LLAMA (Lama glama) ANTISERUM AGAINST SARS-CoV-2

The receptor-binding domain (RBD) of SARS-CoV-2 Spike protein constitutes the key access for the virus inside the host cell. A positive correlation between titers of anti-RBD immunoglobulin G and antibodies (Ab) capable of neutralizing the virus has been demonstrated. In this context, passive immunotherapies such as convalescent plasma and hyperimmune equine serum have gained relevance as therapies against COVID-19. Another promising alternative is the use of polyclonal Ab from llamas (Lama glama), because of their unique features. For this reason, we aimed to obtain recombinant RBD as an immunogen to generate anti-RBD Ab in llamas. To achieve this, HEK 293 cells were transfected and transduced with the RDB encoding sequence, resulting in higher yields with this last method. The RBD was purified by affinity chromatography. An immunization schedule was designed and evaluated on two male animals, which were initially inoculated with RBD, followed by periodical boosters. Exploratory bleedings were performed in order to evaluate the reached titers, and larger bleedings in order to obtain enriched plasma with anti-RBD Ac. Ac quantification was accomplished by an in-house ELISA. Results showed that the immunization scheme was successful, achieving a maximum titer of 168000 at 28 days post-immunization. The results lay the foundations for the production of polyclonal anti-RBD Ab.

PURIFICATION OF ANTI-RBD ANTIBODIES OBTAINED FROM LLAMA (Lama glama) FOR POTENTIAL COVID-19 THERAPY

Despite worldwide efforts to develop different treatments for SARS-CoV-2 COVID-19, the situation remains critical, requiring rapid and effective strategies. In this regard, antibodies (Ab) have demonstrated clinical potential. Among them, camelid nanoAb (VHH) arise as a possible alternative, as they recognize epitopes which are inaccessible to conventional Ab. Further advantages of VHH are their small size, high solubility, high stability, and resistance to low pH. The aim of this work is to describe a purification scheme of different isotypes of anti-SARS-CoV-2 immunoglobulin G (IgG) produced after immunizing two llamas (Lama glama). To achieve this, plasma was injected into an affinity chromatographic column (Protein G), and the resulting fractions were analyzed by SDS-PAGE under non-reducing conditions. The anti-RBD titers were determined by an “in house” ELISA, reaching titers of 52000 and 13000 for IgG1 and IgG3 fractions, respectively. Subsequently, an affinity column (HiTrap NHS-activated) was prepared to separate monospecific anti-RBD polyclonal Ab. RBD produced in our laboratory was covalently coupled to this column, achieving a coupling efficiency of 97%. Different isotypes of monospecific anti-RBD Ab (IgG1: 140 kDa and IgG3: 95 kDa) were obtained. IgG3 represent the starting point for obtaining VHH and/or evaluating their potential use as a therapeutic or preventive alternative, which represents a notable regional contribution in the fight against COVID-19.