ABSTRACT
In December 2019, a beta-coronavirus called SARS-Cov 2 emerged in the Chinese city of Wuhan, causing an outbreak of unusual and severe bilateral pneumonia. The virus managed to spread rapidly, expanding westward with a high contagion rate, unleashing the most important pandemic of the last hundred years. This generated a collapse not only in health systems but also in international trade, cutting the supply chain of medical supplies. The first official case registered in our country occurred at the beginning of March 2020. Faced with this scenario, our laboratory presented a proposal to the National Executive Power for the development and manufacture of molecular diagnostic tests and columns for RNA purification, two critical inputs necessary to meet the growing demand of the national diagnostic network. Thanks to the financing of the Corporación Andina de Fomento (CAF), we established a public-private consortium between IIB-UNSAM, the UNQ molecular biology laboratory, and the companies Productos Bio-Lógicos SA and Chemtest SA who contributed their human and technical resources, and administrative capacities to carry out the task. The consortium with the collaboration of different dependencies of the National State brought from China the critical supplies for the development and production of 700,000 manual and automated RNA purification kits that were distributed throughout the country. Also, an isothermal amplification method of viral genetic material followed by detection of nucleic acid by lateral flow immunochromatographic assay (NALFIA) was developed. The kit, called ELA-CHEMSTRIP, combines bio and nano components developed and manufactured entirely in the country, allowing the detection of the viral genetic material present in a swab sample with a detection limit, sensitivity, and diagnostic specificity equivalent to RT-PCR but without the need for sophisticated thermal cyclers. This technology made it possible to decentralize the COVID 19 diagnosis and implement it even in rural areas where there was no infrastructure for molecular diagnosis. In this way, we took advantage of a unique historical opportunity that allowed us to articulate actions and capacities of both the public and private sectors, converging on a common goal. The challenge for the future is to expand and consolidate these capacities to generate positive feedback that enables the development of a national biotechnology industry facing the challenges of the 21st century.
ABSTRACT
Since SARS-COV-2 virus spread worldwide and COVID-19 turned rapidly into a pandemic illness, the necessity for vaccines and diagnostic tests became crucial. The viral surface is decorated with Spike, the major antigenic determinant and main target for vaccine development. Within Spike, the receptor binding domain (RBD), constitutes the main target of highly neutralizing antibodies found in COVID-19 convalescent plasma. Besides vaccination, another important aspect of Spike (and RBD) is their use as immunogen for the development of poli- and monoclonal antibodies (mAbs) for therapeutic and diagnostic purposes. Here we report the development and preliminary biochemical characterization of a set of monoclonal antibodies against the Spike RBD domain along with the recombinant expression of two mayor COVID-19 protein reagents: the viral Spike RBD domain and the extracellular domain of the human receptor ACE2. RBD and the extracellular domain of ACE2 (aa 1-740) were obtained through transient gene transfection (TGE) in two different mammalian cell culture systems: HEK293T adherent monolayers and Expi293F™ suspension cultures. Due to its low cost and ease scale-up, all transfections were carried with polyethyleneimine (PEI). Expressed proteins were purified from culture supernatants by immobilized metal affinity chromatography. Anti-RBD mAbs were developed from two different immunization schemes: one aimed to elicit antibodies with viral neutralizing potential, and the other with the ability to recognize denatured RBD for routine lab immunoassays. To achieve this, the first group of mice was immunized with RBD in aluminum salts (RBD/Al) and the other with RBD emulsified in Freunds adjuvant (RBD/FA). Polyclonal and monoclonal antibody reactivities against native or denatured RBD forms were then assessed by ELISA. Complete RBD denaturation was followed by intrinsic fluorescence spectral changes upon different physicochemical stress treatments. As expected, RBD/Al immunized mice developed an antibody response shifted to native RBD while those immunized with RBD/FA showed a high response against both forms of the protein. In accordance with the observed polyclonal response, RBD/FA derived mAbs recognize both, native and denatured RBD. On the contrary, hybridomas generated from the RBD/Al protocol mostly recognize RBD in its native state. Further ELISA binding assays revealed that all RBD/FA derived mAbs can form a trimeric complex with ACE2 and RBD, denoting they would not have viral neutralizing activity. ELISA competition assays with the RBD/ACE2 complex aimed to determine the neutralization potential of the RBD/Al derived mAbs are under way. Overall, the anti-Spike RBD mAbs and the recombinant RBD and ACE2 proteins presented here constitute valuable tools for diverse COVID-19 academic research projects and local immunity surveillance testing.