expression of SRLV-rp25 was confirmed by Western Blot. The humoral immune responses in mice showed an increased level of antibodies from day 21 to 35 of the SRLV-rp25-GAL and SRLV-rp25-ISCOM® groups and the cellular immune response showed no significant difference in IL-10 levels (P > 0.05), however a significant difference (P < 0.001) was observed when comparing SRLV-rp25-GAL with SRLV-rp25 groups. Immunoreactivity towards SRLV-rp25 revealed 61% of positive samples from naturally infected goats and sheep.

A Candidate Antigen of the Recombinant Membrane Protein Derived from the Porcine Deltacoronavirus Synthetic Gene to Detect Seropositive Pigs

“…The antibody response was dramatically higher using a Matrix-M TM as an adjuvant. Matrix-M TM selection was according to our research group [ 55 , 72 , 73 ]; we have observed that this immunostimulatory complex is an adjuvant that enhances strong immunogenicity using advanced technology in a nanocarrier to deliver antigens [ 59 ]. These results are in agreement with previously reported studies, which described antigen enhanced with Matrix-M TM as being highly immunogenic and inducing both antibody and cellular immune responses [ 57 ].…”

“…A secondary antibody (mouse anti-IgG-HRP) was used with a dilution of 1:20,000. The chromogenic reaction was developed using 3,3′,5,5-tetramethylbenzidine (TMB) substrates (Sigma-Aldrich, St. Louis, MO, USA), as previously described [ 58 , 59 ] and measured at an absorbance of 450 nm. Statistical analyses were performed using two-way ANOVA to compare immunized groups with protein plus adjuvant versus protein alone on different days.…”

“…An iELISA was performed according to J. S. Cuevas-Romero et al [ 59 ]. Briefly, to standardize the ELISA assay, different concentrations of antigen (25, 50 and 75 ng), dilutions of the sera sample (1:200, 1:300, 1:500, 1:600, 1:800 and 1:1000) and dilutions of the secondary antibody were evaluated (1:10,000, 1:12,000, 1:15,000 and 1:20,000), and 75 ng of antigen, 1:1000 diluted sera and 1:20,000 diluted secondary antibody were the optimal conditions to perform the test.…”

Development of Glycyrrhizinic Acid-Based Lipid Nanoparticle (LNP-GA) as An Adjuvant That Improves the Immune Response to Porcine Epidemic Diarrhea Virus Spike Recombinant Protein

“…They were then centrifuged, pelleted, and solubilized in 5% N-lauroylsarcosine sodium salt and 50 mM Tris-HCl pH 7.5 (250 rpm, 12 h, 25 °C). rNTD-S were purified using immobilized-metal affinity chromatography (IMAC) according to the procedure described by Lara et al [ 29 ], it was dialyzed (Tris-HCl 5 mM, pH 8 buffer), quantified with the Bradford method [ 21 ], and confirmed by SDS-PAGE and WB before immunization of mice. Detection in WB was performed using anti-6x-His-Tag diluted 1:5000 (Invitrogen, Carlsbad, CA, USA) as primary antibody and a mouse anti-IgG conjugated to horseradish peroxidase (dilution 1:5000) (Sigma-Aldrich, St. Louis, MO, USA) as a secondary antibody.…”

“…GA and its derivatives can form intermolecular complexes and micelles containing drug molecules for targeted delivery [ 18 , 19 ]. In addition, formulations based on phospholipids, cholesterol, and GA coupled with a viral antigen generated an increase in immune responses compared to the antigen alone, indicating that GA has promising effects if used within a vaccine antigen delivery system [ 20 , 21 ].…”