COVID-19 Infection Detection and Prevention by SARS-CoV-2 Active Antigens: A Synthetic Vaccine Approach.

COVID-19, a global pandemic causing to date more than 50 million cases and more than a million deaths, has to be controlled. SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) was identified as the causative agent. Controversy about this virus origin and infectious mechanism for adapting to humans remains a matter for discussion. Among all strategies for obtaining safe and potent vaccines, approaches based on attenuated-killed virus and non-replicating RNA viral vectors are demonstrating promising results. However, specificity of viral components targeted by human antibodies so far has not been demonstrated. A consistent strategy for obtaining functional-active antigens from SARS-CoV-2 specific ligands lead us to propose and test a number of synthetic components. From hundreds of starting sequences only fifteen fulfilled the design requirements and were produced as monomer and polymer forms and immuno-chemically tested. The design was based on worldwide representative reported virus genomes. A bioinformatics scheme by conventional methods and knowledge on MHC-I and II antigen processing mechanisms and HLA haplotype-restriction was performed including sensitive and resistant human populations to virus infection. Covid-19 patients' sera reactivity for synthetic SARS-CoV-2-designed components have proven a high recognition of specific molecules, as well as some evidence for a long-lasting humoral immune response.

Vigilancia tecnológica e inteligencia competitiva de un desarrollo tecnológico para la detección de Giardia, una innovación en salud

[RESUMEN]. La giardiasis es una enfermedad causada por Giardia duodenalis, protozoario enteropatógeno que también es responsable de la enfermedad diarreica aguda y la gastroenteritis en humanos. Dadas las características de excreción intermitente propias de este parásito, la probabilidad de obtener resultados falsos negativos durante el proceso de diagnóstico es alta. En el Grupo de Parasitología del Instituto Nacional de Salud de Colombia se identificaron proteínas inmunogénicas presentes en quistes y trofozoítos del parásito en aislamientos colombianos de Giardia con potencial comercial y posibilidad de ser patentadas. Se realizó un estudio exploratorio descriptivo a partir de las características de la técnica en estudio, su estado de desarrollo y la viabilidad comercial de la invención. Se encontraron 390 patentes y se seleccionaron cinco invenciones similares que no representaban ningún riesgo frente a una eventual solicitud de patente. De los 36 estuches para el diagnóstico de Giardia disponibles en el mercado y las 21 licencias sanitarias otorgadas hasta diciembre del 2017, sólo cuatro y tres, respectivamente, proponen técnicas similares a la de la estudiada, pero ninguna de ellas cuenta con un registro de patente relacionado. La técnica en desarrollo es innovadora y puede aplicarse en el campo de la salud pública en la Región de las Américas con beneficios previsibles.

[ABSTRACT]. Giardiasis is a disease caused by Giardia duodenalis, an intestinal protozoan that causes acute diarrhea and gastroenteritis in humans. Given the characteristic intermittent excretion of this parasite, there is a high probability of false negative results during diagnosis. The Parasitology Group of Colombia’s National Institute of Health identified immunizing proteins present in the parasite’s cysts and trophozoites in Colombian Giardia isolates with commercial and patentability potential. A descriptive exploratory study was conducted that focused on characteristics of the technique under study, its state of development, and the commercial viability of the invention. A total of 390 patents were found and five similar inventions were selected that did not pose any risk regarding an eventual patent application. Of the 36 Giardia diagnostic kits available on the market and the 21 health licenses granted as of December 2017, only four and three, respectively, offer techniques similar to the technique studied, but none of them has a related patent on record. The technique under development is innovative and can be applied in the field of public health in the Region of the Americas with foreseeable benefits.

[RESUMO]. A giardíase é uma doença causada pela Giardia duodenais, protozoário enteropatogênico causador de doença diarreica aguda e gastroenterite em seres humanos. Como uma das características deste parasita é a excreção intermitente, a probabilidade de se obter resultados falso-negativos no processo diagnóstico é alta. O Grupo de Parasitologia do Instituto Nacional de Saúde da Colômbia identificou proteínas imunogênicas presentes em cistos e trofozoítos do parasita em isolados colombianos de Giardia com potencial comercial e possibilidade de patenteamento. Foi realizado um estudo exploratório descritivo das características da técnica em estudo, situação de desenvolvimento e viabilidade comercial da invenção. Foram encontradas 390 patentes e selecionadas cinco invenções similares sem risco de eventual pedido de patente. Dos 36 kits de diagnóstico de Giardia comercializados e 21 licenças sanitárias concedidas até dezembro de 2017, apenas quatro kits e três licenças de produtos empregam técnicas semelhantes à estudada, mas nenhum deles tem um registro de patente relacionado. A técnica em desenvolvimento é inovadora e pode ser empregada em saúde pública na Região das Américas com benefícios esperados.

