An engineered SARS-CoV-2 receptor-binding domain produced in Pichia pastoris as a candidate vaccine antigen.

Developing affordable and easily manufactured SARS-CoV-2 vaccines will be essential to achieve worldwide vaccine coverage and long-term control of the COVID-19 pandemic. Here the development is reported of a vaccine based on the SARS-CoV-2 receptor-binding domain (RBD), produced in the yeast Pichia pastoris. The RBD was modified by adding flexible N- and C-terminal amino acid extensions that modulate protein/protein interactions and facilitate protein purification. A fed-batch methanol fermentation with a yeast extract-based culture medium in a 50 L fermenter and an immobilized metal ion affinity chromatography-based downstream purification process yielded 30-40 mg/L of RBD. Correct folding of the purified protein was demonstrated by mass spectrometry, circular dichroism, and determinations of binding affinity to the angiotensin-converting enzyme 2 (ACE2) receptor. The RBD antigen also exhibited high reactivity with sera from convalescent individuals and Pfizer-BioNTech or Sputnik V vaccinees. Immunization of mice and non-human primates with 50 µg of the recombinant RBD adjuvanted with alum induced high levels of binding antibodies as assessed by ELISA with RBD produced in HEK293T cells, and which inhibited RBD binding to ACE2 and neutralized infection of VeroE6 cells by SARS-CoV-2. Additionally, the RBD protein stimulated IFNγ, IL-2, IL-6, IL-4 and TNFα secretion in splenocytes and lung CD3+-enriched cells of immunized mice. The data suggest that the RBD recombinant protein produced in yeast P. pastoris is suitable as a vaccine candidate against COVID-19.

Aplicación del reactivo de Neesler en la cuantificación de amonio para las fermentaciones de productos biotecnológicos / Application of Neesler reagent in the ammonium quantification used in the fermentations of biotechnology product

Las sales de amonio se utilizan en las fermentaciones para suplementar las cantidades deficitarias de nitrógeno y estabilizar el pH del medio de cultivo. El ion amonio en exceso, ejerce un efecto perjudicial en el proceso fermentativo ya que inhibe el crecimiento microbiano. Con el objetivo de monitorear y controlar la concentración de amonio durante el proceso de fermentación, se desarrolló un método con el reactivo de Neesler, para la cuantificación de dicho analito. El ensayo se estandarizó mediante: selección del equipo de medición; tiempo de reacción del ensayo y comparación de las sales estándares de amonio. El método se caracterizó con la evaluación de los parámetros: especificidad; linealidad y rango del sistema, límite de cuantificación, exactitud y precisión. El método demostró ser específico. Se establecieron dos sistemas con curvas que fueron lineales en los rangos de cloruro de amonio (2 a 20 µg/mL) y sulfato de amonio (5 a 30 µg/mL). Los límites de cuantificación fueron los puntos inferiores de cada rango de trabajo. El método resultó ser preciso y exacto. Este ensayo se aplicó a muestras de cultivos de levadura y bacterias del género Saccharomyces y Escherichia. coli respectivamente. Se desarrolló un método novedoso en microplaca para la cuantificación y control analítico del amonio. Mediante este método se controla este componente químico fundamental en las fermentaciones, para optimizar el medio de cultivo. Se logra una adecuada expresión de proteínas recombinantes y la obtención de candidatos vacunales para uso clínico(AU)

The ammonium salts are used in fermentations to supplement the deficient amounts of nitrogen and stabilize the pH of the culture medium. The excess ammonium ion exerts a detrimental effect on the fermentation process inhibiting microbial growth. An analytical method based on Neesler reagent was developed for monitoring and controlling the concentration of ammonium during the fermentation process. The test was standardized, by means of the selection of measuring equipment, and the reaction time as well as comparing standards of ammonium salts. The method was characterized with the evaluation of the next parameters: Specificity, Linearity and Range, Quantification Limit, Accuracy and Precision. The method proved to be specific. Two linear curves were defined in the ranges of concentrations of ammonium chloride salt (2-20 µg/ml) and ammonium sulfate salt (5-30 µg/ml). The limits of quantification were the lowest points of each one. The method proved to be accurate and precise. This assay was applied to samples of the yeast culture and bacteria of the genus Saccharomyces and E. coli respectively . A novel method in micro plate for quantification and analytical control of ammonia was developed. T his method is used to control this fundamental chemical component in the fermentations , to optimize the culture medium. Thus, an appropriate expression of recombinant proteins and proper vaccine candidates for clinical use are achieved(AU)