Monoclonal antibody generated against Nile tilapia (Oreochromis niloticus) IgT heavy chain using a peptide-based strategy.

Teleost IgT/Z plays a principal role in the defense mechanisms against infectious agents in the mucosal compartments and in systemic immunity. Previously, Nile tilapia (Oreochromis niloticus) IgT was discovered and characterized at transcription level. In this work, we generated a monoclonal antibody (mAb) that specifically recognized the Nile tilapia IgT. BALB/c mice were immunized with three synthetic peptides conjugated to KLH. The sequences of these peptides derived from the constant region of the Nile tilapia IgT heavy chain. ELISA and Western blotting confirmed the specificity of the polyclonal sera and the culture supernatant from a positive hybridoma clone. We observed immunoreactivity against a recombinant IgT fragment and native IgT in skin mucus. The anti-IgT mAb did not cross-react with purified tilapia IgM. Direct ELISA analysis allowed the quantification of skin mucus IgM and IgT concentrations. Flow cytometry analysis revealed differences in the percentage of IgT+ B cell populations between juveniles and adults in peripheral blood, head kidney and spleen lymphocytes and among the tissues analyzed. For further validation of the anti-IgT mAb utility, a recombinant vaccine candidate against sea lice (TT-P0 Ls) was injected into juvenile tilapia. Direct ELISA results revealed a differential secretion of skin mucus IgT and IgM after immunostimulation. In addition, the percentages of IgT+ B cells were determined at 7 days after booster and ex-vivo stimulation by flow cytometry. This mAb constitutes an important immunological tool to study the biological function and structural characteristics of tilapia IgT.

SARS-CoV-2 spike RBD-specific IgA and IgG antibodies in breast milk after vaccination with the protein subunit vaccine Abdala.

Background: COVID-19 vaccines that trigger a strong secretory antibody response in breast milk may achieve effective passive protection of vulnerable newborns and breastfed infants of immunized mothers. The aim of this work was to investigate the presence of SARS-CoV-2 spike RBD-specific IgA and IgG antibodies in breast milk, 5 and 9 weeks after vaccination with 3 doses of the protein subunit vaccine Abdala, compared to those found in breast milk from COVID-19-recovered women, collected at least 40 days after the infection. Methods: SARS-CoV-2 spike RBD-specific IgA and IgG antibodies were semi-quantified by indirect ELISA, using a homemade standard generated by pooling twenty breast milk samples with high absorbance values according to preliminary data. The validity of the standard curves was proved following the European Medicines Agency Guideline. Two breast milk samples from 2 unvaccinated women who had not been infected with COVID-19 were included as negative controls. Potentially neutralizing antibodies was assessed by a SARS-CoV-2 surrogate virus neutralization test. Results: High levels of anti-RBD IgA antibodies were detected in breast milk samples 9 weeks after vaccination and anti-RBD IgG antibodies rise from the fifth to the ninth week. In the post-COVID-19 time that was evaluated, the IgG-type response was notably higher compared to both post-vaccination periods. Neutralizing antibody titers were similar in breast milk from vaccinated and COVID-19 recovered women. Conclusions: This is the first report about the immune response in breast milk after the administration of a COVID-19 protein subunit vaccine, which could provide analogous protection to that conferred by SARS-CoV-2 infection. This implies a potential passive immunity that breastfed infants receive from their mothers vaccinated with Abdala.

Murine monoclonal antibodies against RBD of the SARS-CoV-2 spike protein as useful analytical tools for subunit vaccine development and clinical trials.

COVID-19 pandemic poses a serious threat to human health; it has completely disrupted global stability, making vaccine development an important goal to achieve. Monoclonal antibodies play an important role in subunit vaccines strategies. In this work, nine murine MAbs against the RBD of the SARS-CoV-2 spike protein were obtained by hybridoma technology. Characterization of purified antibodies demonstrated that five of them have affinities in the order of 108 L/mol. Six MAbs showed specific recognition of different recombinant RBD-S antigens in solution. Studies of the additivity index of anti-RBD antibodies, by using a novel procedure to determine the additivity cut point, showed recognition of at least five different epitopes. The MAbs CBSSRBD-S.11 and CBSSRBD-S.8 revealed significant neutralizing capacity against SARS-CoV-2 in an ACE2-RBD binding inhibition assay (IC50 = 85.5pM and IC50 = 122.7pM, respectively) and in a virus neutralizing test with intact SARS-CoV-2 (VN50 = 0.552 nM and VN50 = 4.854 nM, respectively) when D614G strain was used to infect Vero cells. Also CBSSRBD-S.11 neutralized the SARS-CoV-2 strains Alpha and Beta: VN50 = 0.707 nM and VN50 = 0.132 nM, respectively. The high affinity CBSSRBD-S.8 and CBSSRBD-S.7 recognized different epitopes, so they are suitable for the development of a sandwich ELISA to quantitate RBD-S recombinant antigens in biomanufacturing processes, as well as in pharmacokinetic studies in clinical and preclinical trials.