Detection of Giardia duodenalis antigen in human fecal eluates by enzyme-linked immunosorbent assay using polyclonal antibodies

The present study developed and standardized an enzime-linked immunosorbent assay (ELISA) to detect Giardia antigen in feces using rabbit polyclonal antibodies. Giardia cysts were purified from human fecal samples by sucrose and percoll gradients. Gerbils (Meriones unguiculatus) were infected to obtain trophozoites. Rabbits were inoculated with either cyst or trophozoite antigens of 14 Colombian Giardia isolates to develop antibodies against the respective stages. The IgG anti-Giardia were purified by sequential caprylic acid and ammonium sulfate precipitation. A portion of these polyclonal antibodies was linked to alkaline phosphatase (conjugate). One hundred and ninety six samples of human feces, from different patients, were tested by parasitologic diagnosis: 69 were positive for Giardia cysts, 56 had no Giardia parasites, and 71 revealed parasites other than Giardia. The optimal concentration of polyclonal antibodies for antigen capture was 40 æg/ml and the optimal conjugate dilution was 1:100. The absorbance cut-off value was 0.24. The parameters of the ELISA test for Giardia antigen detection were: sensitivity, 100 percent (95 percent CI: 93.4-100 percent); specificity, 95 percent (95 percent CI: 88.6-97.6 percent); positive predictive value, 91 percent (95 percent CI: 81.4-95.9 percent); and negative predictive value, 100 percent (95 percent CI: 96.1-100 percent). This ELISA will improve the diagnosis of Giardia infections in Colombia and will be useful in following patients after treatment

Detection of Giardia duodenalis antigen in human fecal eluates by enzyme-linked immunosorbent assay using polyclonal antibodies.

The present study developed and standardized an enzime-linked immunosorbent assay (ELISA) to detect Giardia antigen in feces using rabbit polyclonal antibodies. Giardia cysts were purified from human fecal samples by sucrose and percoll gradients. Gerbils (Meriones unguiculatus) were infected to obtain trophozoites. Rabbits were inoculated with either cyst or trophozoite antigens of 14 Colombian Giardia isolates to develop antibodies against the respective stages. The IgG anti-Giardia were purified by sequential caprylic acid and ammonium sulfate precipitation. A portion of these polyclonal antibodies was linked to alkaline phosphatase (conjugate). One hundred and ninety six samples of human feces, from different patients, were tested by parasitologic diagnosis: 69 were positive for Giardia cysts, 56 had no Giardia parasites, and 71 revealed parasites other than Giardia. The optimal concentration of polyclonal antibodies for antigen capture was 40 g/ml and the optimal conjugate dilution was 1:100. The absorbance cut-off value was 0.24. The parameters of the ELISA test for Giardia antigen detection were: sensitivity, 100% (95% CI: 93.4-100%); specificity, 95% (95% CI: 88.6-97.6%); positive predictive value, 91% (95% CI: 81.4-95.9%); and negative predictive value, 100% (95% CI: 96.1-100%). This ELISA will improve the diagnosis of Giardia infections in Colombia and will be useful in following patients after treatment.

Detección de antígeno de Giardia en materia fecal de Meriones unguiculatus (Gerbil) por ELISA utilizando anticuerpos policlonales / Detection of Giardia antigen in Meriones unguiculatus (Gerbil) faeces by ELISA using policlonal antibodies

La identificación de Giardia lamblia en heces puede fracasar debido a la eliminación intermitente de quiste y/o trofozoítos del parásito. Actualmente, puede recurrirse a la detección de antígeno del parásito en materia fecal comprobando que el huésped está infectado y/o enfermo circunstancia que ofrece una gran ventaja para el tratamiento oportuno de paciente y de manera indirecta para el control de la enfermedad de las personas que se encuentran alredodor de éste. En este trabajo, se estandarizó y evaluó el ensayo inmunoenzimático ELISA utilizando anticuerpos policlonales anti-quiste y antitrofozoíto de cepas colombianas de Giardia desarrollados en conejo para la detección de antígeno del parásito en heces de gerbils (Meriones unguiculatus), modelo animal para estudios de giardiasis, como paso previo para la detección de antígeno en heces humanas. Para ello, se purificaron quistes de Giardia a partir de heces humanas mediante gradientes de sucrusa y percoll para infectar gerbils y obtener periódicamente quistes y trofozoítos del parásito. Posteriormente con la finalidad de obtener anticuerpos policlonales anti-quiste y anti-trofozoítos del parásito, se inocularon conejos independientemente con antígeno de quiste y trofozoíto de Giardia. Se realizó una mezcla de anticuerpos policlonales anti-quiste y anti-trofozoíto de Giardia, en una proporción 1:2 respectivamente, previa purificación de éstos mediante precipitación secuencial con ácido caprílico y sulfato de amonio y se eleboró un conjugado con parte de los anticuerpos policlonales uniéndole a éstos fosfatasa alcalina. Finalmente se realizó el diagnóstico parasitológico a 47 heces de gerbils infectados previamente con quistes de giardia y 55 heces de gerbils no infectados. Se observó quistes de Giardia en las 47 heces de gerbils infectados (muestras positivas) y ningún parásito intestinal en los animales no infectados (muestra negativas). La concentración óptima de anticuerpos policlonales fue de 40 µgr/ml y la dilución óptima de conjugado fue de 1:400. El valor de absorbancia (punto de corte) que diferenció una muestra negativa de una positiva fue de 0.142. Los parámetros de la prueba fueron: sensibilidad: 91 por ciento, especificidad: 93 por ciento, valor predictivo positivo: 91 por ciento y valor predictivo negativo: 93 por ciento. El ELISA estandarizado y evaluado servirá como base para la detección de antígeno de Giardia en heces humanas